2024-03-28T11:49:11Zhttps://kuscholarworks.ku.edu/oai/requestoai:kuscholarworks.ku.edu:1808/42542018-01-31T20:08:08Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Blevins, Melanie
2008-10-06T22:08:39Z
2008-10-06T22:08:39Z
2008-05-05
http://dissertations.umi.com/ku:2538
http://hdl.handle.net/1808/4254
The cytochrome P450 (CYP) superfamily of enzymes plays the predominant role in human phase I xenobiotic metabolism. The CYP2 family, in particular, is known for it extensive Phase I metabolism of a majority of the xenobiotic compounds [1]. The goal of this project is to determine the structural foundation for the substrate selectivities of the CYP2A6 and CYP2A13 enzymes versus CYP2E1. Because these enzymes metabolize both common, as well as unique, small molecule substrates, it is likely that only few key residue-substrate interactions are responsible for those metabolic capabilities that differ between them. Amino acid residues in regions of the CYP2E1 protein likely to contact ligands and that differ between CYP2E1 and the CYP2A enzymes were examined by site-directed mutagenesis. The resulting mutated CYP2E1 proteins were characterized for their ability to hydroxylate the reportedly selective CYP2E1 substrates p-nitrophenol (pNP) [2] and chlorzoxazone (CZN) [3], but none showed significant differences in activity from the CYP2E1 wild type enzyme. However, in contrast to previous literature reports [4], both CYP2A6 and CYP2A13 were observed to metabolize both CYP2E1 substrates pNP and CZN with catalytic efficiencies equal to or greater than CYP2E1. These unexpected activities of the CYP2A enzymes with CYP2E1 substrates demonstrate that the human CYP2A and CYP2E enzymes are more functionally similar than previously believed. References 1 Rendic, S. and Di Carlo, F. J. (1997) Human cytochrome P450 enzymes: a status report summarizing their reactions, substrates, inducers, and inhibitors. Drug Metab Rev 29, 413-580 2 Koop, D. R., Laethem, C. L. and Tierney, D. J. (1989) The utility of p-nitrophenol hydroxylation in P450IIE1 analysis. Drug Metab Rev 20, 541-551 3 Peter, R., Bocker, R., Beaune, P. H., Iwasaki, M., Guengerich, F. P. and Yang, C. S. (1990) Hydroxylation of chlorzoxazone as a specific probe for human liver cytochrome P-450IIE1. Chem Res Toxicol 3, 566-573 4 Zerilli, A., Ratanasavanh, D., Lucas, D., Goasduff, T., Dreano, Y., Menard, C., Picart, D. and Berthou, F. (1997) Both cytochromes P450 2E1 and 3A are involved in the O-hydroxylation of p-nitrophenol, a catalytic activity known to be specific for P450 2E1. Chem Res Toxicol 10, 1205-1212
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Chemistry
Biochemistry
Cytochrome p450 2e1
Cytochrome p450 2a13
Cytochrome p450 2a6
P-nitrophenol
Chlorzoxazone
Human Cytochrome P450 2E1: Functional Comparison to Cytochrome 2A13 and 2A6
Thesis
oai:kuscholarworks.ku.edu:1808/97442020-07-14T14:37:22Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Brandt, Gary E. L.
2012-06-03T14:43:40Z
2012-06-03T14:43:40Z
2011-12-31
http://dissertations.umi.com/ku:11918
http://hdl.handle.net/1808/9744
Abstract: The 90 kDa heat shock protein (Hsp90) is a molecular chaperone that is critical cellular survival and growth under both typical and stressful conditions. Hsp90 is responsible for the maturation and stability of more than 200 client proteins involved in a diverse assortment of cellular processes. Disruption of Hsp90's chaperoning activity causes client protein degradation and ultimately leads to cytostasis and/or apoptosis. While this phenomenon is observed in normal cells, the effects of Hsp90 inhibition are more pronounced in oncogenic cell lines as a result of higher expression levels and increased cellular dependence on Hsp90 activity. As such, targeting Hsp90 inhibition with small molecules has emerged as a powerful strategy for the development of anticancer chemotherapeutics. Several small molecule Hsp90 inhibitors are currently under evaluation in FDA sanctioned clinical trials for the treatment of various cancers, however, some undesired side effects have been observed. All of the Hsp90 targeting small molecules involved in these trials are ATP competitive inhibitors that bind at the N-terminal ATP binding domain. Inhibitors of this class elicit non-specific client protein degradation and cause the induction of the heat shock response that results in an upregulation of Hsp90 and other Hsp expression levels following incubation within cells. As a result, untoward toxicological effects are observed and the determination of appropriate dosing schedules to mitigate the heat shock response is highly complicated. A new strategy for Hsp90 inhibition capable of targeting specific client proteins for therapeutic efficacy that avoids heat shock response induction is desired. Presented herein are preliminary studies that investigate potential strategies to target the selective degradation of Hsp90 client proteins while avoiding the heat shock response. Specifically, small molecule natural products that elicit Hsp90 co-chaperone disruption are considered and the chemical and biological results are discussed. These studies provide the first steps toward developing a second generation of Hsp90 inhibitors that circumvent the detrimental effects observed for clinically evaluated inhibitors.
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Chemistry
Pharmaceutical sciences
Biochemistry
Co-chaperone disruption
Cruentaren a
Gedunin
Heat shock response
Hsp90
Trienomycin a
The Pursuit of Unshocking Hsp90 Inhibitors: Development of Gedunin and Cruentaren A as Chemical Leads
Dissertation
oai:kuscholarworks.ku.edu:1808/275772020-10-13T13:45:14Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Charaschanya, Manwika
2019-01-01T20:13:23Z
2019-01-01T20:13:23Z
2018-05-31
http://dissertations.umi.com/ku:15848
http://hdl.handle.net/1808/27577
https://orcid.org/0000-0002-2773-240X
This dissertation comprises three chapters, which focus on the development of new synthetic methodologies and the construction of a screening collection. An Application of the Schmidt Reaction: Construction of an Azasteroid Library. Ring expansion chemistry is a powerful way of introducing a heteroatom substituent into carbocyclic frameworks. However, such reactions are limited by the tendency of a given substrate to afford only one of the two rearrangement products or fail to achieve selectivity at all. These limitations may prove critical when seeking to carry out late-stage functionalization of natural products as starting points in drug discovery. In this chapter, a stereoelectronically controlled ring expansion sequence towards selective and flexible access to complementary ring systems derived from commercial or readily synthesized steroidal substrates of the A- and D-rings is described. A requisite intermediate in the reaction was leveraged to afford over one hundred isomerically pure analogs with spatial and functional diversity. This regiodivergent rearrangement, and the concept of using chiral reagents to effect regiocontrol in chiral natural products, adds value to late-stage natural product diversification programs. New Variations of the Schmidt Reaction: A strong hydrogen-bond-donating solvent, hexafluoro-2-propanol (HFIP), was found improve the intermolecular reaction of ketones with trimethylsilyl azide and hydroxyalkyl azides. This study prompted the hypothesis for interrupting the classic Schmidt reaction with an added nucleophile reagent in HFIP. An extensive acid screen identified aluminum tribromide as a promoter for intercepting the Schmidt reaction iminium ion intermediate and combining it with subsequent reaction with 1,3,5-trimethoxybenzene to form substituted imines, enamides, and amines. This new variation of the Schmidt reaction provided access to unique heterocycles. Enabling Chemistry Technologies: High-Temperature and High-Pressure Continuous Flow Chemistry. The synthetic applications of a high-temperature and high-pressure flow reactor were investigated. The Gould-Jacobs reaction, nucleophilic aromatic substitution reaction with amine nucleophiles, and tert-butyloxycarbonyl deprotection in flow were explored. The protocols developed were applied to the high-throughput preparation of small-molecule libraries, as well as reaction telescoping, automation, and scaling.
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Chemistry
Pharmaceutical sciences
Diversity-oriented synthesis
Drug discovery
Flow chemistry
Medicinal chemistry
Organic chemistry
Synthetic methodology
Advances in Heterocyclic Synthesis through Ring Expansions and Flow Chemistry
Dissertation
oai:kuscholarworks.ku.edu:1808/239232018-02-02T21:51:13Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Yoo, Euna
2017-05-07T16:30:29Z
2017-05-07T16:30:29Z
2015-05-31
http://dissertations.umi.com/ku:13902
http://hdl.handle.net/1808/23923
Toll-like receptors (TLRs)-7/-8 are among pathogen recognition receptors (PRRs) present in the endosomal compartment that are activated by viral single-stranded RNA (ssRNA) as well as synthetic small molecules. TLR7/8 agonists hold promise as potential vaccine adjuvants, since they directly activate antigen-presenting cells and enhance T helper 1-driven immune responses. A general introduction to TLRs, with an emphasis on the role of TLR7/8 activation in innate and adaptive immune responses is presented in Chapter 1. Structure-activity relationship (SAR) studies in small molecule TLR8/7-agonistic ligands showed that thiazolo[4,5-c]quinolines display mixed TLR8/7 agonistic activities with the optimal C2-alkyl chain length being butyl (Chapter 2). In an ongoing search toward exploring alternative chemotypes, furo[2,3-c]pyridines with pyridoxal as the aldehyde component in a one-pot multicomponent Groebke-Blackburn-Bienaymé reaction were obtained and found to exhibit TLR8-dependent NF-kB activation and strong adjuvanticity without proinflammatory cytokine induction (Chapter 3). Combinatorial libraries using the Groebke-Blackburn-Bienaymé reaction have also yielded TLR7/8-inactive, but antibacterial imidazo[1,2-a]pyridines (Chapter 4). Based on the previously reported SARs on imidazoquinolines, the syntheses and biological evaluation of novel imidazo[4,5-c]pyridine analogues were undertaken, with modifications at the N4- and C6 positions, which afforded strong Type I IFN inducers in conjunction with attenuated proinflammatory profiles (Chapter 5). With the goal of defining structural requisites governing activity and selectivity at TLR7 and/or TLR8, we undertook scaffold-hopping approach, quantum chemical calculations followed by linear discriminant analyses that permitted the classification of inactive, TLR8-active, and TLR7/8 dual-active compounds, confirming the critical role of partial charges in determining biological activity (Chapter 6). Molecular conjugation of TLR7/8 agonists to hyaluronic acid (HA) was evaluated to enhance selective and targeted delivery of vaccine construct to draining lymph nodes while limiting systemic exposure. The superior adjuvanticity evoking affinity-matured high-avidity immunoglobulins after a single boost was observed with HA conjugate bearing dual TLR7/8 agonist (Chapter 7). Extensive SAR investigations in several TLR7/8 agonistic scaffolds and exploration as vaccine adjuvant candidates have incrementally improved our understanding of how these molecules activate innate and adaptive immune responses and also catalyzed novel approaches to vaccine design and development.
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Pharmaceutical sciences
Immunology
Exploration of Toll-like Receptor 7 and 8 Agonists as Potential Vaccine Adjuvants
Dissertation
oai:kuscholarworks.ku.edu:1808/62802020-07-30T13:52:30Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Bi, Lei
2010-06-09T02:48:59Z
2010-06-09T02:48:59Z
2010-02-26
http://dissertations.umi.com/ku:10740
http://hdl.handle.net/1808/6280
Tyloindicine F is a potent anticancer natural product first isolated from the Himalaya region of India. It demonstrated a unique profile in the NCI60 human tumor cell line anticancer drug screen. Due to its scarcity from natural sources, a total synthesis of tyloindicine F is desirable. Two attempts at the synthesis of this natural product have been conducted and are discussed. β-enaminones are a group of push-pull olefins whereas C-H functionalization/C-C coupling is a highly efficient method of constructing new carbon-carbon bonds. β-enaminones are highly polarized and their β; position is suitable for C-H functionalization by means of electropalladation. A novel methodology for the oxidative Hiyama coupling of β-enaminones has been developed and two new bifunctional activators/reoxidants have been found. Furthermore, a non-oxidative version for β-enaminone C-H functionalization and the decarboxylative coupling of β-enaminones were also investigated.
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Organic chemistry
Pharmaceutical chemistry
Β-enaminone
C-h functionalization
Methodology
Natural products
Total synthesis
Tyloindicine f
Part I. Approaches Toward the Total Synthesis of Tyloindicine F; Part II. Palladium-Catalyzed C−H Functionalization of β-enaminones
Dissertation
oai:kuscholarworks.ku.edu:1808/81742020-08-19T14:15:45Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Poole, Jennifer Lynn
2011-10-09T13:25:56Z
2011-10-09T13:25:56Z
2011-08-31
http://dissertations.umi.com/ku:11713
http://hdl.handle.net/1808/8174
The research presented herein describes the development of methods and applications of flow technology in discovery chemistry. The first two chapters highlight the use of several beneficial features offered by flow technology to increase the throughput, safety, and convenience of organic synthesis. The last two chapters describe the use of microfluidic technology as a platform for rapid reaction discovery. Working with researchers at Abbott Laboratories, a droplet-based library method was developed. This approach allowed for the preparation of a theoretically unlimited number of compounds in a single run using minimal amounts of material. The universal nature of this approach was subsequently demonstrated in the preparation of two 20-membered libraries based around thiazole and pyrazole cores. A second methodology study, also completed with researchers at Abbott Laboratories, took advantage of the intrinsic closed environment of flow systems which enabled the creation, reaction, and removal of noxious chemicals in situ. Using these features an in situ synthesis of isocyanides, reagents notorious for their unpleasant smell, was developed. Coupling this method to the Ugi four-component reaction, a series of medicinally relevant amides was synthesized. Reactions performed in the flow system experienced an overall reduction in transformation time from two-days to two-hours and gave yields that were generally higher than those for the same reactions performed on the benchtop or in the microwave. Expanding the application of this technology toward the discovery and development of new synthetic methodologies we partnered with the laboratory of Dr. John A. Porco, Jr. at Boston University to explore transformations of multifunctional substrates. Given the variant nature of these reactions, both a simple iminium ether and a densely functionalized iminium ether derived from a bicyclo[3.2.1]octanoid scaffold were explored. Multidimensional reaction screening on an automated microfluidic platform was employed to facilitate the simultaneous investigation of multiple reaction variables. While the majority of products obtained from the study resulted from expected modes of O- and N- alkylation, several interesting transformations were uncovered. These included the pseudo-dimerization of homophthalic anhydride, an unusual integration of the van Leusen sulfone, and an unexpected carbon-carbon bond forming event of ethyl diazoacetate and acetonitrile. Finally, in a follow-up study, collaboration with Boston University was continued to explore additional reactivity of the bicyclo[3.2.1]octanoid scaffold. Preliminary reaction screens uncovered the synthesis of a series of densely functionalized donor-acceptor cyclopropanes which resulted from the photochemical rearrangement of the bicyclic scaffolds. Expansion of the photochemical screening to a polycyclic iminium ether led to the first example of an aza-di-pi reaction of a charged iminium species. Subjection of the new cyclopropane scaffolds to a variety of reaction conditions led to the discovery of additional rearrangement reactions affording several structurally diverse chemotypes including a fused dihydropyran, a fused pyrrole, a bicyclic imide, and a complex cyclic imine.
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This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Organic chemistry
Pharmaceutical sciences
Aza-schmidt of hydroxyalkyl azides
Bicyclo[3.2.1]octanoid chemistry
Droplet-based library synthesis
Flow technology
In-situ generation of isocyanides
Reaction discovery
Diverse Applications of Flow Technology in Discovery Chemistry
Dissertation
oai:kuscholarworks.ku.edu:1808/216242020-10-22T13:21:34Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Caspers, Michael J.
2016-10-11T15:08:58Z
2016-10-11T15:08:58Z
2013-05-31
http://dissertations.umi.com/ku:13212
http://hdl.handle.net/1808/21624
While many ligands are known to interact with the opioid receptor system, most can be traced back to the common morphine scaffold. In several cases ligands have been developed that are selective for either the mu, kappa, or delta opioid receptors. Selective kappa opioid receptor agonists and antagonists have shown potential to be used to decrease cocaine self-administration and be utilized in the treatment of relapse. The emergence of salvinorin A as a novel scaffold, that selectively interacts with the kappa opioid receptor, retains therapeutic potential with a reduced side effect profile to the current treatments. Traditional mu opioid ligands, such as morphine, have life threatening side effects such as respiratory depression and constipation among other drawbacks like tolerance and dependence. To fully investigate the potential of salvinorin A as a viable alternative for therapeutic treatment it must be isolated in high purity for pharmacological evaluation. In efforts to isolate high purity salvinorin A, a reactive handle was generated that was shown to undergo cycloaddition to the furan ring of salvinorin A. This handle was developed using the reactivity limitations that were also investigated for the Diels-Alder cycloaddition of electron deficient alkenes and alkynes with the salvinorin A furan ring. In order to understand the unique mode of interaction salvinorin A imparts on the opioid receptor system, investigations into the pharmacological profile of salvinorin A were explored. Analytical methods were developed for the identification and quantification of salvinorin A from non-human primate cerebrospinal fluid and human plasma. This method was then exploited to develop a time-course graph for the measurement of in vivo concentrations of salvinorin A in various biological fluids which could be correlated to subjective and biological observations.
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openAccess
Copyright held by the author.
Chemistry
LC-MS/MS
Natural Product
Opioid
Opioid Receptor
Salvinorin A
Solid Support Reagent
Investigations Of Salvinorin A: Synthetic Isolation, Quantification, And In Vivo Characteristics
Thesis
oai:kuscholarworks.ku.edu:1808/62812020-07-30T14:11:33Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Niphakis, Micah James
2010-06-09T03:01:12Z
2010-06-09T03:01:12Z
2010-04-08
http://dissertations.umi.com/ku:10757
http://hdl.handle.net/1808/6281
This work is directed towards the development of safe phenanthropiperidines for the treatment of cancer. It focuses on synthetic methodologies that facilitate their preparation and biological studies to better understand the neurological side-effects of the only alkaloid within this class to enter clinical trials: tylocrebrine. The preparation of cyclic enaminones in high enantiomeric purity is made possible through a one-flask, two-step protocol that uses mild Boc-deprotection conditions to suppress racemization. Elaboration of the enaminone scaffold was made possible with a direct palladium(II)-catalyzed arylation using aryltrifluoroborates as coupling partners. These methods give access to enantiomerically pure 3-arylpiperidines which were used as precursors of phenanthropiperidine alkaloids. A small phenanthropiperidine library was prepared and studied to elucidate the cause of tylocrebrine's neurological side-effects. Although the causes remain elusive, tylocrebrine and several of its analogs were found to bind to key biogenic amine receptors, disclosing another potential risk factor for their therapeutic use.
EN
openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Organic chemistry
Pharmaceutical chemistry
Enaminone
Palladium
Phenanthropiperidine
Tylocrebrine
Tylophora indica
Tylophorine
Phenanthropiperidine Alkaloids: Methodology Development, Synthesis and Biological Evaluation
Dissertation
oai:kuscholarworks.ku.edu:1808/261592021-09-29T15:37:28Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Forsberg, Leah Kathleen
2018-03-09T22:44:41Z
2018-03-09T22:44:41Z
2017-12-31
http://dissertations.umi.com/ku:15671
http://hdl.handle.net/1808/26159
The heat shock proteins are a highly conserved protein family that are constitutively expressed and function as molecular chaperones. Molecular chaperones 90kDa Heat Shock Protein (Hsp90) and 70kDa Heat Shock Protein (Hsp70) have emerged as promising therapeutic targets for both cancer and neurodegenerative diseases. Hsp90 is responsible for the maturation of more than 300 nascent polypeptides, “clients”. These client proteins are involved in the oncogenic process, as many are associated with all 10 hallmarks of cancer. Hsp70 is involved in protein folding and maintenance of protein homeostasis. Targeting molecular chaperones, Hsp70 and Hsp90, is a viable therapeutic strategy for various neurodegenerative diseases as they prevent protein aggregation via refolding denatured proteins and solubilizing protein aggregates. Therefore, small molecules that interact with Hsp90 are sought, as modulation of Hsp90 can impact the cellular function of Hsp70. Hsp90 contains a traditional N-terminal ATP-binding site and a C-terminal dimerization domain, which contains an additional binding site. Targeting the Hsp90 C-terminus with inhibitors derived from novobiocin is one approach to modulating molecular chaperones. Structure activity relationship studies on novobiocin have led to the development of either neuroprotective or cytotoxic compounds. Segregation of the pro-survival heat shock response from a cytotoxic response due to client protein degradation is unique to C-terminal inhibitors. Described herein is the design, synthesis and biological evaluation of small molecules that target the C-terminus of Hsp90, for the continued development of potential therapeutics for the treatment of cancer or neurodegenerative diseases.
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openAccess
Copyright held by the author.
Chemistry
The Development of Small Molecules that Modulate Molecular Chaperones Hsp90 and Hsp70
Dissertation
oai:kuscholarworks.ku.edu:1808/298422021-10-28T19:35:01Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Gao, Zhe
2019-12-10T20:41:30Z
2019-12-10T20:41:30Z
2019-08-31
http://dissertations.umi.com/ku:16696
http://hdl.handle.net/1808/29842
https://orcid.org/0000-0002-4468-9773
A key enabling technology in biological sciences involves fluorescent probes. These probes are typically small molecules, proteins, or nucleic acids that either possess intrinsic fluorescence or are linked to a fluorophore that emits photons and can be detected by techniques such as fluorescence spectroscopy, imaging, or flow cytometry. In early-stage drug discovery projects, fluorescent probes can be used to sort and differentiate particular types of cells, conduct high-throughput screening campaigns, and image subcellular compartments. In this dissertation, I describe the use of fluorescent probes to study microtubules and mitochondria in living cells. These structures and organelles are of substantial interest in fundamental cellular biology and as targets of anticancer agents. One of these projects is focused on the anticancer agent Paclitaxel (Taxol). This small molecule that binds microtubules and is one of the most effective treatment for patients with breast, ovarian, and lung cancers. Remarkably, although Taxol can shrink slow-growing tumors in some patients, this drug spares rapidly proliferating cells such as bone marrow cells. This inconsistency has been termed the proliferation rate paradox and is not well understood. To probe the mechanism of action of Taxol, we designed and synthesized a drug-like fluorescent probe termed PB-Gly-Taxol. This compound recapitulates many aspects of the biological properties of Taxol in cells, and provides a new tool to study proliferation rate paradox. In a second project, I describe the discovery of a small molecule termed 2,7-difluoropyronin B that accumulates in hyperpolarized mitochondria of cancer cells. When irradiated with visible blue light, this probe depolarizes mitochondrial membranes, offering a new chemical tool for photochemical control over mitochondrial biology.
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openAccess
Copyright held by the author.
Biochemistry
Fluorescent probes
Microtubules
Mitochondria
P-glycoprotein
Pyronin
Taxol
Synthesis and Evaluation of Fluorescent Tools for Studies of Cancer Biology
Dissertation
oai:kuscholarworks.ku.edu:1808/148422020-10-07T14:15:15Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Joshi, Anand Anant
2014-07-28T02:32:53Z
2014-07-28T02:32:53Z
2013-08-31
http://dissertations.umi.com/ku:12896
http://hdl.handle.net/1808/14842
https://orcid.org/0000-0003-4940-7998
We are interested in the development of potent and highly selective dynorphin (Dyn) analogs targeting kappa opioid receptors (KOR) and studying the pharmacokinetic and physicochemical properties of kappa opioid peptides. In contrast to the extensive structure-activity relationships (SAR) studies on Dyn A, there is minimal SAR information on Dyn B. Therefore we performed an alanine scan of Dyn B amide. The results indicated that Tyr1 and Phe4 residues are critical, while Arg7 is important for maintaining the KOR affinity of Dyn B amide. There is also minimal SAR information for the selective KOR antagonist zyklophin [Nα-benzylTyr1-cyclo(D-Asp5,Dap8)]Dyn A(1-11)NH2 which is active in vivo after systemic administration. Hence, we synthesized linear and cyclic zyklophin analogs using solid phase peptide synthesis. To synthesize cyclic analogs the 5-11 linear fragments were selectively deprotected and cyclized, followed by extension of the peptide chain, coupling of the N-terminal N-alkyl amino acids and cleavage from the resin. We modified a key synthetic step to avoid potential racemization of the N-terminal residue. Pharmacological results suggested that the residue in position 5 has a greater influence on the KOR affinity of zyklophin than the residue in position 8. While Phe4 and Arg6 residues in zyklophin were important for maintaining KOR affinity, surprisingly Tyr1 and Arg7 were not important. The natural product CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) and its D-Trp isomer exhibited KOR activity after oral administration. Therefore, we are investigating the pharmacokinetic and physicochemical properties of these lead peptides. In Caco-2 cell monolayer permeability studies the D-Trp isomer exhibited 7-fold higher permeability in the apical to basolateral direction than CJ-15,208, and the natural product appeared to be an efflux substrate. The permeability of the D-Trp isomer was only 2-fold lower than that of the high permeability compound caffeine. We also examined various agents to solubilize these hydrophobic peptides that are compatible with in vivo studies. The D-Trp isomer had 5- to 40-fold higher solubility than the L-Trp isomer irrespective of the type of solubilizing enhancer used. These studies will help guide the future design of peptidic KOR ligands.
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openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Pharmaceutical sciences
Caco-2
Dynorphins
Kappa opioid receptors
Macrocyclic tetrapeptides
Sar
Zyklophin
Synthesis and biological evaluation of dynorphin analogs and, Caco-2 permeability of opioid macrocyclic tetrapeptides
Dissertation
oai:kuscholarworks.ku.edu:1808/113072018-11-20T17:26:35Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Blake, Linda Cherise
2013-06-21T22:29:19Z
2013-06-21T22:29:19Z
2012-05-31
http://dissertations.umi.com/ku:12059
http://hdl.handle.net/1808/11307
Cytochrome P450 (CYP) is a superfamily of heme-containing monooxygenase enzymes that metabolize a variety of endogenous and exogenous substrates. These transformations can be advantageous in the role of homeostasis or clearance of foreign compounds. However, aberrant CYP activity or biotransformations of procarcinogens can be detrimental to human health. Thus cytochrome P450 enzymes can be both therapeutic targets and counter-targets. In the process of drug discovery, in vitro evaluation of both the efficacy and selectivity of drug candidates is necessary before in vivo studies can be pursued. In the case of the xenobiotic-metabolizing cytochrome P450 2A13 (CYP2A13), in vitro analysis was used to identify and evaluate selective inhibitors for reducing the risk of lung cancer in tobacco users. Additionally, in vitro biochemical analysis of the steroidogenic cytochromes P450 21A2 (CYP21A2) and 11B1 (CYP11B1) is being pursued for counter-target evaluation in the development of selective CYP17A1 inhibitors for the treatment of prostate cancer and the rational design of selective CYP11B1 inhibitors for the treatment of cortisol-dependent diseases. Lung cancer is the leading cause of all cancer related deaths and results in 6 million annual deaths worldwide. Since 80% of all lung cancer incidence is attributed to tobacco use but tobacco cessation methods are unsuccessful in 95% of users, an increased emphasis has been placed on lung cancer chemoprevention. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most prevalent procarcinogens compounds in tobacco and is selectively activated by CYP2A13 metabolism in the respiratory tract. The resulting diazonium ions are able to form DNA adducts and initiate lung cancer. Therefore, the selective inhibition of CYP2A13 offers a novel therapeutic strategy in the chemoprevention of lung cancer. High throughput screening identified the benzylmorpholine scaffold, and a small library was evaluated for both binding (Kd) and inhibition (Ki) of CYP2A13 versus the 94% identical hepatic cytochrome P450 CYP2A6 (CYP2A6), which does not efficiently metabolize NNK. These investigations identified the structural features of benzylmorpholine analogs responsible for selective binding and inhibition of CYP2A13 versus CYP2A6, leading to the determination of structure-activity relationships for the benzylmorpholine scaffold. Docking and X-ray crystallography studies were further employed to identify the atomic-level interactions between benzylmorpholine analogs and CYP2A13 but were hampered by apparent binding in multiple orientations. Nevertheless, these results could be used to design additional selective and potent CYP2A13 inhibitors for reducing the risk of lung cancer in tobacco users who are unable, unwilling, or in the process of ceasing tobacco use. In a similar pursuit to identify inhibitors of CYP17A1 for the treatment of prostate cancer, it became important to evaluate the selectivity of potential drug candidates against obvious counter-targets. CYP21A2 is involved in the biosynthesis of glucocorticoids and mineralocorticoids and has overlapping substrates with CYP17A1. CYP11B1 follows CYP21A2 in the steroid biosynthetic pathway and is also a counter-target for the development of CYP17A1 inhibitors. Additionally, CYP11B1's crucial role in cortisol production also presents this enzyme as an independent therapeutic target for the treatment of Cushing's disease resulting from cortisol overproduction. However, biochemical studies for both human CYP21A2 and CYP11B1 have been limited by protein availability. Human CYP21A2 was successfully cloned, expressed, purified, and crystallized for the first time, which allows for structural and functional studies of the human enzyme. CYP11B1 was also successfully cloned and expressed, but more optimization is necessary for consistent large-scale expression and purification. This work provides the necessary groundwork for a biochemical and biophysical understanding of both CYP21A2 and CYP11B1 for the evaluation of these enzymes as counter-targets. In addition these studies could lead to the rational design of CYP11B1 inhibitors for the treatment of cortisol dependent diseases.
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Biochemistry
Chemistry
Chemoprevention
Cytochrome p450
Lung cancer
Steroidogenesis
CYTOCHROMES P450 AS THERAPEUTIC TARGETS AND COUNTER-TARGETS FOR THE PREVENTION OF LUNG CANCER AND TREATMENT OF STEROIDOGENIC DISEASES
Dissertation
oai:kuscholarworks.ku.edu:1808/63012020-07-30T15:39:30Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Gu, Xingxian
2010-06-09T04:08:16Z
2010-06-09T04:08:16Z
2010-04-26
http://dissertations.umi.com/ku:10933
http://hdl.handle.net/1808/6301
The iminosugar N-butyl-1-deoxynojirimycin (nB-DNJ) has reversible, nonhormonal
contraceptive effects on C57B/L6 mice at micromolar concentrations.
In order to increase the potency and bioavailability of this lead compound, a
series of novel iminosugars was synthesized as inhibitors of two potential target
enzymes – ceramide-specific glucosyltransferase (CGT) and β-glucosidase 2
(GBA2). The new derivatives were shown to be inactive as inhibitors of CGT and
are awaiting testing in a GBA2 assay. Efforts were made to identify additional
protein targets of the iminosugars by preparing two iminosugar affinity labels that
were used to isolate a potential new iminosugar target.
A method was developed to functionalize 2,3-dihydropyridin-4(1H)-ones
by taking advantage of the nitrogen-induced nucleophilicity of the beta-carbon
(C5) of the enamine moiety. Reaction of 2,3-dihydropyridin-4(1H)-ones with
aliphatic and aromatic aldehydes under acidic conditions furnished 5,5’-
(methylene)bis(2,3-dihydropyridin-4(1H)-ones). In the presence of the reducing
agent triethylsilane, the same reaction provided C5 alkylated derivatives. This
chemistry was extended to 4-(pyrrolidin-1-yl)furan-2(5H)-one, an enaminone with
an exocyclic nitrogen. A three-component Mannich aminomethylation of 2,3-
dihydropyridin-4(1H)-ones and 4-(pyrrolidin-1-yl)furan-2(5H)-one, carbamates,
and formaldehyde was achieved when lithium perchlorate was present in the
reaction mixture. This chemistry was extended to the reaction of exocyclic
enaminones with formaldehyde and malonates to furnish the corresponding
methylmalonates. Mechanist studies suggest that this reaction proceeds via the
formation of 2-methylenemalonates (Knoevenagel condensation), which is
followed by a nucleophilic (Michael) addition of the enaminone to the
methylenemalonates. The methylmalonate reaction products were cyclized
under acidic conditions to form bicyclic lactams (octahydroquinoline-3-
carboxylates and cyclopenta[b]pyridine-3-carboxylates). Oxidation of the
octahydroquinoline-3-carboxylates furnished 2,5-dioxo-1,2,5,6,7,8-
hexahydroquinoline-3-carboxylates, a class of compounds known to possess ionotropic properties. In a related reaction, 3-aminocyclohex-2-enones,formaldehyde and methyl cyanoacetate directly furnished the corresponding bicyclic 2,5-dioxo-1,2,3,4,5,6,7,8-octahydroquinoline-3-carbonitriles. In this case
the reaction was catalyzed by a phosphine, which promoted both, the Knoevenagel reaction and the bicyclic lactam formation.
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Pharmaceutical chemistry
Organic chemistry
Enaminone
Iminosugar
Lithium perchlorate
Male contraceptive effect
Multicomponent reaction
Synthesis and Evaluation of Novel Iminosugars as Potential Male Contraceptive Agents; and the Chemistry of 2,3-Dihydropyridin-4-(1H)-ones and Related Enaminones in Multicomponent Reactions
Dissertation
oai:kuscholarworks.ku.edu:1808/61842018-01-31T20:08:09Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Dresner, Kendra Nelson
2010-05-03T01:07:45Z
2010-05-03T01:07:45Z
2009-01-22
http://dissertations.umi.com/ku:10191
http://hdl.handle.net/1808/6184
Our research focuses on the development of kappa opioid receptor (KOR) antagonists. KOR antagonists have a variety of possible therapeutic applications; these compounds have shown anxiolytic activity, anti-depressive activity, and have potential uses in the treatment of opioid and cocaine addiction. Dynorphin A (Dyn A) is an endogenous agonist at KOR. Dyn A has been used as a lead for the development of KOR antagonists including arodyn, an acetylated Dyn A analog identified in our laboratory. Arodyn is a potent and selective KOR antagonist that has shown promise in the treatment of stress-induced relapse of cocaine seeking behavior. However, arodyn is rapidly metabolized in rat brain homogenate and slices. The objective of this research was to synthesize arodyn analogs with greater metabolic stability and to test these analogs in metabolism studies. Previous research in our laboratory had identified the sites of cleavage in arodyn and these sites were targeted for modification. Analogs with extended sequences, N-methylarginine replacements and reduced amide bond stabilizations were synthesized. In metabolism studies in washings from rat brain slices, several analogs were identified that showed increased stability over arodyn.
EN
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Pharmaceutical chemistry
Arodyn
Drug addiction
Kappa opioid receptors
Opioid antagonists
Peptide drugs
DESIGN, SYNTHESIS AND METABOLISM OF ARODYN ANALOGS
Thesis
oai:kuscholarworks.ku.edu:1808/61772020-07-28T13:59:30Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Trivedi, Rushi
2010-05-03T00:43:56Z
2010-05-03T00:43:56Z
2009-12-15
http://dissertations.umi.com/ku:10695
http://hdl.handle.net/1808/6177
The thesis details the development of a decarboxylative synthesis of aryl ethers using a relatively new Iron catalyst and a novel decarboxylative coupling of coumarins catalyzed by palladium. Aryl allyl carbonates underwent facile decarboxylative coupling in presence of a new iron catalyst. The allylation is found to be regioselective rather than regiospecific. This suggests that the allylation proceeds through π-allyl iron intermediates in contrast to related allylations of carbon nucleophiles that proceed via σ-allyl complexes. The reason we looked at iron as a catalyst was to offset the cost of expensive palladium catalyst usually used in these types of reactions. The second project involved allyl esters of 3-carboxylcoumarins, which underwent facile decarboxylative coupling at just 25−50 °C in moderate to good yields. This represents the first extension of decarboxylative C−C bond-forming reactions to the coupling of aromatics with sp3-hybridized electrophiles. Finally, the same concept can be applied to the sp2−sp3 couplings of pyrones and thiocoumarins. A variety of biologically important heteroaromatics can be readily functionalized without the need for strong bases or stoichiometric organometallics that are typically required for more standard cross-coupling reactions.
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Organic chemistry
3-allyl coumarins
Decarboxylation
Hieber anion
Iron catalysis
Prenylation
Sp2-sp3 coupling
Iron-catalyzed decarboxylative cross coupling reactions and palladium-catalyzed sp2-sp3 coupling of coumarins.
Thesis
oai:kuscholarworks.ku.edu:1808/102192018-01-31T20:08:10Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Stephens, Eva Susanne
2012-10-27T10:49:01Z
2012-10-27T10:49:01Z
2012-08-31
http://dissertations.umi.com/ku:12213
http://hdl.handle.net/1808/10219
The cytochrome P450 (P450) superfamily of mixed function oxidase enzymes catalyze the metabolism of a variety of endogenous and exogenous biochemicals, including steroids, fatty acids, vitamins, eicosanoids, drugs, pesticides, and toxins. P450-mediated oxidative metabolism often serves a beneficial role in the clearance of foreign compounds and the regulation of endogenous molecules, both of which are necessary for the maintenance of homeostasis. However, the reactions catalyzed by P450 enzymes also have the potential to promote disease and injury, whether it be in the activation of procarcinogens or by undermining the therapeutic efficacy of a drug. Altogether, these processes make P450 enzymes important subjects of interest for the prediction of chemical toxicology and the development of therapeutic agents. The focus of this thesis research is the characterization of the CYP26 and CYP2A families of P450 enzymes in an effort to contribute to an understanding of how substrates and inhibitors specifically interact with the individual enzymes within these families. The functional enzymes of the human CYP2A family include CYP2A6 and CYP2A13. CYP2A6 is primarily a hepatic enzyme, while CYP2A13 is mainly expressed within the respiratory tract. Though these two enzymes are primarily localized to different tissues within the body, they share many substrates in common as a result of their 94% amino acid sequence identity. These substrates include: nicotine, cotinine, para-nitrophenol, and coumarin. However, CYP2A13 appears to preferentially activate a number of procarcinogens, including the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), into reactive intermediates that can result in DNA adducts and the initiation or promotion of carcinogenesis. In order to evaluate the relative involvement of CYP2A6 activity versus that of CYP2A13 in the metabolism and/or activation of potentially harmful chemical agents in vivo, selective inhibitors are needed. A number of compounds were reported to selectively inhibit CYP2A6 before CYP2A13 was determined to be a functional member of the CYP2A family, or have simply not been tested against CYP2A13. This work examined such compounds including: phenethyl isothiocyante (PEITC), 4-dimethylaminobenzaldehyde (DMABA), 8-methoxypsoralen (8-MOP), tranylcypromine, tryptamine, pilocarpine, (S)-nicotine, (R)-(+)-menthofuran, and β-nicotyrine. The relative impact of these inhibitors on CYP2A6 and CYP2A13 function was evaluated by determining Ki values and modes of inhibition for each of the compounds against both enzymes, followed by the calculation of a selectivity factor. The results of these studies serve as the first explicit determination of the selectivity of these compounds for enzymes within the CYP2A family and indicate that only (R)-(+)-menthofuran and tranylcypromine demonstrate even a 10-fold preference for CYP2A6 inhibition over CYP2A13. This information can be used as a guide for the selection of inhibitors with the greatest potential for determining whether CYP2A6 or CYP2A13 is responsible for the metabolism or activation of procarcinogenic compounds, such as tobacco-derived NNK in the human respiratory tract. Collectively, the CYP26 family of P450 enzymes serve as important mediators of retinoic acid (RA) catabolism in the body. In humans, the CYP26 family of enzymes consists of three isoforms: CYP26A1, CYP26B1, and CYP26C1. As a result of RA's endogenous role in regulating cellular growth and differentiation, geometric isomers of RA such as all-trans-RA (atRA) and 13-cis-RA represent attractive targets for cancer therapy and the treatment of dermatological conditions. Unfortunately, RA resistance is often experienced in patients undergoing prolonged RA-based therapy. This failure has been suggested to be the result of up-regulation of P450 enzymes, particularly the enzymes of the CYP26 family, resulting in enhanced RA metabolism in vivo. As a result, a significant amount of work has been conducted for the development of chemical agents that inhibit P450-mediated metabolism of atRA, commonly referred to as retinoic acid metabolism blocking agents (RAMBAs). The development of safe and effective RAMBAs could be greatly facilitated by more detailed structural and functional characterizations of the CYP26 family of enzymes, since very little is known about these enzymes aside from their dominant role in atRA hydroxylation. However, the fact that the human CYP26 enzymes are membrane-bound proteins, a characteristic shared by all human P450 enzymes, represents a major challenge to the recombinant expression and purification of these enzymes in sufficient quantities to enable detailed biochemical and biophysical characterization studies. To address this challenge, a new method for the production of pure CYP26 enzymes was developed based on techniques that have proven successful in the expression and purification of other membrane-bound P450 enzymes in E. coli, while incorporating the use of detergents and stabilizing ligands specific to the CYP26 enzymes. The combination of these approaches led to the successful generation of mouse CYP26A1 and CYP26B1 proteins in yields of 200-400 nmol per 2.25 L of E. coli expression media and the ability to recover 20-50 nmol following a single chromatographic step. Unfortunately, this protein appeared to be inactive according to reduced carbon monoxide difference spectrum analysis and assays of RA metabolism. As a result, further optimization will be required to reach the original goal set forth to generate pure recombinant CYP26 enzymes for basic kinetic and structural determination studies.
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Biochemistry
Pharmaceutical sciences
Cancer
Cyp26
Cyp2a
Cytochrome p450
Retinoic acid
Cytochromes P450: Inhibition of CYP2A Enzymes Involved in Xenobiotic Metabolism and Generation of CYP26 Enzymes Involved in Retinoic Acid Metabolism
Thesis
oai:kuscholarworks.ku.edu:1808/96982020-09-01T14:01:18Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Singh, Gurpreet
2012-06-03T13:31:15Z
2012-06-03T13:31:15Z
2011-21-31
http://dissertations.umi.com/ku:11916
http://hdl.handle.net/1808/9698
This thesis describes the development of libraries inspired by the natural product sparteine. Multi-gram synthesis of important intermediate representing sparteine core has been carried out. A scalable process has been developed based on the previously reported total synthesis of (+)-sparteine from Aubé's group. Libraries were produced via parallel synthesis and submitted to NIH Molecular Libraries-Small Molecule Repository for biological screening.
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This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Organic chemistry
Development of libraries inspired by sparteine
Thesis
oai:kuscholarworks.ku.edu:1808/55812020-07-27T13:53:01Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Sinha, Bhaswati
2009-11-02T20:39:58Z
2009-11-02T20:39:58Z
2009-07-17
http://dissertations.umi.com/ku:10493
http://hdl.handle.net/1808/5581
Narcotic analgesics produce pain relief generally through activation of &mu opioid receptors (MOR), but the use of these analgesics is limited by their side effects, namely respiratory depression, tolerance, physical dependence and constipation. Understanding receptor-ligand interactions at the molecular level could facilitate the design of novel opioid ligands potentially with less deleterious side effects. This task is challenging since there is no crystal structure available for opioid receptors. With the aim of understanding MOR-ligand interactions, we designed novel MOR selective peptide ligands containing a reactive affinity label group. Affinity labels that interact with the receptor in a non-equilibrium manner can provide information about specific receptor-ligand interactions. We selected two MOR selective peptides: dermorphin, an endogenous ligand present in South American frog skin, and the synthetic enkephalin analog DAMGO ([D-Ala2,NMePhe4,glyol]enkephalin), for developing electrophilic affinity label derivatives. We substituted D-Orn or D-Lys in position 2 (in place of D-Ala) in both dermorphin and DAMGO, and attached a bromoacetamide or an isothiocyanate group as the electrophilic functionality to the side chain amines of the D-amino acids. For the dermorphin derivatives, we successfully identified several affinity labels with high MOR affinity (IC50 = 0.1-5 nM) and high selectivity for MOR that exhibit wash-resistant inhibition of binding to these receptors. Among these, [D-Lys(=C=S)2]dermorphin was further modified to include a purification tag (d-desthiobiotin) and a fluorescent tag (Oregon Green or 5-carboxyrhodamine B). This multifunctional affinity label peptide was synthesized successfully using an Fmoc-solid phase synthetic strategy. Initial fluorescent microscopy studies suggest irreversible labeling of MOR expressed on SH-SY5Y cells by this multifunctional peptide, thus demonstrating the utility of the fluorescent tag. For the DAMGO series of analogs, the bromoacetamide derivatives exhibited subnanomolar binding affinity (IC50 = 0.45 nM) to MOR. However, the isothiocyanate derivatives resulted in the formation of an unexpected cyclic O-alkyl thiocarbamate side product. This side reaction was successfully overcome by replacing the glyol in DAMGO by the glycylamide, yielding affinity label derivatives that exhibited subnanomolar affinity (IC 50 = 0.3-0.8 nM) and wash-resistant inhibition of MOR binding. These high affinity peptide-based affinity labels will be useful pharmacological tools to study MOR.
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Pharmaceutical chemistry
Affinity labels
Damgo
Dermorphin
Multifunctional
Opioids
Peptides
Design, Synthesis and Evaluation of Peptide-Based Affinity Labels for Mu Opioid Receptors
Dissertation
oai:kuscholarworks.ku.edu:1808/293142020-07-09T21:44:39Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Mishra, Sanket
2019-06-12T03:06:49Z
2019-06-12T03:06:49Z
2018-05-31
http://dissertations.umi.com/ku:15789
http://hdl.handle.net/1808/29314
Heat shock protein 90 kDa (Hsp90) is a member of the molecular chaperone family of proteins that processes newly synthesized polypeptides into their three-dimensional and biologically active form. In addition, Hsp90 assists in the stabilization, trafficking and refolding of denatured proteins. Many Hsp90-dependent proteins are critical to the pathogenesis of cancer, neurodegeneration and/or viral infection. As a result, Hsp90 has garnered attention as a chemotherapeutic target and has resulted in the development of more than 17 Hsp90 inhibitors that that have been evaluated in clinical trials. However, these inhibitors exhibit pan-inhibitory activity against all four Hsp90 isoforms: Hsp90alpha, Hsp90beta, Grp94 and Trap1, which results in various side effects, including, hepatotoxicity, cardiotoxicity, and renal toxicity. Therefore, the development of isoform-selective Hsp90 inhibitors has been proposed to delineate the contribution of each Hsp90 isoform towards these toxicities. The cytosolic Hsp90 isoforms, alpha and beta modulate the activity of numerous Hsp90-dependent proteins that regulate cancer progression. Hydrolysis of ATP by the N-terminal nucleoside-binding site provides the energy required for the maturation of client protein substrates, and all four Hsp90 isoforms share 85% identity within this region. Between cytosolic Hsp90 isoforms (alpha and beta), the N-terminal ATP-binding site exhibits 95 % identity. As a result, the design of inhibitors that selectively target individual cytosolic Hsp90 isoforms has been challenging. Described herein is the development of Hsp90alpha- and Hsp90beta-selective inhibitors using a structure-based approach that has produced compounds that exhibit both high selectivity and affinity. The efficacy of these inhibitors has been evaluated against an array of cancer cell lines and revealed an Hsp90 isoform-dependent cancer profile.
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Pharmaceutical sciences
Anticancer
Drug Discovery
Hsp90
Medicinal Chemistry
Structure-based Design
A New Generation of Isoform Selective Hsp90 Inhibitors: Targeting the Cytosolic Hsp90 Isoforms
Dissertation
oai:kuscholarworks.ku.edu:1808/216292020-10-16T14:40:06Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Vasiljevik, Tamara
2016-10-11T15:40:05Z
2016-10-11T15:40:05Z
2013-12-31
http://dissertations.umi.com/ku:13078
http://hdl.handle.net/1808/21629
Agents that exert their effects in the central nervous system are among the most important in medicinal practice. One in three Americans will have a disease affecting the nervous system in their lifetime exceeding $600 billion dollars per year in cost. Side effects, lack of efficacy, and lack of selectivity hamper their clinical use. Thus, additional agents need to be developed and new chemical tools are needed to further elucidate the mechanisms of CNS disorders. Salvinorin A, the main component of the hallucinogenic mint Salvia divinorum is a novel and potent ê opioid (KOP) receptor agonist that is the first naturally occurring opioid ligand that lacks a basic nitrogen atom. In efforts to attain a greater understating for the interatctions of the furan ring binding pocket within the KOP receptor, several modifications were made at the C-13 position of the salvinoirn A scaffold. Pharmacological evaluations of the synthesized analogues indicated that there is a prefferred orientation of the furan O-atom in that the cis alkene analogue (236) had a greater binding affinity than the trans alkene analogue (235). However, despite its similar binding affinity to that of salvinorin A the cis analogue showed to 34-fold less active than salvinorin A. It was also hypothesized that a combination of the salvinorin A scaffold with a similar terpene based family of marine natural products, namely the nakijiquinones, will alter the biological activity of salvinorin A and yield a useful biological probe. With this hypothesis in mind, several quinone containing salvinoirn A analogues were synthesized and upon pharmacological evaluation it was determined that these analogues did not exhibit any significant KOP receptor activity, indicative of a change in the biological activity of the salvinorin A scaffold. Furthermore, these analogues proved to be the first salvinorin A based analogues that exhibit antiproliferative activity. In the search for novel CNS biological probes, the aminoalkylindole class of synthetic cannabinoids was also investigated. Due to its cannabinoid 1 receptor (CB1R) antagonist activity, JWH-073-M4 was shown to be a valuable lead molecule. The hypothesis was that the design and synthesis of JWH-073-M4 based analogues would lead to compounds with dual CB1R antagonist/CB2R agonist activity that may have potential as alcohol abuse therapies. Upon in vitro pharmacological investigation, two analogues 461 and 444 were shown to have the most promise and were further subjected to in vivo pharmacological evaluation in animal models of alcohol abuse, namely ethanol self-administration and ethanol conditioned place preference. From these studies it was determined that analogues 461 and 444 exhibit dual CB1R antagonist/CB2R agonist activity and represent potential leads in the ongoing search for novel alcohol abuse therapies.
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Chemistry
Pharmaceutical sciences
Pharmacology
alcohol abuse
biological probes
CNS disorders
KOP receptor
salvinoirn A
synthetic cannabinoids
Design and Synthesis of Novel CNS Biological Probes
Dissertation
oai:kuscholarworks.ku.edu:1808/66302020-07-27T13:12:14Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Wu, Wenyan
2010-09-03T03:05:08Z
2010-09-03T03:05:08Z
2009-08-31
http://dissertations.umi.com/ku:10549
http://hdl.handle.net/1808/6630
The toxicity of Gram-negative bacterial endotoxin (lipopolysaccharide, LPS) resides in its structurally highly conserved glycolipid component called lipid A. The major goal of the first project was to further explore structure-activity relationships in small-molecules that would sequester LPS by binding to the lipid A moiety, so that it may find application in the prophylaxis or adjunctive therapy of Gram-negative sepsis. Several guanylhydrazones had earlier been identified in rapid-throughput screens as potent LPS binders. It was desirous to examine if grafting the guanylhydrazone functionality on the scaffold of a lead N-alkyl polyamine compound would afford greater LPS sequestration potency. In the first project, a congeneric set of guanylhydrazone analogues were synthesized and evaluated for LPS-sequestering potency. It was found that a C16 alkyl substitution was optimal in the N-alkylguanylhydrazone series; a homospermine analogue with the terminal amine N-alkylated with a C16 chain with the other terminus of the molecule bearing an unsubstituted guanylhydrazone moiety was marginally more active suggesting very slight, if any, steric effects. Neither C16 analogue was significantly more active than the N-C16-alkyl or N-C16-acyl compounds that we had characterized earlier, indicating that basicity of the phosphate-recognizing cationic group is not a determinant of LPS sequestration activity. The N-terminus of bacterial lipoproteins are acylated with a (S)-(2,3-bisacyloxypropyl)cysteinyl residue. Lipopeptides derived from lipoproteins activate innate immune responses by engaging Toll-like receptor 2 (TLR2), and are highly immunostimulatory and yet without apparent toxicity in animal models. The lipopeptides may therefore be useful as potential immunotherapeutic agents. Previous structure-activity relationships in such lipopeptides have largely been obtained using murine cells and it is now clear that significant species-specific differences exist between human and murine TLR responses. In the second project, I have examined in detail the role of the highly conserved Cys residue as well as the geometry and stereochemistry of the Cys-Ser dipeptide unit. (R)-diacylthioglycerol analogues were maximally active in reporter gene assays using human TLR2. The Cys-Ser dipeptide unit represents the minimal part-structure, but the stereochemistry of neither amino residues of the dipeptide was found to be a critical determinant of activity. The thioether bridge between the diacyl and dipeptide units was determined to be crucial, and replacement by an ether bridge resulted in a dramatic decrease in activity. Moreover, the replacement of the two ester-liked C16 hydrocarbons by ether or amide linkages led to a dramatic loss in activity, while the replacement of one of the ester-linked fatty acyl moiety (internal) with an amide linkage remained partially active.
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This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Pharmaceutical chemistry
Lipopeptide
Lps
Pam2csk4
Tlr
Modulators of Toll-like Receptors-4 and -2
Thesis
oai:kuscholarworks.ku.edu:1808/216382020-06-26T20:55:39Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Pashikanti, Srinath
2016-10-11T16:14:09Z
2016-10-11T16:14:09Z
2014-05-31
http://dissertations.umi.com/ku:13261
http://hdl.handle.net/1808/21638
Part I. HIV Protease Targeted Synthesis of Conformationally–Rigid Hydroxyethylene Dipeptide Isosteres: A Combinatorial approach With the success of Highly Active Anti-Retro Viral therapy (HAART), the life expectancy of HIV patients has dramatically increased. However, emergence of drug resistance, toxicity, pharmacokinetic issues, and side effects etc. are some of the concerns still associated with the currently available drugs. To address these issues, there is a continuous need for the development of novel and improved anti-HIV agents. Drugs acting via potent inhibition of the critical enzyme HIV protease constitute a major class of drugs in AIDS therapy. Currently available HIV protease inhibitors utilize peptide hydrolysis transition-state isosteres as the key element for their anti-HIV activity. Hydroxyethylene, dihydroxyethylene, hydroxyethyl amine, and methyleneamine moieties are some of the strategic nonhydrolyzable transition state isosteres that have been employed in the construction of various HIV protease inhibitors. As part of our research towards medicinal chemical studies of bioactive compounds, the present research investigates the design and synthesis of conformationally rigid hydroxyethylene dipeptide mimics. Among the various structural motifs useful in imparting conformational rigidity, cyclopropane rings remain a popular choice in drug discovery endeavors. Accordingly, in an as yet unreported investigation, synthesis of cyclopropane ring-constrained dipeptide isosteres containing a hydroxyethylene `warhead' has been initiated. Utilizing a multifunctional enantiopure aminobutenolide (developed in our research group) as a `second-generation' chiral building block, the synthesis involved an initial diastereoselective cyclopropanation to construct a strategic [3,5]-bicyclic lactone. Utilizing two diversification sites in the target analogs, and employing select sets of amines and amino acids as the diversity elements, a combinatorial parallel synthetic approach has been followed towards rapid construction of a demonstration library of cyclopropane ring containing hydroxyethylene dipeptide isosteres. The synthetic route designed also offers additional flexibility in terms of further diversification in future studies. Part II. Total Synthesis and Structure_Activity Relationship Studies of the Cytotoxic Ahydrophytosphingosine Jaspine B Jaspine B, also known as Pachastrissamine, is a cytotoxic marine natural product isolated from the sponges Pachastrissa sp. and Jaspis sp. In biological assays, Jaspine B exhibited sub-micromolar cytotoxicity (IC50 ≤0.5 μM) against several different cancer cell lines (murine B16, human Sk-Mel28 melanoma etc.). Exposure of these cells to Jaspine B triggered cell death by typical apoptosis, as indicated by phosphatidylserine externalization, the release of cytochrome C, and caspase processing. Recent studies have indicated that interference with ceramide metabolism via inhibition of sphingomyelin synthase is most probably responsible for the apoptotic effects of this natural product. Despite its impressive biological activity, detailed structure-activity relationship (SAR) investigations of Jaspine B are relatively limited. The present research describes an efficient and practical total synthetic route to Jaspine B, and application of the method thereof in the structure-activity relationship (SAR) studies of this bioactive natural product. Starting from the previously mentioned chiral aminobutenolide as an advanced building block, a key reaction in our synthetic route involved a stereocontrolled synthesis of the cis-fused [5,5]-bicyclic lactone. Subsequent functional group transformations of this lactone led to a short-step total synthesis of enantiopure Jaspine B. Easy amenability of various advanced chiral intermediates from the total synthesis route has also allowed us to perform strategic structural modifications to access a number of unique Jaspine B analogs for biological investigations. Subsequent in vitro cytotoxicity assays of the analogs thus obtained have provided important SAR information, which is expected to provide useful direction in the potential development of new anti-cancer agents, based on the Jaspine B structural lead. Simultaneously to the above studies, a previously reported synthetic route to Jaspine B from our group has also been reinvestigated in order to help clarify some unanswered questions. Gratifyingly, the results from the above study have also confirmed the utility of the previous route in stereoselective synthesis of enantiopure Jaspine B. Part III. A Rapid Approach Towards Diastereoselective Synthesis of Azacarbohydrate Scaffolds of Biological Significance A wide range of monocyclic and bicyclic polyhydroxylated alkaloids (azacarbohydrates/iminosugars) have been isolated from various plants, insects and microorganisms. Many of these compounds have been found to be potent inhibitors of glycosidase and glycosyl transferase enzymes of clinical importance. Advancements involving stereoselective synthesis, structural modification, and biological evaluation of these compounds resulted in therapeutic agents Glyset® and Zavesca® for use in patients with type II diabetes and type I Gaucher's disease respectively. In further application of the L-serine-derived chiral aminobutenolide as an advanced building block, a rapid stereoselective synthesis of azacarbohydrate structural scaffolds have been investigated in the present study. The key reaction steps involved, utilization of an enone in the starting material towards stereoselective installation of key functional groups, and an intramolecular cyclization of an appropriately located amine into the lactone carbonyl resulting in the formation of the desired azacarbohydrate structural scaffolds. Subsequent reduction and deprotection provided an efficient and short-step route to variously functionalized azacarbohydrates.
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Pharmaceutical sciences
Organic chemistry
Anti-cancer
Azacarbohydrate
Conformationally rigid hydroxyethylene dipeptide isostere
HIV Protease Inhibitors
Jaspine B
Sphingolipid
A Serine-Derived Butenolide as a Versatile Chiral Building Block: Applications in the Synthesis of Natural and Nature-Like Compounds of Biological Significance
Dissertation
oai:kuscholarworks.ku.edu:1808/239242018-01-31T20:07:48Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Petrunak, Elyse Marie
2017-05-07T16:32:37Z
2017-05-07T16:32:37Z
2015-08-31
http://dissertations.umi.com/ku:14253
http://hdl.handle.net/1808/23924
Cytochromes P450 (CYP450) are heme-containing monooxygenase enzymes that perform a variety of functions in humans, including xenobiotic metabolism and the production of endogenous signaling molecules. Six isoforms of cytochrome P450 including cytochrome P450 17A1 (CYP17A1) and cytochrome P450 21A2 (CYP21A2), are responsible for the generation of steroid hormones, making these enzymes crucial for development and homeostasis. However in certain pathological conditions, inhibition of steroidogenic cytochrome P450 enzymes can be therapeutically useful. For prostate cancers dependent on androgens such as dihydrotestosterone for tumor growth and proliferation, inhibition of CYP17A1, a required enzyme in androgen biosynthesis, is a promising treatment strategy. Although inhibition of CYP17A1 has been successful clinically, its additional roles in the biosynthesis of other steroid hormones and its similarity to other steroidogenic P450 enzymes can bring about off-target effects. An understanding of the structure and function of CYP17A1, as well as other steroidogenic countertarget enzymes such as CYP21A2, could therefore be fundamental to developing improved inhibitors of this enzyme for the treatment of prostate cancer. CYP17A1 performs two different reactions in the same active site to generate androgens, a hydroxylation reaction followed by a carbon-carbon bond cleavage (or lyase) reaction. While the second, carbon-carbon bond cleavage reaction commits a steroid to the androgen pathway, the initial hydroxylation reaction is also necessary for the production of glucocorticoids. Human CYP17A1 performs the hydroxylation reaction on two structurally similar substrates, Δ4-progesterone and Δ5-pregnenolone, which only exhibit differences on the A ring on the opposite end of the steroid from the carbon which is hydroxylated by CYP17A1. Following hydroxylation, Δ5-17α-hydroxypregnenolone will undergo the second lyase reaction, but the Δ4-17α-hydroxyprogesterone does not. To elucidate a structural basis for preferential lyase turnover of Δ5 steroids by human CYP17A1, the crystal structure of the enzyme containing a background mutation was determined in the presence of both hydroxylase substrates and both lyase substrates. These structures reveal some similarities in binding among all four substrates but also differences in positions relative to the heme iron between hydroxylase substrates, the poor lyase substrate 17α-hydroxyprogesterone, and the efficient lyase substrate 17α-hydroxypregnenolone. Observed differences in distances between 17α-hydroxyprogesterone or 17α-hydroxypregnenolone and the heme iron may reflect differential stabilization of the proposed intermediate for the 17,20-lyase reaction. In addition to substrates, steroidal inhibitors of CYP17A1 can have different configurations of the A ring. X-ray crystal structures of a series of inhibitors with such modifications were also determined with CYP17A1 to compare active site interactions among A-ring modified steroidal inhibitors. Modifications to the A ring of steroidal inhibitor abiraterone did not alter direct contacts with CYP17A1, but did change indirect contacts with the enzyme through active site water networks. The structure of CYP17A1 with one A-ring modified inhibitor was resolved to 2.0 Å, making it the highest resolution crystal structure of CYP17A1 to date, and revealed an additional steroid binding site in the periphery of enzyme that had not been fully appreciated in previous CYP17A1 structures. CYP17A1 and redox partner proteins were recombinantly expressed in E. coli and purified, providing a well-defined and well-controlled system for evaluation of CYP17A1 function and inhibition. This strategy was employed to compare hydroxylase and lyase deficiencies among clinically-reported mutants of the enzyme, as well as steroidal and non-steroidal clinical inhibitors for selective inhibition of the lyase reaction compared to the hydroxylase reaction. Most clinical inhibitors of CYP17A1 demonstrated 1- to 3- fold selectivity for 17,20-lyase inhibition over 17α-hydroxylase inhibition. Only one of the four inhibitors to reach clinical trials, S-orteronel, demonstrated 3- to 5- fold selectivity for 17,20-lyase inhibition compared to inhibition of progesterone and pregnenolone 17α-hydroxylase reactions. However, its enantiomer, R-orteronel demonstrated 8- to 11-fold selectivity. X-ray crystal structures of CYP17A1 with non-steroidal inhibitors reported to selectively inhibit the lyase reaction were also determined to investigate the structural basis for lyase selectivity. Finally, some CYP17A1 inhibitors have also been shown to interact with another P450 responsible for steroid biosynthesis, CYP21A2. The physiological consequences of off-target CYP21A2 inhibition by compounds developed to target CYP17A1 are complex. The crystal structure of human CYP21A2 was determined to provide a structural comparison to CYP17A1 and potentially aid in the design of inhibitors more selective for CYP17A1 over CYP21A2. Some structural features of the CYP17A1 active site, including a hydrophobic pocket over the I helix, are not conserved in CYP21A2. Exploitation of this pocket is a potential strategy for the development of inhibitors with reduced affinity for CYP21A2. In aggregate, the studies described herein use structural information coupled with functional analysis to better understand steroidogenic cytochromes P450. These enzymes act as targets and countertargets in the treatment of hormone dependent diseases including prostate cancer. More detailed knowledge of how these enzymes interact with both substrates and inhibitors could inform the development of better prostate cancer therapeutics.
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Copyright held by the author.
Biochemistry
Structural and Functional Evaluation of Steroidogenic Cytochrome P450 Enzymes
Dissertation
oai:kuscholarworks.ku.edu:1808/44442018-01-31T20:08:08Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
DeVore, Natasha M.
2009-03-24
2009-03-24
2008-01-01
http://dissertations.umi.com/ku:10014
http://hdl.handle.net/1808/4444
The goal of this research was to identify the differential structure-activity relationships between cytochromes P450 (CYP) 2A13 and 2A6 and their substrates. Cytochromes P450 2A13 and 2A6 are very closely related, having 94% amino acid sequence identity. Both proteins metabolize drugs, toxins, and procarcinogens, including the nicotine derivative 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and aflatoxin B1. Yet CYP2A13 has a much higher rate of metabolism than CYP2A6 for both compounds. Combined with its increased affinity and expression in the respiratory tract, CYP2A13 is a candidate for breakdown of NNK into the reactive metabolites that can form DNA adducts in the lung, leading to the development of lung cancer (1). Thus, understanding differential metabolism is important. Due to the high identity of CYP2A13 and CYP2A6, we hypothesized that a few amino acids found within the active site are responsible for CYP2A substrate specificity. Of the 32 amino acids which differ between CYP2A13 and CYP2A6, ten are located in regions of the protein that often determine substrate recognition in cytochromes P450 (2). Crystallization of CYP2A13 by the Scott lab verified that these amino acids were in or near the active site. The goal of this project was to determine which of these ten amino acids were vital for substrate selectivity in the CYP2A enzymes. Several different methods were employed throughout this study including site-directed mutagenesis, ligand binding assays, metabolism assays, molecular modeling, and crystallography. Site-directed mutagenesis was used to construct ten single residue CYP2A13 mutants with each mutation substituting the residue found in CYP2A6 in that position. The consequences of these ten mutations to the binding of coumarin, 2′-methoxyacetophenone, phenyl isothiocyanate, and phenacetin were determined using spectral ligand binding assays. The amino acids in positions 117, 208, 300, 301, 365, and 369 caused significant changes in ligand affinity. To determine if these amino acids were also essential for substrate metabolism, phenacetin was used as a structural probe since CYP2A6 converts phenacetin to acetaminophen with decreased catalytic efficiency compared to CYP2A13 (3). Employing this assay, four mutations (S208I, F300I, A301G, and G369S) were identified that diminished CYP2A13 phenacetin O-deethylation to near CYP2A6 activity. Two mutants, CYP2A13 A117V and L366I, both increased enzyme activity more than four-fold. Construction of the reverse mutant series, a CYP2A6 protein incorporating the residues found at the corresponding active site positions in CYP2A13, confirmed that positions 208, 300, 301, and 369 were jointly responsible for phenacetin metabolism and binding. While all single 2A6 mutants had very low-level activity, the double, triple, and a quadruple mutant with changes at these four positions increasingly conferred phenacetin metabolism to CYP2A6. A structural basis for the effects of these four mutations on phenacetin binding and metabolism was explored with molecular docking studies using Surflex-Dock (4). The results suggested that the ability of CYP2A13 to bind and metabolize phenacetin was due to steric variations among key residues in the CYP2A13 and CYP2A6 active sites. This was confirmed with the crystal structure of the CYP2A6 I208S/I300F/G301A/G369S complexed with phenacetin. Finally, this study characterized the effects of naturally occurring CYP2A13 polymorphisms on the binding of the well-known CYP2A ligand coumarin (5). All of these polymorphisms were located on the exterior portions of the protein. None of the polymorphisms examined resulted in a significant change in ligand affinity, indicating that having one of these variants would not substantially alter an individual's ability to metabolize CYP2A13 substrates. Thus, the research presented in this thesis provides structural and functional insight into CYP2A13 and CYP2A6 substrate selectivity, which is largely modulated by the steric effects mediated by the differential amino acids at positions 208, 300, 301, and 369. References 1. Su, T., Bao, Z. P., Zhang, Q. Y., Smith, T. J., Hong, J. Y., and Ding, X. X. (2000) Cancer Res. 60(18), 5074-5079 2. Zhang, J. Y., Wang, Y., and Prakash, C. (2006) Curr Drug Metab 7(8), 939-948 3. Bieche, I., Narjoz, C., Asselah, T., Vacher, S., Marcellin, P., Lidereau, R., Beaune, P., and de Waziers, I. (2007) Pharmacogenetics and Genomics 17(9), 731-742 4. Nishimura M Fau - Yaguti, H., Yaguti H Fau - Yoshitsugu, H., Yoshitsugu H Fau - Naito, S., Naito S Fau - Satoh, T., and Satoh, T. (0031-6903 (Print)) 5. Schlicht, K. E., Michno, N., Smith, B. D., Scott, E. E., and Murphy, S. E. (2007) Xenobiotica, 1-11
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This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Chemistry
Biochemistry
Cyp2a13
Cyp2a6
Cytochrome p450
Site-directed mutagenesis
X-ray crystallography
The human cytochrome P450 2A family: Comparisons and identification of amino acids essential for substrate recognition
Thesis
oai:kuscholarworks.ku.edu:1808/67322020-08-05T12:34:44Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Hartsock, Wendy Jeanne
2010-10-03T03:23:58Z
2010-10-03T03:23:58Z
2010-07-23
http://dissertations.umi.com/ku:11079
http://hdl.handle.net/1808/6732
The δ opioid receptor (DOR) is involved in the modulation of μ opioid receptor (MOR) agonist mediated side effects such as the development of tolerance, and MOR and DOR may interact to form heterodimers capable of unique pharmacological signaling. Understanding the molecular interactions involved in ligand recognition by opioid receptors will aid in the design of novel therapeutics that target the opioid receptors with fewer side effects. The aim of this research was to develop and use opioid peptide ligands to examine ligand-receptor interactions at the molecular level. We designed a multifunctional peptide-based affinity label derivative of the high affinity DOR selective peptide antagonist TIPP (Tyr-Tic-Phe-Phe, Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) to irreversibly interact with DOR. The multifunctional peptide also contains a fluorescent group and multiple tags to aid in the detection and isolation of labeled DOR. The initial solid phase peptide synthesis of the multifunctional peptide generated multiple side products and required extensive evaluation and optimization to increase the efficiency of peptide synthesis. A model peptide that contained only the affinity label and a fluorescent tag (at 65 nM) demonstrated 95% wash resistant inhibition of binding to DOR suggesting that this peptide is an affinity label for DOR. We also report the synthesis and initial binding analysis of a heterobivalent peptide-based affinity label targeting the proposed MOR-DOR heterodimer. This peptide demonstrated nanomolar affinity to both DOR and MOR in radioligand competition assays, and at 96 nM exhibited 94% wash resistant inhibition of binding to DOR, suggesting that the heterobivalent peptide is an affinity label for DOR. Thus, we successfully prepared two series of novel opioid peptide ligands which appear to be affinity labels for DOR. One series of peptides contains multiple functional groups to aid in DOR isolation, detection and analysis. The second peptide consists of two pharmacophores incorporated into a single entity to study proposed MOR-DOR heterodimers. These peptides will be useful pharmacological tools to study opioid receptors.
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openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Organic chemistry
Chemistry
Biochemistry
The Design, Synthesis and Evaluation of Peptide Ligands to Study Opioid Receptors
Dissertation
oai:kuscholarworks.ku.edu:1808/217302018-01-31T20:07:52Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Reedy, Christianna Lee
2016-10-13T18:59:14Z
2016-10-13T18:59:14Z
2015-12-31
http://dissertations.umi.com/ku:14305
http://hdl.handle.net/1808/21730
We are exploring analogs of the macrocyclic tetrapeptide natural product CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) as both potential analgesics and kappa opioid receptor (KOPr) antagonists. KOPr agonists exhibit analgesic activity but do not cause some of the adverse side effects associated with mu opioid agonists. Additionally, both KOPr agonists and antagonists have demonstrated the ability to block reinstatement of cocaine-seeking behavior under different conditions. CJ-15,208 has shown both opioid agonist (antinociceptive) activity and KOPr antagonist activity in vivo, while its D-Trp isomer, [D-Trp]CJ-15,208, has shown predominantly KOPr antagonist activity in vivo. Both compounds penetrate the central nervous system following oral administration and are stable to proteases in plasma and whole blood. However, studies in liver microsomes suggest that these peptides are susceptible to CYP P450 metabolism. Analogs of CJ-15,208 with modification on one of the aromatic residues were synthesized to study and improve pharmacokinetic properties. Additionally, CJ-15,208 and [D-Trp]CJ-15,208 were studied with in vitro biological barrier models using Madin-Darby canine kidney (MDCK) cells, both wild type and a line transfected with the MDR1 gene coding for the efflux protein P-glycoprotein (P-gp). The peptides’ inhibition of P-gp was assessed using rhodamine 123 efflux, and their efflux was assessed by analyzing bidirectional transport across MDCK-MDR1 and MDCK-WT monolayers. Evidence of efflux was observed for CJ-15,208 in the transport studies, but the similar results in the experiments using MDCK-MDR1 and MDCK-WT cells make it unclear which efflux proteins are involved. Preliminary studies suggested that [D-Trp]CJ-15,208 may also be an efflux substrate, but additional studies are need to confirm this. The results of these studies will help guide the design and evaluation of new analogs of these lead peptides.
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openAccess
Copyright held by the author.
Pharmaceutical sciences
Organic chemistry
Biochemistry
ADME
kappa opioid receptor
MDCK
peptide
Assessing ADME properties of CJ-15,208: synthesis of new analogs and examination of P-glycoprotein interactions
Thesis
oai:kuscholarworks.ku.edu:1808/278232020-10-08T15:37:25Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Crowley, Rachel S.
2019-05-10T15:35:16Z
2019-05-10T15:35:16Z
2017-08-31
http://dissertations.umi.com/ku:15477
http://hdl.handle.net/1808/27823
https://orcid.org/0000-0001-6366-5577
In the search for effective methods to mitigate the increasing rates of abuse and addiction of illicit substances, a variety of neurological pathways have been explored. Towards this goal of reducing drug abuse and ultimately overdose-related deaths, two avenues of research have emerged: 1) a preventative approach, the development of pain-relieving medications without the abuse and addiction liabilities associated with current therapies, and 2) a responsive approach, the development of medications for people suffering from drug abuse and addiction. The natural product salvinorin A can be manipulated towards both of these research avenues through the development of opioid receptor (MOR) agonists for treating pain with reduced abuse liabilities as well as through the development of opioid receptor (KOR) agonists with improved pharmacokinetic properties towards the development of therapies that attenuate relapse and withdrawal. Previous structure-activity relationship (SAR) studies of salvinorin A identified that replacing the C2-acetate with a C2-benzoate results in a compound that is 4-fold selective for MORs over KORs. In an effort to increase this selectivity, to allow for probing the physiological effects induced upon activation of MORs with this non-morphine scaffold, a potent and selective MOR agonist kurkinorin (MOR EC50 = 1.2 ± 0.6 nM, and KOR > 10,000nM) was identified. Upon in vivo evaluation, kurkinorin was determined to elicit centrally-mediated antinociception with similar potency to morphine and a reduced tolerance, sedation, and reward profile in comparison to morphine. Through a SAR campaign, a variety of kurkinorin analogues were synthesized and evaluated in vitro for their ability to activate G-proteins and recruit β-arrestin-2 upon MOR activation. Through these studies, compounds that are more potent than kurkinorin at MORs, compounds that are biased towards β-arrestin-2 recruitment, and compounds that are biased towards G-protein activation have been identified. Salvinorin A suffers from poor pharmacokinetic properties, including low water solubility and rapid metabolism. In an effort to address this issue of water solubility, we sought to identify a point on the molecule through which its water solubility could be modified without sacrificing KOR activity. Previous studies indicated that salvinorin A’s lactone functionality was not necessary for KOR activity; therefore, the lactone was modified to further explore its necessity and tolerance to modification. Analogues that varied in chain length, stereochemistry, and polarity at the lactone position were synthesized and evaluated for their ability to activate KORs. Overall, small linear moieties were well-tolerated, while bulkier groups were not. Salvinorin A analogues that are potent KOR agonists with polar, ionizable moieties in the C17-position have been identified, and the lactone position has been validated as a position on the molecule through which the pharmacokinetic properties can be manipulated without significant loss of KOR activity. Salvinorin A has a very short half-life in humans ( 10,000nM) was identified. Upon in vivo evaluation, kurkinorin was determined to elicit centrally-mediated antinociception with similar potency to morphine and a reduced tolerance, sedation, and reward profile in comparison to morphine. Through a SAR campaign, a variety of kurkinorin analogues were synthesized and evaluated in vitro for their ability to activate G-proteins and recruit β-arrestin-2 upon MOR activation. Through these studies, compounds that are more potent than kurkinorin at MORs, compounds that are biased towards β-arrestin-2 recruitment, and compounds that are biased towards G-protein activation have been identified. Salvinorin A suffers from poor pharmacokinetic properties, including low water solubility and rapid metabolism. In an effort to address this issue of water solubility, we sought to identify a point on the molecule through which its water solubility could be modified without sacrificing KOR activity. Previous studies indicated that salvinorin A’s lactone functionality was not necessary for KOR activity; therefore, the lactone was modified to further explore its necessity and tolerance to modification. Analogues that varied in chain length, stereochemistry, and polarity at the lactone position were synthesized and evaluated for their ability to activate KORs. Overall, small linear moieties were well-tolerated, while bulkier groups were not. Salvinorin A analogues that are potent KOR agonists with polar, ionizable moieties in the C17-position have been identified, and the lactone position has been validated as a position on the molecule through which the pharmacokinetic properties can be manipulated without significant loss of KOR activity. Salvinorin A has a very short half-life in humans (<15 minutes upon inhalation). SAR work in the past has focused on developing analogues with reduced metabolic liabilities, specifically through replacement of the acetate moiety, while maintaining KOR activity. However, the metabolism of many of these compounds had not been directly compared, to one another or to salvinorin A. Therefore, we developed a method to analyze the metabolic profiles of salvinorin A and its analogues in liver microsomes. Through screening salvinorin A and its analogues, several modifications that increase metabolic stability in comparison with salvinorin A have been identified.
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openAccess
Copyright held by the author.
Pharmacology
Organic chemistry
Drug addiction
Metabolism
pain
Pharmacology
salvinorin A
Semisynthesis
Exploration of Salvinorin A for the Development of Pain and Addiction Therapies
Dissertation
oai:kuscholarworks.ku.edu:1808/278462020-07-09T21:40:49Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Crowley, Vincent Matthew
2019-05-10T16:35:18Z
2019-05-10T16:35:18Z
2017-12-31
http://dissertations.umi.com/ku:15631
http://hdl.handle.net/1808/27846
Molecular chaperones are responsible for the maturation of nascent polypeptides and the re-maturation of denatured proteins. One chaperone family that has emerged as an attractive therapeutic target is the 90 kDa heat shock proteins (Hsp90). Hsp90 is responsible for the maturation of proteins associated with all ten hallmarks of cancer, and its inhibition results in a multidirectional attack on cancer. More than seventeen small molecule inhibitors have entered clinical trials; however, concerns have risen due to toxicities, dosing and scheduling issues, and lack of efficacy as a single agent. Therefore, alternative strategies for Hsp90 inhibition are needed to take advantage of the unique biological role of Hsp90. All of the molecules evaluated in clinical trials exhibit pan-Hsp90 inhibition and target all four Hsp90 isoforms with similar affinities. Humans express four different Hsp90 isoforms: Hsp90α and Hsp90β reside in the cytosol, Grp94 is localized to the endoplasmic reticulum, and TRAP1 is found in the mitochondria. Emerging evidence suggests that each Hsp90 isoform plays a unique role in cancer progression. Therefore, to further study the individual roles of each isoform, the development of Hsp90 isoform-selective inhibitors is highly desirable. However, the development of such inhibitors is hindered by the fact that the N-terminal ATP-binding pocket is 85% identical among all four isoforms. Described herein is the development of isoform-selective inhibitors of the ER- and mitochondria-localized Hsp90 isoforms, Grp94 and TRAP1, respectively. Grp94-selective inhibitors were evaluated in models of myocilin-associated open angle glaucoma, cancer metastasis, and multiple myeloma. TRAP1-selective inhibitors were evaluated for their ability to induce apoptosis in cancer cells. These isoform-selective inhibitors will serve as invaluable tools to continue to study the roles played by these isoforms in cancer as well as other indications.
en
openAccess
Copyright held by the author.
Organic chemistry
Biochemistry
cancer
Grp94
Hsp90
medicinal chemistry
TRAP1
The Development of Organelle-localized Hsp90 Isoform-selective Inhibitors
Dissertation
oai:kuscholarworks.ku.edu:1808/99932018-11-15T17:05:50Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Araya, Juan Jose
2012-07-22T18:19:53Z
2012-07-22T18:19:53Z
2012-05-31
http://dissertations.umi.com/ku:12089
http://hdl.handle.net/1808/9993
https://orcid.org/0000-0003-4525-7291
For decades, chemists and medicinal chemists have found in nature the source of inspiration for drug discovery and development. This work describes several aspects of the interaction between the fields of natural products and medicinal chemistry, from isolation and characterization of bioactive molecules to semi-synthetic analogs preparation. A new phase-trafficking approach for acidic, basic, and neutral compounds separation from organic plant extracts was developed, validated and successfully applied to crude plant extracts. This new method could be applied to natural extracts of diverse origin in order to generate better quality samples for initial bioassays. Furthermore, this new catch-and-release methodology allowed the isolation and identification of three compounds new to the literature from the extensively studied ginger rhizomes. Using a more traditional bioassay guided fractionation, we have identified six small-molecules from Rollinia emarginata that modulate organic anion transporting polypeptide´s (OATPs) function. The results of this study show that diverse plant materials are a promising source for the isolation of OATP modulating compounds, and that a bioassay-guided approach can be used to efficiently identify selective OATP modulators. In addition, a 1H NMR-based metabolomic approach was used as a dereplication tool to study the effect of aqueous green tea extracts on OATP1B1-mediated uptake of estrone-3-sulfate. Our findings suggested that not only the gallate catechins were important for the observed uptake inhibition, but also compounds theogalline and 3-p-cumaroyl quinic acid could have been involved. A screening against breast cancer cell line Hs578T was conducted with ten plant species from the Asclepiadaceae family and, based on our findings, three plants were selected for detailed investigation: Asclepias verticillata, Asclepias syriaca, and Asclepias sullivantii. As a result, a total of 46 compounds were isolated and identified, half of which represented novel structures. The isolates showed a wide variety of structures including pregnane and cardiac glycosides, pentacyclic triterpenes, glycosylated flavonoids and lignans, among others. Furthermore, a group of cardiac glycosides were found to have strong cytotoxicity selected breast cancer cell lines. Finally, using a semi-synthetic approach, cardiac glycoside analogs with modifications in the butenolide ring were pursued in order to better understand their SAR. Starting from the commercially available trans-aldosterone, the cardiac glycoside core was built up using a microwave-promoted allylic oxidation using SeO2 (Riley oxidation). In addition, a microwave-promoted Miyaura-Suzuki cross-coupling was utilized to obtain the desired 17β-aryl analogs.
en
openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Organic chemistry
Asclepias
Cardenolides
Natural products
Nmr
Oatp
Phase-trafficking
Phase-Trafficking Methods in Natural Products, Modulators of Organic Anion Transporting Polypeptides from Rollinia emarginata, and Pregnane and Cardiac Glycosides from Asclepias spp.
Dissertation
oai:kuscholarworks.ku.edu:1808/261602018-04-19T20:23:23Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Salyer, Alex Christopher Dean
2018-03-09T22:50:00Z
2018-03-09T22:50:00Z
2017-08-31
http://dissertations.umi.com/ku:15579
http://hdl.handle.net/1808/26160
Small-molecule agonists have been identified for Toll-like Receptors (TLR) 2, TLR4, TLR7 and TLR8 thus far, and chemotypes other than those of canonical ligands are yet to be explored for a number of innate immune receptors. The discovery of novel immunostimulatory molecules would enhance the repertoire of tools available for interrogating innate immune effector mechanisms, and provide additional venues for vaccine adjuvant development. It is with this in mind that we aimed to identify novel immunostimulatory compounds by high-throughput screening, characterize transcriptomal ‘signatures’ of innate immune stimulation and explore mechanisms of adjuvanticity for TLR2, TLR2/7 and TLR8 agonists. A multiplexed, reporter gene-based high-throughput assay capable of detecting agonists of TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, nucleotide-binding oligomerization domain-like receptors (NOD) 1 and NOD2 was utilized in screening 123,943 compounds, in which amphotericin B (AmpB) and nystatin were identified as prominent hits. The polyene antifungal agents act as TLR2- and TLR4-agonists. The TLR4-stimulatory activity of AmpB was similar to that of monophosphoryl lipid A, suggestive of TRIF-biased signaling. The adjuvantic activity of AmpB, at a dose of 100 micrograms, was comparable to several other candidate adjuvants in rabbit models of immunization. (Chapter 2) We sought to identify transcriptomal signatures of innate immune stimulating molecules using next-generation RNA sequencing with the goal of being able to utilize such signatures in identifying novel immunostimulatory compounds with adjuvantic activity. We observed that the CC family of chemokines, particularly CC chemokines 1, 2, 3, 4, 7, 8, 17, 18, 20, and 23, were broadly upregulated by most TLR and nucleotide-binding domain and leucine-rich repeat–containing receptors (NLR) stimuli, while the CXC chemokine family appeared to show distinctions in upregulation. Extracellular receptors such as TLR2, TLR4 and TLR5 induced the transcription of CXC chemokines including CXCL5, CXCL6 and CXCL8, whereas intracellular receptors such as TLR7 and TLR8 upregulated CXC chemokines 11 and 12. A comparison of a variety of TLR agonists in a standardized rabbit immunization model indicated prominent adjuvantic activity for TLR2 agonists. Strong chemokine induction by TLR2 agonists was observed in human peripheral blood mononuclear cells. In addition, human foreskin fibroblasts stimulated with TLR2/6 agonists, but not TLR1/2 agonists resulted in chemokine production, which was consistent with strong expression of TLR2 and TLR6, but not of TLR1, in fibroblasts. TLR2/6 stimulated fibroblasts demonstrated functional chemotactic responses to human T cell and natural killer cells subsets. (Chapter 3) We hypothesized that an ESAT-6-based subunit vaccine adjuvanted with a TLR2/7 hybrid would induce balanced T helper (Th) 1/Th2 responses capable of conferring protection against M. tuberculosis. We therefore covalently linked a potent TLR2 agonist with a dual TLR7/8 agonist, and observed that the resulting TLR2/7 hybrid molecules remained active, though less potent, against TLR2 and TLR7. The TLR2/7 hybrid was equipotent to the two individual TLR agonists in a standardized rabbit immunization model, but induced higher ‘quality’ antibodies as measured by surface plasmon resonance. Linear epitope mapping revealed that the hybrid induced immunoreactivity to more contiguous epitopes in a model antigen. The hybrid molecule was able to induce increases in ESAT-6-specific interferon-γ spot-forming units in the lungs of mice, and reduce the mycobacterial burden in the lungs following M. tuberculosis challenge. (Chapter 4) Part-structures of the 2-aminobenzimidazole scaffold were examined with a view to identifying structural requisites corresponding to the smallest possible fragment of the benzimidazole core that would allow for retention of TLR8-agonistic activity. TLR8-specific agonistic activity was retained in 1-pentyl-4-phenyl-1H-imidazol-2-amine. The crystal structure of this compound bound to TLR8 ectodomain displayed binding interactions that are common to other TLR8 agonists. This compound showed markedly attenuated proinflammatory properties in ex vivo human blood models. Structure-activity relationship (SAR) studies revealed that 4-(2-(benzyloxy)phenyl)-1-pentyl-1H-imidazol-2-amine inhibited TLR signaling in a variety of TLR reporter cell lines, as well as in pharmacologically-relevant human blood model systems. A kinase screen of this compound showed relative specificity for calmodulin kinases. (Chapter 5) The effects of TLR8 agonists on innate immune function suggest that these compounds could potentially be useful as vaccine adjuvants in neonatal vaccines. We examined how TLR8 agonists influence processing of soluble antigens by antigen presenting cells. TLR8-active compounds were unique in inducing pyroptosis-like death in monocytes, leading to the formation of CD14+ extracellular vesicles (ECV) of 100-400 nm diameter. ECV formation was dependent on myeloid differentiation primary response gene 88 (MyD88), interleukin-1 receptor-associated kinases (IRAK) 1 and 4, and p38 mitogen-activated protein kinase (MAPK). The monocyte-derived ECVs contain near-intact soluble antigens, and stimulate antigen-specific recall responses in autologous CD4+ T lymphocytes. The formation of antigen-loaded, monocyte-derived ECVs may be a distinct mechanism underlying the adjuvantic activities of TLR8 agonists. (Chapter 6) The results presented here highlight the applicability of high-throughput screens for the identification of novel innate immune stimuli, and identified transcriptomal profiles to aid in determining adjuvanticity of new compounds, as well as aiding in target identification. The insight gained into mechanisms of adjuvanticity for the TLR2, TLR2/7, and TLR8 agonists highlights the utility of TLR agonists as vaccine adjuvants, and justifies the continued study of small-molecule innate immune stimuli for applications in vaccines.
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openAccess
Copyright held by the author.
Immunology
Chemistry
Adjuvanticity
Immunity
Toll-like Receptors
Vaccine Adjuvants
Vaccines
Exploration of the Mechanisms of Adjuvanticity for Toll-like Receptor Agonists
Dissertation
oai:kuscholarworks.ku.edu:1808/277552019-06-27T21:42:04Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Ma, Huiyong
2019-04-19T20:42:19Z
2019-04-19T20:42:19Z
2017-05-31
http://dissertations.umi.com/ku:15267
http://hdl.handle.net/1808/27755
Development of Bisamides as Kappa Opioid Receptor Agonists. The structure-activity relationship (SAR) expansion was carried out on bisamides KOR agonists. Previous four-step linear synthetic route was replaced by Ugi multicomponent reaction, affording final compound in one step. Parallel synthesis was adopted using Bohdan MiniBlock synthesis platform in combination with subsequent purification with MS-directed HPLC. A total of 80 analogues with diverse substitutions were prepared, including three pairs of enantiomers obtained by chiral HPLC separation of racemic precursors. All of the final compounds were tested in [35S]GTPγS functional assay. Enantiopure analogues were also accessed by arrestin2 imaging assay. Several analogues with improved potency and bias toward G-protein signaling were obtained. A useful SAR was established based on the biological results obtained, which would direct the study of this chemotype in future. Potency Enhancement of Sulfonamide-based Kappa Opioid Receptor Antagonists. Structural modification on a sulfonamide-based KOR antagonists was accomplished. A total of 34 analogues were prepared through linker replacement, constraint manipulation, and substitution introduction. All of the final compounds were assayed using a DiscoveRx PathHunter -arrestin assay platform. One compound with four-fold increase of potency (IC50 = 18.9 ± 4 nM) was obtained, compared with the lead compound (IC50 = 83.5 ± 20.3 nM). A putative binding mode of sulfonamide analogues with the KOR were generated based on the data obtained previously and this study. The enriched SAR and putative binding mode provide insights into the interactions between sulfonamide analogues and the KOR which will direct further study on this chemotype. Asymmetric Acyl Transfer Reactions Catalyzed by a Cyclic Peptide. Kinetic resolution of secondary alcohols by a cyclic peptide was described. The cyclic peptide was designed as a modified version of Miller’s peptide catalyst, which was synthesized in five steps. Single crystal X-ray experiments demonstrated that it adopted a conformation close to type II -turn. Selectivity of this proposed catalyst was examined on five secondary alcohols, with best selectivity factor as about 24.
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openAccess
Copyright held by the author.
Pharmaceutical sciences
agonist
antagonist
catalysis
cyclic peptide
kappa opioid receptor
I. Development of Bisamides as Kappa Opioid Receptor Agonists. II. Potency Enhancement of Sulfonamide-based Kappa Opioid Receptor Antagonists. III. Asymmetric Acyl Transfer Reactions Catalyzed by a Cyclic Peptide.
Dissertation
oai:kuscholarworks.ku.edu:1808/102152020-09-16T14:04:39Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Smith, Katherine Marie
2012-10-27T10:44:24Z
2012-10-27T10:44:24Z
2012-08-31
http://dissertations.umi.com/ku:12334
http://hdl.handle.net/1808/10215
Kappa opioid (KOP) receptors have been suggested as an alternative target to the mu opioid (MOP) receptor for the treatment of pain because KOP activation is associated with fewer negative side-effects (respiratory depression, constipation, tolerance, and dependence). The KOP receptor has also been implicated in several abuse-related effects in the central nervous system (CNS). KOP ligands have been investigated as pharmacotherapies for drug abuse; KOP agonists have been shown to modulate dopamine concentrations in the CNS as well as attenuate the self-administration of cocaine in a variety of species, and KOP antagonists have potential in the treatment of relapse. One drawback of current opioid ligand investigation is that many compounds are based on the morphine scaffold and thus have similar properties, both positive and negative, to the parent molecule. Thus there is increasing need to discover new chemical scaffolds with opioid receptor activity. The flavonoid class of natural products has been identified as a potential source of novel opioid ligands. In particular, dioclein (86) and dioflorin (87) have been reported to have an antinociceptive effect in rodent models of pain, although there has been no in vitro pharmacological evaluation to date. Dioclein and several simplified analogs of dioflorin were synthesized in order to develop structure activity relationships (SAR) for the flavonoid scaffold at opioid receptors. The analogs were pharmacologically evaluated in several cell-based assays (radioligand binding, fluorescent calcium mobilization, and luminescent PathHunterTM beta-arrestin) and found to be inactive at both opioid and cannabinoid receptors. The novel KOP receptor agonist and neoclerodane diterpene salvinorin A was also investigated. Salvinorin A is the main active component of the hallucinogenic plant Salvia divinorum and is the first opioid ligand reported that lacks a basic nitrogen atom in the structure. Ether analogs at the C-2 position of salvinorin A have been reported to have improved affinity and potency over the parent molecule. As alkyl chain ethers have a high degree of flexibility and the oxygen atom may allow for extra hydrogen bonding interactions in the receptor, modifications were made at the C-2 position in order to develop analogs to elucidate the molecular basis for this improved affinity and potency. Tetrahydropyranyl ether 220, ether 223a, and methyltetrahydropyranyl ether 231 were found to have similar KOP affinity and potency to salvinorin A in radioligand binding, [35S]GTP-gamma-S functional, and fluorescent calcium mobilization assays. Tetrahydropyranyl ether 220 was further evaluated for its effects on the cocaine-primed reinstatement of extinguished cocaine self-administration in rats; 220 (1 mg/kg) was found to attenuate cocaine self-administration comparably to salvinorin A (0.3 mg/kg), previously reported to be effective in this animal model. This represents the first report of a salvinorin A derivative with demonstrated anti-addictive capability.
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openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Organic chemistry
Pharmaceutical sciences
Flavonoid
Natural products
Neoclerodane diterpene
Opioid receptors
Investigation of Natural Product Scaffolds for the Development of Opioid Receptor Ligands
Dissertation
oai:kuscholarworks.ku.edu:1808/239222018-01-31T20:07:47Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Hall, Jessica Ann
2017-05-07T16:27:33Z
2017-05-07T16:27:33Z
2015-05-31
http://dissertations.umi.com/ku:14019
http://hdl.handle.net/1808/23922
Heat shock proteins (Hsps) are molecular chaperones that facilitate the conformational maturation of newly synthesized and unfolded cellular proteins (termed “clients”) to maintain protein homeostasis. Heat shock protein 90 (Hsp90) is a chaperone that folds client substrates, many of which drive signal transduction pathways associated with cellular proliferation and differentiation. Consequently, Hsp90 can facilitate oncogenic transformation and can sustain the proper functioning of signaling pathways that have been hijacked during cancer formation and progression. Hsp90 functions as a homodimer and facilitates client maturation via the Hsp90 chaperone cycle. During this cycle, Hsp90 forms a heteroprotein complex with additional proteins (e.g., co-chaperones, partner proteins, immunophilins, etc.) that assist in client folding at different stages of the cycle. However, if this cycle is disrupted and a client is unable to reach conformational maturity, the immature client is ubiquitinylated and degraded via the proteasome. Given Hsp90’s role in cancer progression, Hsp90 inhibition has emerged as a viable strategy for the development of anticancer chemotherapeutics. Classic Hsp90 inhibitors compete with ATP at the Hsp90 N-terminus, of which ATP-binding and hydrolysis is crucial for client maturation. However, these N-terminal inhibitors lead to induction of the pro-survival heat shock response via activation of the transcription factor, heat shock factor-1 (HSF-1; resides at the Hsp90 N-terminus). HSF-1 activation ultimately increases the cellular concentration of Hsps, including Hsp90. Therefore, alternative strategies to inhibit Hsp90 function and/or client maturation that avoid HSF-1 activation (i. e. increased Hsp90 levels) have been pursued. An alternative strategy to inhibit Hsp90-dependent client maturation is to target the Hsp90 C-terminus. The natural products novobiocin and (-)-epigallocatechin-3-gallate (EGCG) were previously identified as Hsp90 C-terminal inhibitors and provide platforms for elucidation of structure-activity relationship studies for the Hsp90 C-terminus. These natural products led to development of analogs that exhibited potent anti-proliferative activity across several different cancer cell lines, many of which decreased the cellular levels of Hsp90 clients and did not affect Hsp levels. Another strategy to prevent Hsp90 client maturation is to disrupt components of the heteroprotein complex, specifically the interactions between Hsp90 and its co-chaperones. The known F1F0 ATP synthase inhibitor, cruentaren A, was shown to disrupt interactions between the co-chaperone F1F0 ATP synthase and Hsp90 via F1F0 ATP synthase inhibition. Disruption of this interaction led to decreased client levels and no increase in Hsp levels. Other disruptors of Hsp90 client maturation include members of the cucurbitacin class of natural products, specifically cucurbitacin D and 3-epi-isocucurbitacin D. These cucurbitacins led to decreased client protein levels without HSF-1 activation. However, only cucurbitacin D disrupted interactions between Hsp90 and the co-chaperones, Cdc37 and p23, similar to the known heteroprotein complex disruptor, gedunin.
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openAccess
Copyright held by the author.
Cellular biology
Biochemistry
Pharmaceutical sciences
Co-chaperone
Heat shock response
Hsp90
Natural Products
Inhibition of Heat Shock Protein 90 Machinery for the Treatment of Cancer: Progress in the Development of Alternative Strategies
Dissertation
oai:kuscholarworks.ku.edu:1808/216472018-07-26T16:50:03Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Fehl, Charles Joseph
2016-10-11T17:09:22Z
2016-10-11T17:09:22Z
2014-12-31
http://dissertations.umi.com/ku:13705
http://hdl.handle.net/1808/21647
https://orcid.org/0000-0001-6182-0879
Selective Probes for Cytochrome P450 17A1 Suggest a Design Strategy Toward Improved Breast and Prostate Cancer Agents. Sex steroids stimulate the growth of hormone-responsive breast and prostate tumors, the two most commonly diagnosed cancers in America. Inhibiting the sex steroid biosynthetic pathway is a promising strategy to halt the progression of these cancers. Cytochrome P450 17A1 (CYP17A1) is a validated target in prostate cancer, but clinical agents fail to achieve selectivity over the highly similar CYP21A2, involved in mineralocorticoid production and blood pressure regulation. In this work, we devised and confirmed a strategy to allow the selective blockade of sex steroidogenesis with the continued production of corticosteroid hormones. To rationally achieve this aim, several co-crystal structures were solved and an assay to evaluate CYP17A1/CYP21A2 selectivity was developed to guide the synthesis of new compounds. A class of these probes achieved significant gains in selectivity over the currently marketed drug abiraterone and galaterone and orteronel, still in clinical trials. This design strategy represents a step toward selectively targeting sex steroidogenesis as a chemotherapeutic tactic for prostate and possibly breast cancers. Spatiotemporal Control of Reactivity via Visible Light-Mediated C-H Activation Creates Chemically Patterned Carbohydrate Surfaces. Reactivity in photochemistry can be restricted to a desired area of a reaction medium by controlling the exposure of reagents to light. This can create patterned surfaces of chemical functionality, which can be used in numerous applications in chemical biology. To demonstrate this utility, we formulated a light-mediated photo-Meerwein arylation reaction to occur on a surface encoded with the reaction substrate. In this proof-of-concept study, reactants were chosen that would undergo a visible color change upon successful reaction. Potential surfaces to display these reactants were then tested. Paper sheets proved optimal for visualization, in which the cellulose C6-position was covalently modified by functionalized coumarin reactants. Use of a photomask allowed these surfaces to be selectively modified by exposure to visible light in the presence of various aryl diazonium reagents and a photocatalyst. These reactions resulted in a strikingly visible color change over the target area. This represents a rapid, cost-effective strategy to selectively encode desired chemical functionality on cellulose medium. This technology could be applied toward surfaces bearing protein capture resins, biosensors, or other agents for chemical biology. Studies Toward Overcoming Product Inhibition in Catalysis of the Intramolecular Schmidt Reaction. Transformations that efficiently generate molecular complexity are useful in drug design, polymer chemistry, and natural product synthesis. The intramolecular Schmidt reaction allows access to amides and lactams from ketone starting materials, and has seen extensive use in the above applications. This reaction is promoted by strongly acidic conditions. Since the reaction creates amide products with increased Lewis basicity over the ketone reactants, most acids are readily sequestered upon successful reaction. In this chapter, we describe the optimization of conditions that promote the intramolecular Schmidt reaction with substoichiometric Sc(OTf)3, which turns over to allow a catalytic cycle in response to heat. The scope, temperature dependence, and kinetics of this transformation were characterized. Additionally, several strategies to expand this strategy were screened. This work, with additional results from a co-worker, ultimately led to the development of vastly improved conditions and scope for catalytic Schmidt reactions.
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openAccess
Copyright held by the author.
Organic chemistry
Biochemistry
Oncology
abiraterone analogs
organic synthesis
photolithographic organic surface patterning
Schmidt reaction catalysis
selective CYP17A1 inhibitors
structure based drug design
I. Selective Probes for Cytochrome P450 17A1 Suggest a Design Strategy Toward Improved Breast and Prostate Cancer Agents II. Spatiotemporal Control of Reactivity via Visible Light-Mediated C-H Activation Creates Patterned Carbohydrate Surfaces III. Toward Catalytic Intramolecular Schmidt Reactions
Dissertation
oai:kuscholarworks.ku.edu:1808/43342020-07-22T12:43:52Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Beckman, Karen
2009-02-02T05:49:17Z
2009-02-02T05:49:17Z
2008-09-04
http://dissertations2.umi.com/ku:2726
http://hdl.handle.net/1808/4334
This thesis describes the rationale, design, and syntheses of derivatives of isatin (1-H-indole-2,3-dione). Isatin was identified, during a high throughput screen of 10,000 compounds, as a potential scaffold for microtubule-destabilizing agents. Additional screening of purchased isatin derivatives gave rise to four substitution patterns of interest, 7-arylisatins, 5-methyl-N¬-alkyl/aryl isatins, 5-chloro-N-alkyl/aryl isatins and 5,7-dichloro-N-alkylated isatins. Series of compounds with the substitutions of interest were synthesized to further probe the structure-activity relationship (SAR) of isatin. The SAR study showed that substitutions in the 5- and 7- positions of the aromatic ring combined with N-substitutions increased the disruption of microtubule assembly. The 7-phenylisatin and N-arylisatin derivatives were inactive in the biological assay. Several of the 5-chloro-N-alkylisatins and the 5,7-dichloro-N-alkylisatins were cytotoxic in both MCF-7 and NCI/ADR-RES cell lines. 5,7-Dichloro-N-(4-bromobenzyl)isatin was the most active compound against MCF-7 cells, IC50 = 2.1 µM. To date the most cytotoxic compound tested is 5-methyl-N-(1-propyl)isatin, with an IC50 value of 52 nM (microtubule assembly IC50 = 2.6 µM) in the drug resistant cancer cell line NCI/ADR-RES.
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openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Organic chemistry
Chemistry
Biochemistry
Medicinal chemistry
Isatin Derivatives as Inhibitors of Microtubule Assembly
Thesis
oai:kuscholarworks.ku.edu:1808/98012020-06-25T20:49:39Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Duerfeldt, Adam S.
2012-06-03T15:57:13Z
2012-06-03T15:57:13Z
2011-12-31
http://dissertations.umi.com/ku:11798
http://hdl.handle.net/1808/9801
https://orcid.org/0000-0002-3130-9890
The 90 kDa heat shock proteins (Hsp90) are molecular chaperones that are upregulated in response to cellular stress and are responsible for the conformational maturation, activation and/or stability of more than 200 client proteins. Many of these clients are oncogenic and facilitate the progression of cancer. Disruption of Hsp90's inherent ATPase activity renders the chaperone inactive, leading to degradation of substrates and ultimately, apoptosis. Consequently, Hsp90 has become a highly sought after anti-cancer target and numerous pharmaceutical companies and academic labs are expending efforts to develop novel methods to regulate the Hsp90-mediated protein folding process. Included within the Hsp90 family are four isoforms, each of which exhibits a unique cellular localization, expression, function and clientele. Hsp90α (inducible) and Hsp90β (constitutive) both localize to the cytoplasm and share similar functions; however, recent studies have identified isoform specific substrates. Tumor necrosis factor receptor-associated protein (TRAP-1) is the Hsp90 isoform localized to the mitchondria and to date, no specific clients or selective inhibitors have been identified. The fourth isoform is glucose-regulated protein 94 kDa (Grp94), which is localized to the endoplasmic reticulum and is responsible for the maturation and stability of specific secreted and membrane bound proteins. Currently identified Hsp90 inhibitors exhibit pan-inhibition, resulting in the disruption of all four isoforms' ability to bind and hydrolyze ATP. This activity is believed responsible for the undesired toxicities related to Hsp90 inhibition in the clinic, as proteins that are critical to cardio function and the central nervous system are dependent upon yet to be determined Hsp90 isoforms. Another detriment arising from N-terminal Hsp90 inhibition is induction of the pro-survival, heat shock response. Specifically, induction of the target, Hsp90, has resulted in therapeutic resistance and complications with dosing and administration protocols. Presented herein is rationale for the development of Hsp90 isoform selective inhibitors and the first irreversible inhibitor of Hsp90 that mitigates induction of Hsp90; thus, providing key advancements towards addressing the detriments associated with Hsp90 inhibitors currently under clinical investigation.
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openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Chemistry
Medicine
Cancer
Grp94
Hsp90
Inflammation
Isoform selectivity
Small molecule
A New Generation of Hsp90 Inhibitors: Addressing Isoform Selectivity and Heat Shock Induction
Dissertation
oai:kuscholarworks.ku.edu:1808/257732019-01-17T17:03:05Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Lee, Molly Marie
2018-01-29T17:50:36Z
2018-01-29T17:50:36Z
2016-12-31
http://dissertations.umi.com/ku:15063
http://hdl.handle.net/1808/25773
Early-stage drug discovery and chemical biology projects often use fluorescence-based assays to obtain information about biological interactions and cellular processes. However, many of the best and brightest fluorophores suffer from major limitations such as very high cost and/or restrictive chemical properties that hinder their utility for studies of living biological systems. To create improved fluorescent molecular probes of receptor-ligand interactions and dynamic cellular processes, a major focus of this dissertation is on the bright coumarin-derived fluorophore Pacific Blue. Although Pacific Blue is commercially available, its high cost has restricted its utility as a building block for preparation of small molecule-derived and peptide-derived molecular probes. To overcome this limitation, we developed a new synthetic route that readily allows access to gram quantities of this fluorophore. This synthetic route is superior to previously published routes, and it can facilitate access to a wide variety of fluorescent ligands of receptors, biosensors, and related cellular probes. Quantification of receptor-ligand interactions is important for screening of both on-target and off-target affinities of chemical probes and drug candidates. These assays must be cost-effective, efficient, and high-throughput to keep up with fast-paced needs of drug discovery projects. Toward this end, we characterized a new FRET pair, comprising the endogenous fluorescent amino acid tryptophan and Pacific Blue. We demonstrate that these fluorophores allow quantification of interactions between small molecules and tryptophan-containing proteins in vitro. We also synthesized and evaluated Pacific Blue derivatives of paclitaxel (Taxol) as tools to label microtubules, detect cellular efflux by P-glycoprotein (P-gp), and potentially explore some of the paradoxical clinical responses associated with the parent anticancer drug. We also characterized two other new FRET pairs, Pacific Blue-Pennsylvania Green and Pacific Blue-Oregon Green, and investigated the stability of disulfide linkers both in vitro and in living cells as models of drug delivery systems. Finally, using an alternative detection platform of fluorescence polarization, we describe the development of methods for the characterization of inhibitors of a protein-protein interaction involved in iron homeostasis in the pathogenic bacterium Pseudomonas aeruginosa. This research extends the utility of Pacific Blue and related fluorophores as tools for studies of chemical biology and drug discovery.
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openAccess
Copyright held by the author.
Chemistry
Chemical biology
Fluorescence
FRET
Receptor-ligand interactions
An Improved Synthesis of the Pacific Blue Fluorophore and Fluorescence-based Studies of Receptor-Ligand Interactions
Dissertation
oai:kuscholarworks.ku.edu:1808/73862020-07-23T13:19:30Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Porubsky, Patrick R.
2011-04-25T19:15:06Z
2011-04-25T19:15:06Z
2009-04-09
http://dissertations.umi.com/ku:10229
http://hdl.handle.net/1808/7386
Cytochrome P450 (CYP) is the predominate superfamily of enzymes responsible for Phase I metabolism of drugs and other xenobiotics. Understanding the structural reasons for the substrate selectivity of these enzymes is important for both pharmacological and toxicological reasons. Two isoforms of interest from this enzyme superfamily that are CYP2A13 and CYP2E1. Cytochrome P450 2A13 (CYP2A13) is a lung specific enzyme known to activate the potent tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) into two carcinogenic metabolites. CYP2A13 has been crystallized and X-ray diffraction experiments illuminated the structure of this enzyme, but with an unknown ligand present in the enzyme active site. This unknown ligand was suspected to be indole but a selective method had to be developed to differentiate among indole and its metabolites in the protein sample. We successfully modified a microbiological colorimetric assay to spectrophotometrically differentiate between indole and a number of possible indole metabolites in nanomolar concentrations by derivatization with p-dimethylaminocinnamaldehyde (DMACA). Further differentiation of indoles was made by mass spectrometry (HPLC-UV/vis-MS/MS) utilizing the chromophore generated in the DMACA conjugation as a UV signature for HPLC detection. The ligand in the crystallized protein was unambiguously identified as unsubstituted indole, which facilitated refinement of two alternate conformations of indole in the CYP2A13 crystal structure active site. Human cytochrome P450 2E1 (CYP2E1) is a xenobiotic metabolizing enzyme that is highly conserved among mammals. In addition to small molecular weight exogenous drugs like the analgesic acetaminophen and the volatile anesthetic halothane, CYP2E1 is also involved in endogenous fatty acid metabolism. To more fully understand the structural factors that contribute to the substrate selectivity of CYP2E1, it has been cocrystallized with two structurally different heme-binding compounds: indazole, a small molecular weight inhibitor and ω-imidazolyl-decanoic acid, a fatty acid analog. Comparison of the CYP2E1 structures shows that only small side chain movements are required for the accommodation of the much larger fatty acid analog. Rotation of the side chain of F298 causes a change in the active site volume from 190 Å3 in the indazole-bound structure to 440 Å3 in the ω-imidazolyl-decanoic acid-bound structure. Future work will be focused on cocrystal structures of CYP2E1 with both longer and shorter chain analogs to better understand the ability of the enzyme to metabolize a variety of fatty acids substrates.
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openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Chemistry
Biochemistry
Crystal structure
Cyp2a13
Cyp2e1
Cytochrome p450
Indole
Structural biology
A TALE OF TWO ENZYMES: IDENTIFICATION OF AN UNKNOWN LIGAND BOUND TO CYTOCHROME P450 2A13 AND UNDERSTANDING SUBSTRATE SELECTIVITY OF CYTOCHROME P450 2E1
Thesis
oai:kuscholarworks.ku.edu:1808/19912020-07-15T12:43:13Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Biery, Jennifer J.
2008-03-01T17:25:53Z
2008-03-01T17:25:53Z
2007-12-18
http://dissertations.umi.com/ku:2302
http://hdl.handle.net/1808/1991
MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) catalyzes the first committed step in the biosynthesis of cell wall peptidoglycan. Because this pathway is absent in mammals, MurA is an attractive target for the development of antimicrobial agents. While Escherichia coli and all other gram-negative bacteria possess only one copy of the MurA gene, analyses of the genomes of several gram-positive bacteria reveal the existence of two MurA genes, termed MurA1 and MurA2. The research presented here focuses on the cloning, expression, purification and kinetic characterization of the MurA enzymes from two gram-positive organisms: Staphylococcus aureus and Bacillus subtilis. Sequence analysis indicates that all genes are complete and that the enzymes contain the important catalytic residues previously identified in E. cloacae MurA. Kinetic characterization revealed that the enzymes from both organisms are active and are inhibited by fosfomycin.
EN
openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Molecular biology
Chemistry
Biochemistry
Mura
B. subtilis
S. aureus
Gram-positive
Probing the Antibiotic Target MurA from S. aureus and B. subtilis
Thesis
oai:kuscholarworks.ku.edu:1808/261502018-04-26T19:30:43Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Ambler, Brett R.
2018-03-09T22:21:28Z
2018-03-09T22:21:28Z
2017-05-31
http://dissertations.umi.com/ku:15229
http://hdl.handle.net/1808/26150
Trifluoromethanes play an important role in medicinal chemistry, and methods that enable the rapid synthesis of trifluoromethanes from common functional groups are essential for the synthesis of bioactive compounds. We describe a series of Cu-catalyzed decarboxylative trifluoromethylation reactions that enable the conversion of alcohols to trifluoromethanes. These reactions rely on the efficient generation of nucleophilic “Cu–CF3”, and Chapter 1 provides background on the synthesis, stability, and reactivity of this organometallic species. In addition, we discuss the use of halodifluoroacetates as common, inexpensive, and green precursors to “Cu–CF3”. Cu-catalyzed trifluoromethylation of electrophiles was an appealing, but underdeveloped strategy for accessing fluorinated compounds. Chapter 2 describes our entry into Cu-catalyzed decarboxylative trifluoromethylation of bromodifluoroacetates. We discovered that ligand and catalyst activation played critical roles in the development of an efficient Cu-based catalyst system. Trifluoroethylarenes are commonly found in bioactive compounds, and in Chapter 3, we describe a straightforward Cu-catalyzed strategy to access this motif from benzylic bromodifluoroacetates. A key aspect of this reaction involved the generation of active electrophilic species in situ. In Chapter 4, we describe the ability of ligands to alter the regioselectivity of Cu-catalyzed trifluoromethylation reactions. Propargylic bromodifluoroacetates are converted into a mixture of propargylic trifluoromethanes and trifluoromethylallenes using “Cu–CF3”; however, the use of 1,10-phenanthroline inverts the typical regioselectivity, and provides trifluoromethylallenes in high yield and selectivity. This is the first example of ligands controlling the regioselectivity of Cu-based trifluoromethylation reactions.
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openAccess
Copyright held by the author.
Chemistry
Organic chemistry
Chemistry
Copper
Fluorine
Medicinal Chemistry
Organic Chemistry
Trifluoromethylation
Copper-Catalyzed Decarboxylative Trifluoromethylation Reactions
Dissertation
oai:kuscholarworks.ku.edu:1808/61792020-07-28T13:44:16Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Schneider, Christopher Mark
2010-05-03T00:48:26Z
2010-05-03T00:48:26Z
2009-10-20
http://dissertations.umi.com/ku:10632
http://hdl.handle.net/1808/6179
The benzolactone enamide natural products are identified by three structural characteristics: a salicylate arene, a 12- or 15-membered macrolactone, and an enamide side chain. These natural products exert their biological activity by inhibiting the vacuolar-(H+)-ATPase (V-ATPase) enzyme. The benzolactone enamide oximidine II has been synthesized twice previously with only moderate yields realized for the key macrocyclization step. Following a previous Georg group strategy, we envisioned performing the ring-closure using a Castro-Stephens reaction. While optimizing this copper-mediated macrocyclization, we discovered an unprecedented copper-mediated reductive coupling reaction. The enamide side chain of these natural products is postulated to be critical for biological activity. To probe the importance of this acid-sensitive moiety, we synthesized an allylic amide homolog of oximidine II and tested both oximidine II and this analog in melanoma cancer cells. The bacterial enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the first committed step of cell wall biosynthesis. Using highthroughput screening, 5 scaffolds were identified with MurA inhibitory activity. Analog development of the pyrrole-benzoic acid scaffold failed to generate compounds with improved potency. We then turned to structure-based drug design to investigate new MurA inhibitors. Using computer-modeling software, low molecular weight molecules were docked into various MurA crystal structures. Evaluation of these docking studies revealed 4 small molecules as potential leads for further optimization. Dioxins are environmental pollutants that cause a range of biological effects in a dose-dependent manner. The exact mechanism of action for dioxins is not fully understood. 2,3,7,8-Tetrachlorophenothiazine (TCPT) was designed to probe potential mechanisms of action and biological effects of dioxin analogs. Utilization of Buchwald-Hartwig coupling methodology produced TCPT in 37% yield. Preliminary biological testing of TCPT has shown favorable pharmacokinetic properties.
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Organic chemistry
2,3,7,8-tetrachlorophenothiazine
Copper hydride
Medicinal chemistry
Oximidine ii
Udp-n-acetylglucosamine enolpyruvyl transferase
Studies on Oximidine II - Total Synthesis by an Unprecedented Reductive Coupling
Dissertation
oai:kuscholarworks.ku.edu:1808/293152020-10-08T15:00:26Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Hunt, Jordan Robert
2019-06-12T03:12:18Z
2019-06-12T03:12:18Z
2018-05-31
http://dissertations.umi.com/ku:15767
http://hdl.handle.net/1808/29315
https://orcid.org/0000-0001-5443-147X
The 1H-Imidazo-[4,5-c]quinolines are a class of compounds that are agonists towards Toll-like receptor 7 and 8 (TLR7/8). For example, Imiquimod and Resiquimod have been shown to act as vaccine adjuvants, enhancing antigen-specific antibody production and skewing the immunity towards a Th1 response. Activation of TLR7/8 have been shown to stimulate dendritic cells to secrete cytokines, upregulate costimulatory molecule expression and enhance antigen presentation to T cells. Imidazoquinoline compounds have demonstrated vaccine adjuvant properties in several animal models. The adjuvant effects can be enhanced by measures that localize the drug with the vaccine without quickly entering the systemic circulation. Clinical studies demonstrate that local immune activation is useful; imiquimod is approved for the topical treatment of basal cell carcinomas. However, injection or oral routes of administration of imidazoquinolines are not therapeutically beneficial and possibly dangerous due to systemic and non-specific activation of the immune system. To take advantage of the adjuvant property of imidazoquinolines, they need to be formulated or designed to allow for local immune activation without induction of systemic cytokines. This study focuses on developing a new site on the phenyl ring to lead to better imidazoquinolines that could be easily formulated in future drug delivery studies.
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Organic chemistry
Immunology
Adjuvants
Imidazoquinolines
Structure Activity Relationship
Toll-like Receptor 7
Vaccines
Investigation of the Phenyl Ring of Imidazoquinolines
Thesis
oai:kuscholarworks.ku.edu:1808/81862020-08-07T14:24:30Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Shukla, Nikunj M.
2011-10-09T14:40:49Z
2011-10-09T14:40:49Z
2011-05-31
http://dissertations.umi.com/ku:11438
http://hdl.handle.net/1808/8186
Toll-like receptors (TLR)-7/-8 are innate immune receptors present in the endosomal compartment that are activated by single-stranded RNA (ssRNA) molecules of viral as well as nonviral origin, inducing the production of inflammatory cytokines necessary for the development of adaptive immunity, and are thus useful as vaccine adjuvants. A general introduction to TLRs, with an emphasis on the role of TLR7 activation in mobilizing innate and adaptive immune responses is presented in Chapter 1. Synthetic small molecule agonists of TLR7 include the imidazoquinoline class of compounds such as Gardiquimod [1-(4-amino-2-((ethylamino)methyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol]. En route to the synthesis of gardiquimod, its des-amino 3H regioisomer was found to be antagonistic at TLR7 (Chapter 2). Using the 3H imidazoquinoline as a lead, the syntheses, characterization, and biological evaluation of a pilot library of 3H imidazoquinolines was undertaken, which led to the identification of a dual TLR7/TLR8 antagonist (Chapter 3). With the goal of developing more potent TLR7 agonists as adjuvants, derivatives of gardiquimod were synthesized and a detailed SAR study on the imidazoquinoline chemotype was performed, which led to the discovery of a highly potent, lipophilic, human TLR7 agonist (Chapter 4). Further exploration on the imidazoquinoline chemotype led us to a highly active TLR7/8 dual agonistic molecule bearing a free primary amine on the N1 substituent. This molecule was utilized to synthesize fluorescent imidazoquinoline analogues that retained TLR7/8-agonistic activity, and were used to study the distribution of TLR7 and also to examine its differential uptake in lymphocytic subsets (Chapter 5). Homodimers of imidazoquinolines were synthesized in order to test whether such constructs would behave as modulators of TLR3 (Chapter 6). The TLR7/8 dual agonistic molecule was also used as a convenient precursor for the synthesis of isothiocyanate and maleimide derivatives, enabling its direct conjugation to protein and polysaccharide antigens to make self adjuvanting vaccine constructs. The isothiocyanate derivative was covalently coupled to a model antigen, alpha-lactalbumin, and this self-adjuvanting alpha-lactalbumin construct induced robust, high-affinity immunoglobulin titers in murine models of vaccination (Chapter 7).
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Pharmaceutical sciences
Immunology
Adjuvant
Autoimmune diseases
Hiv
Imidazoquinoline
Toll-like receptors
Vaccine
Modulators of Toll-like Receptors -7 and -8
Dissertation
oai:kuscholarworks.ku.edu:1808/216522018-11-01T16:50:44Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Vekariya, Rakesh H.
2016-10-11T18:45:25Z
2016-10-11T18:45:25Z
2016-08-31
http://dissertations.umi.com/ku:14828
http://hdl.handle.net/1808/21652
https://orcid.org/0000-0001-8292-9793
Cycloaddition of allylic azides and alkynes. The 1,3-dipolar Huisgen azide-alkyne cycloaddition is a significant area of interest in modern chemistry. The use of allylic azides as dynamic reaction partners represent a novel variant of this chemistry as they undergo facile 1,3-allylic azide rearrangement, which is also known as the Winstein rearrangement. We combined such an allylic azide rearrangement with an intramolecular Huisgen cycloaddition to afford substituted triazoles in a diastereoselective fashion. Although modest diastereoselectivity was observed in most cases, the majority of diastereomeric pairs were separable. Also, depending on the conditions, a difference in the reactivity of various allylic azides was noticed. Under thermal conditions, vinyl-substituted triazoloxazines were formed, in contrast to copper(I)-catalyzed conditions which afforded dimerized macrocyclic products. Hexafluoro-2-propanol promoted Friedel–Crafts acylation reactions. The Friedel–Crafts acylation is one of the most important reactions in both academia and industry for the synthesis of aromatic ketones. The reaction is typically promoted by stoichiometric or greater amounts of acids, such as AlCl3, FeCl3, or H2SO4, which activate the carbonyl for attack by an aromatic group. A drawback of this extremely versatile acylation reaction is the generation of large amounts of corrosive aqueous waste following post-synthesis workup. We have shown that hexafluoro-2-propanol (HFIP) promotes both intramolecular and intermolecular FC acylation without additional catalysts or reagents. This solvent-promoted acylation is practically simple and accommodates a broad substrates scope. Our preliminary kinetic studies reflects involvement of 3 molecules of HFIP in rate determing step.
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Pharmaceutical sciences
Organic chemistry
Chemistry
allylic azide rearrangement
azide-alkyne cycloaddition
copper(I)-catalyzed azide-alkyne cycloaddition
Friedel–Crafts acylation
hexafluoro-2-propanol
HFIP
Studies and Synthetic Methodology: 1. Cycloaddition of Allylic Azides and Alkynes, 2. Hexafluoro-2-propanol-promoted Friedel–Crafts Acylation Reactions
Dissertation
oai:kuscholarworks.ku.edu:1808/102182020-09-16T13:57:55Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Peterson, Laura B.
2012-10-27T10:47:41Z
2012-10-27T10:47:41Z
2012-08-31
http://dissertations.umi.com/ku:12224
http://hdl.handle.net/1808/10218
The heat shock proteins represent an important class of pro-survival proteins that are intimately involved in cell survival, adaptation to cellular stress, and protein management. Heat shock protein 90 kDa (Hsp90) is a molecular chaperone responsible for the post-translational maturation of nascent polypeptides. Many of the Hsp90-dependent client proteins are involved in oncogenic processes, and accordingly, Hsp90 has emerged as a promising target for anti-cancer therapies. Unfortunately, the clinical evaluation of Hsp90 inhibitors has been met with dosing, scheduling, and toxicity issues. The Hsp90 inhibitors that have reached clinical trials bind to the Hsp90 N-terminal ATP-binding site and demonstrate pan-Hsp90 inhibition, as they bind to and inhibit all four human Hsp90 isoforms. This characteristic may rationalize the undesired toxicities related to Hsp90 inhibition. Interestingly, the identification and characterization of isoform specific client proteins has not been extensively explored. In addition, N-terminal Hsp90 inhibition results in the induction of the heat shock response, whereby the expression of Hsp90 and other heat shock proteins is induced. This attribute of N-terminal inhibitors results in the clinically observed dosing and scheduling detriments as levels of Hsp90 increase with administration of the drug. Described herein is the design, synthesis and biological evaluation of novel Hsp90 inhibitors that avoid the above mentioned therapeutic liabilities of currently known Hsp90 inhibitors. The identification and characterization of an Hsp90-isoform dependent client protein and an Hsp90-isoform selective inhibitor is also presented.
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This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Pharmaceutical sciences
Curcumin
Grp94
Hsp90
Isoform
Novobiocin
Investigation of the Hsp90 C-terminal Binding Site, Novel Inhibitors and Isoform-Dependent Client Proteins
Dissertation
oai:kuscholarworks.ku.edu:1808/257682018-09-20T19:38:09Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Khandelwal, Anuj
2018-01-28T23:13:07Z
2018-01-28T23:13:07Z
2016-08-31
http://dissertations.umi.com/ku:14787
http://hdl.handle.net/1808/25768
https://orcid.org/0000-0003-4492-4400
The 90 kDa heat shock proteins (Hsp90) are critical for the maintenance of cellular homeostasis and mitigate the effects of cellular stress and therefore, play an important role in cell survival. Hsp90, as a molecular chaperone, folds nascent polypeptides and denatured proteins to their biologically relevant conformations. Many of the proteins dependent upon Hsp90 are essential to the growth and proliferation of cancer cells. In fact, proteins associated with all ten hallmarks of cancer are dependent upon the Hsp90 protein folding machinery. Consequently, inhibition of Hsp90 represents a combinatorial approach for the treatment of cancer. 17 small molecule inhibitors of Hsp90 have entered clinical trials, all of which bind Hsp90 N-terminus and exhibit pan-inhibitory activity against the four Hsp90 isoforms: Hsp90, Hsp90, Grp94, and Trap1. However, lack of isoform selectivity with current clinical candidates appears detrimental as more than 20 clinical trials have failed, citing hepatotoxicity, cardiotoxicity, and peripheral neuropathy amongst other side effects. Additionally, pan-inhibition of Hsp90 induces the pro-survival heat shock response, requiring the escalation of patient doses to overcome increased Hsp90 expression. Therefore, alternative approaches for Hsp90 modulation are highly sought after. Isoform-selective inhibition of Hsp90 provides an opportunity to address the aforementioned detriments associated with pan-Hsp90 N-terminal inhibitors. Hydrolysis of ATP by the N-terminal nucleoside binding pocket is required for the maturation of client protein substrates, and all four Hsp90’s share 85% identity within this region. Consequently, the discovery of isoform-selective inhibitors has been challenging. Described herein is the rationale for development of isoform selective inhibitors and the identification of the first isoform selective inhibitors of Hsp90 and Hsp90-isoforms. Unlike the N-terminus, inhibition of the Hsp90 C-terminus does not induce the heat shock response and hence, C-terminal inhibitors manifest the desired cytotoxic affect against cancer cells. However, absence of a co-crystal structure and lack of lead compounds, have resulted in limited success towards the development of Hsp90 C-terminal inhibitors. Recently, EGCG, a green tea polyphenol, was shown to bind at the C-terminus of Hsp90. Structure activity relationships studies were conducted on EGCG for improved Hsp90 inhibition and are also presented.
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Organic chemistry
Medicine
Pharmaceutical sciences
EGCG
heat shock proteins
Hsp90
Isoform-selective Hsp90 inhibitors
Natural Products
Unfolding the Hsp90 Foldasome: Structure-Activity Relationship Studies on EGCG and Development of Isoform-Selective Inhibitors
Dissertation
oai:kuscholarworks.ku.edu:1808/98162018-01-31T20:07:59Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Meyer, Angelica Michelle
2012-06-03T16:27:25Z
2012-06-03T16:27:25Z
2011-12-31
http://dissertations.umi.com/ku:11911
http://hdl.handle.net/1808/9816
Marine flora and fauna have provided numerous alluring natural products, some of which contribute to treating diseases. The genus Clavelina is the source of a variety of tricyclic alkaloids, including the lepadiformine and cylindricine families. Novel approaches to synthesizing these molecules are sought after to increase their accessibility and for analogue development. In this dissertation, reaction sequences involving an intramolecular Schmidt transformation, which can quickly build up the molecular architecture associated with these targets is described. In one approach, the Lewis acid promoted intramolecular Schmidt reaction is combined in series with a Prins reaction to afford an interesting tricyclic lactam. This methodology culminates in a formal synthesis of lepadiformine A and a total synthesis of lepadiformine C. In another project, a tandem Diels-Alder/Schmidt reaction is utilized to prepare a similar tricyclic lactam. This process is applied toward an asymmetric total synthesis of (-)-cylindricine C. The preparation for the optically active starting material for the synthesis of (-)-cylindricine C is also discussed, as it is a prominent figure in the preparation of prostaglandins.
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Organic chemistry
Pharmaceutical sciences
Cylindricine
Lepadiformine
Schmidt reaction
Tandem reactions
Total synthesis
Synthetic Strategies for the Lepadiformines and Cylindricine C via Tandem Schmidt Reactions
Dissertation
oai:kuscholarworks.ku.edu:1808/148302020-06-22T19:29:19Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Liu, Ruzhang
2014-07-28T02:05:10Z
2014-07-28T02:05:10Z
2012-12-31
http://dissertations.umi.com/ku:12528
http://hdl.handle.net/1808/14830
https://orcid.org/0000-0002-9569-2622
The research presented herein describes synthetic applications of an allylic azide rearrangement, mainly focusing on its combination with the intramolecular Schmidt reaction to afford vinyl-substituted bicyclic lactams. Undesired stereochemistry was obtained in the initial synthetic route to an advanced intermediate of natural product pinnaic acid, in which a home-made electrochemistry apparatus was involved in the key step to modify the tricyclic lactam. The combination of allylic azide rearrangement and intramolecular Schmidt reaction afforded the target with desired stereochemistry, and the synthesis of the cyclobutanone was achieved by an asymmetric [2+2] cycloaddition. Prior to its combination with the intramolecular Schmidt reaction, the rearrangement of allylic azides was studied from different perspectives such as substrate, time, temperature, and Lewis acid. Next, the combined reaction was studied with the cyclohexanone-based allylic azide, whose stereochemical outcomes were rationalized by the conformational analysis and computational calculations. Different substituents on the cyclic ketone ring system were found to have different impacts on the diastereoselectivity. During this process, a chloro-Prins reaction was found as the major side-reaction, and carbocation-mediated allylic azide rearrangement was confirmed. Dihedral angle was utilized to explain why and in which cases the six-membered intermediate proceeds through chair or boat conformation for fused bicyclic system. This reaction was also utilized to produce twisted amides via cation-π interaction, along with the use of other by-products from different Lewis acids. The utilization of this methodology to finish the total synthesis of alkaloid 205B is underway. Initial studies of the combination of allylic azide rearrangement and alkyne-azide cycloaddition were conducted. Also the reorganization of allylic azides was utilized to generate 2-azadiene species, which may undergo Diels-Alder reactions or electrocyclizations.
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This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Organic chemistry
Alkaloid 205b
Allylic azide rearrangement
Conformationl analysis
Intramolecular schmidt reaction
Pinnaic acid
Stereocontrol
A Combined Allylic Azide Rearrangement and Intramolecular Schmidt Reaction - Discovery, Development, and Application
Dissertation
oai:kuscholarworks.ku.edu:1808/298482021-10-28T19:35:29Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Smith, Tomas Joseph
2019-12-10T20:54:27Z
2019-12-10T20:54:27Z
2019-08-31
http://dissertations.umi.com/ku:16770
http://hdl.handle.net/1808/29848
Fluorescence-based assays play key roles in drug discovery and development. These assays are widely used due to the widespread availability of fluorescent probes and highly sensitive detection platforms. This method is a mainstay of high-throughput drug screening (HTS) campaigns, where simple and inexpensive assays are preferred for scalability and repeatability. This approach can identify novel chemotypes that may lead to new methods to treat disease. To develop a new phenotypic assay for drug discovery, we investigated a fluorescent mimic of the anticancer drug Taxol, termed Pacific Blue-Gly-taxol (PBGT). This molecular probe binds cellular microtubules and is a highly sensitive substrate of the cellular efflux transporter P-glycoprotein (P-gp). When HeLa cervical carcinoma cells are cotreated with PBGT (1 μM) and the P-gp inhibitor verapamil (25 μM), cellular fluorescence increases by ~ 10-fold as analyzed by confocal microscopy or flow cytometry. Because of the simplicity and sensitivity of this assay of P-gp activity, we envisioned that it could be optimized in a 96-well plate format to provide a useful method to investigate cellular efflux mediated by this protein transporter. To provide a proof of concept, 1584 diverse compounds obtained from the National Cancer Institute (NCI) were screened using automated pipetting and flow cytometry. The primary screen yielded more than 23 hit compounds with equivalent or of higher activity than verapamil (25 μM). Among these hits, we identified diarylureas that do not appear to associate directly with P-gp but rather disrupt the typical rod-like structure of mitochondria. These compounds may inhibit P-gp indirectly by affecting mitochondria or via a target that additionally affects this organelle. These results demonstrate that PBGT is a highly sensitive probe for discovery of inhibitors of P-gp and may allow identification of alternative mechanisms of inhibition of this major drug transporter.
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Biochemistry
Chemistry
Pharmacology
assay development
drug resistance
fluorescence
high-content screening
P-glycoprotein
taxol
Discovery of Novel Inhibitors of Cellular Efflux by High-Content Screening with a Fluorescent Mimic of Taxol
Thesis
oai:kuscholarworks.ku.edu:1808/295502021-10-28T19:24:32Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Orsi, Douglas
2019-09-06T19:37:54Z
2019-09-06T19:37:54Z
2019-05-31
http://dissertations.umi.com/ku:16585
http://hdl.handle.net/1808/29550
https://orcid.org/0000-0001-6731-5494
Fluorination is an important strategy for perturbing the biophysical properties of compounds in medicinal chemistry. Specifically, fluorination modulates both the pharmacodynamic and pharmacokinetic properties of bioactive molecules in generally beneficial ways. However, fluorination similarly manipulates the reactivity of compounds in synthetic chemistry, leaving many traditional synthetic methods unable to perform as expected in organofluorine chemistry. Chapter 1 provides background on the effects of fluorine on medicinal and synthetic chemistry, and specifically discusses the effects of fluorine upon alkenes. Gem-difluoroalkenes are an appealing substructure for nucleophilic addition reactions, as they readily react with nucleophiles. However, upon nucleophilic addition defluorination reactions occur, providing fluoroalkene products. Reactions of difluoroalkenes which retain both fluorine atoms would provide access to underexplored difluorinated substructures. To this end, Chapter 2 describes the development of organocatalyzed addition of thiols to gem-difluoroalkenes to provide a,a-difluorophenethyl thioethers. Alcohol nucleophiles possess similar reactivity to thiols, including with gem-difluoroalkenes. Moreover, in medicinal chemistry ethers are a more common substructure than thioethers. Thus, Chapter 3 describes the addition of phenolic nucleophiles across gem-difluoroalkenes in a hydrophenolation reaction to provide a,a-difluorophenethyl arylethers. Gem-difluoroalkenes also possess unusual reactivity with transition metal catalysts. Typically, transition metals perform oxidative addition to C–halogen bonds to initiate cross coupling chemistry. However, the high C–F bond strength generally precludes oxidative addition, enabling alternate mechanistic pathways. Chapter 4 discusses the development of a Co-catalyzed deoxygenation reaction of gem-difluoroalkenes with phenol nucleophiles and O2 to provide b-phenoxy-b,b-difluorobenzyl alcohols. This reaction proceeds by an unusual radical reaction pathway in which superoxide oxidizes phenol to phenoxyl radical, which adds to gem-difluoroalkenes to provide a benzyl radical that quenches with peroxide anion. Finally, Chapter 5 discusses the ongoing work on metal-catalyzed dioxygenation reactions of gem-difluoroalkenes. This work covers the further development of b-phenoxy-b,b-difluorobenzyl alcohols under Pt catalysis, specifically to expand the reaction scope to heteroaryl alcohols, aliphatic alcohols, and aliphatic gem-difluoroalkenes. Further, Cu-catalysis enables the production of b-phenoxy-b,b-difluorobenzyl ketones.
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Organic chemistry
Chemistry
Cobalt Catalysis
Difluorination
Dioxidation
Fluorine
Organocatalysis
Strategies for the Fluorine-Retentive Functionalization of Gem-Difluoroalkenes
Dissertation
oai:kuscholarworks.ku.edu:1808/82032020-08-19T14:36:58Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Tong, Xiaoqin
2011-10-09T15:20:40Z
2011-10-09T15:20:40Z
2011-08-31
http://dissertations.umi.com/ku:11690
http://hdl.handle.net/1808/8203
In order to identify anti-proliferative compounds from selected Latin American plants, approximately 200 plant extracts were screened by the MTS assay (3-[4,5-dimethylthiazol-2yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H tetrazolium). Vassobia breviflora (Solanaceae), was identified as the most active of the species tested in this study. Following a bioassay-guided approach, withaferin A was isolated and characterized. This withanolide-type steroidal lactone showed anti-proliferative activities with IC50 values from 0.5 to 2.2 μM against head and neck squamous cell carcinoma (HNSCC) MDA1986, JMAR, UM-SCC-2 and JHU011. A mechanistic study showed that withaferin A induced apoptosis and cell death in HNSCC cells as well as a shift from G0/G1 to G2/M arrest in cell cycle studies. Western data demonstrated that the anti-proliferative action of withaferin A could be in part explained through degradation of total Akt levels as well as decrease in activation of Akt levels. In order to establish anticancer structure activity relationships, three analogues were semi-synthesized from withaferin A, and ten related withanolides, including a new chlorinated withanolide 6α-chloro-5β,17α-dihydroxywithaferin, were isolated from Withania somnifera (Solanaceae). All structures were elucidated on the basis of spectroscopic methods (IR, MS, and 1D/2D NMR). X-ray crystallography confirmed the absolute configuration of the new withanolide. A method for large-scale isolation of withaferin A from W. somnifera was also developed and presented.
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This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Chemistry
Pharmaceutical sciences
Anti-proliferative
Cancer
Vassobia breviflora
Withaferin a
Withania somnifera
Withanolide
ANTI-PROLIFERATIVE WITHANOLIDES FROM VASSOBIA BREVIFLORA AND WITHANIA SOMNIFERA
Thesis
oai:kuscholarworks.ku.edu:1808/43352020-07-22T12:52:01Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Kim, An Na
2009-02-02T05:51:14Z
2009-02-02T05:51:14Z
2008-09-09
http://dissertations2.umi.com/ku:2730
http://hdl.handle.net/1808/4335
2,3-Dihydropyridin-4(1H)-ones were utilized as scaffolds for the syntheses of libraries of 5-arylethynyl-2,3-dihydropyridin-4(1H)-ones and 2-(3-formyl-5-arylfuran-2-yl)ethylcarbamates. 2,3-Dihydropyridin-4(1H)-ones were prepared from piperidones, ynones, and pyridones and used for the synthesis of a library of 5-arylethynyl-2,3-dihydropyridin-4(1H)-ones employing a Sonogashira reaction. Further reaction of these compounds using an Au(III)-catalyzed cyclization method yielded formylfurans. N-Boc and N-benzyl protected 2,3-dihydropyridin-4(1H)-ones were prepared for the Sonogashira reaction. N-Boc-protected 5-iodo-2,3-dihydropyridin-4(1H)-ones provided tert-butyl 5-arylethynyl-4-oxo-3,4-dihydropyridine-1(2H)-carboxylates in moderate to excellent yields while the N-Bn-protected enaminones provided low yields of 5-arylethynyl-1-benzyl-2,3-dihydropyridin-4(1H)-ones. Furan formation was achieved by Au(III)-catalyzed and Cu-mediated cyclizations. (±)tert-Butyl 1-(3-formyl-5-phenylfuran-2-yl)propan-2-ylcarbamates were obtained during the Sonogashira reactions catalyzed by Cu(I), while (±)tert-Butyl 1-(3-formyl-5-phenylfuran-2-yl)-3-phenylpropan-2-ylcarbamates were formed by the Au(III)-catalyzed cyclization. A library of 16 compounds of highly substituted furans was synthesized in moderate to excellent yields.
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openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Pharmaceutical chemistry
Development of a Parallel Strategy for the Synthesis of a Library of 2-(3-Formyl-5-arylfuran-2-yl)ethylcarbamates from Dihydropyridinones
Thesis
oai:kuscholarworks.ku.edu:1808/19922020-07-15T12:47:55Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Shrestha, Anurupa
2008-03-01T17:26:23Z
2008-03-01T17:26:23Z
2007-12-19
http://dissertations.umi.com/ku:2349
http://hdl.handle.net/1808/1992
Endotoxins, or Lipopolysaccharides (LPS) present on the surface of Gram negative bacteria play a key role in the pathogenesis of septic shock, a common clinical problem and a leading cause of mortality in critically ill patients, for which no specific modalities are available at the present time. The toxic moiety of LPS is a glycolipid called Lipid A, which is composed of a bis-phosphorylated diglucosamine backbone bearing up to seven acyl chains in ester and amide linkages. Lipid A is structurally highly conserved in Gram negative bacteria, and is therefore an attractive target for developing anti-endotoxin molecules designed to sequester, and thereby neutralize, the deleterious effects of endotoxin. The anionic and amphipathic nature of Lipid A enables the interaction of a widevariety of cationic amphiphiles with the toxin. A systematic evaluation of several structural classes of cationic amiphiphiles both peptidic and non-peptidic small molecules, in the broader context of recent efforts aimed at developing novel antiendotoxin strategies. The derivation for the pharmacophore for LPS recognition has led to the identification of novel, nontoxic, structurally simple molecules, the lipopolyamines. The lipopolyamines bind and neutralize LPS in in vitro experiments as well as in animal models of endotoxicity, and thus present novel and exciting leads for rational, structure-based development of LPS sequestering agents of potential clinical value.
EN
openAccess
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Chemistry
Design, Syntheses, and Evaluation of Lipopolyamines as Anti-Endotoxin Agents
Thesis
oai:kuscholarworks.ku.edu:1808/69532018-01-31T20:08:15Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Urban, Michael Joseph
2010-12-31T03:51:11Z
2010-12-31T03:51:11Z
2010-07-30
http://dissertations.umi.com/ku:11101
http://hdl.handle.net/1808/6953
Background: Current Hsp90 inhibitors are therapeutically problematic. Although they induce a pro-survival heat shock response that promotes the refolding of damaged proteins, a confounding issue is that at these concentrations the inhibitors are cytotoxic, due to their ability to decrease the maturation of newly synthesized client proteins. KU-32 contains a novobiocin-based scaffold that binds to the C-terminal of Hsp90 and induces a pro-survival heat shock response at a concentration ~10,000 fold lower than that needed to induce neurotoxicity. This creates an optimal therapeutic window in which to operate, providing promise towards the development of novel neuroprotective agents. Objective: To evaluate whether the induction of the heat shock response through Hsp90 modulation could decrease or reverse the pathophysiological progression of diabetic peripheral neuropathy in Type-1 diabetic mice. Hypothesis: A small molecule modulator of Hsp90 will improve experimental diabetic neuropathy. Methods: After 8-12 weeks of diabetes induced by streptozotocin, the effects of weekly doses of KU-32 on several standard indices of diabetic neuropathy were measured. Results: Initial toxicity studies employing the weekly intraperitoneal administration of 2 or 20 mg/kg KU-32 to non-diabetic mice over 6 week duration did not alter motor or sensory nerve conduction velocity (MNCV/SNCV), mechanical or thermal sensitivity, or intra-epidermal nerve fiber density. Thus, the drug alone had no effect on altering common measures of neuropathy. In a 12-week intervention study, wild-type C57 Bl/6 animals receiving a weekly treatment regimen of 20 mg/kg KU-32 for 6 weeks exhibited a steady recovery to control levels in thermal and mechanical sensitivity, MNCV, and SNCV. KU-32 did not alter metabolic control. As Hsp70 is hypothesized to be a major target for KU-32, its necessity in neuroprotection was examined using Hsp70 double knockout mice (Hsp70.1/Hsp70.3). In a 12-week intervention study, Hsp70 knockout mice receiving a weekly treatment regimen of 20 mg/kg for 6 weeks displayed no improvements in thermal and mechanical sensitivity, MNCV, and SNCV. In 8-week intervention studies, animals demonstrated recoveries in sensory hypoalgesia and nerve conduction velocity deficits in a dose-dependent manner. KU-32 did not alter sensory nerve fiber innervation. Conclusions: These data suggest that hyperglycemia may adversely impact the ability of neurons to promote refolding or decrease unfolding of mildly damaged proteins. C-terminal Hsp90 modulators can improve several standard clinical indices of negative symptoms associated with small and large fiber dysfunction in the absence of improving overall metabolic control. The affects of KU-32 appear to be dose-dependent and require the presence of inducible Hsp70 for efficacy. Inducible Hsp70 is not required for the pathophysiological progression of diabetic neuropathy.
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Pharmaceutical chemistry
Biology
Neurosciences
Molecular biology
Diabetes
Heat shock
Hsp70
Hsp90
Neuropathy
Novobiocin
A Small Molecule Modulator of Hsp90 Improves Experimental Diabetic Neuropathy
Thesis
oai:kuscholarworks.ku.edu:1808/216492018-12-14T17:34:20Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Mishra, Sanket J.
2016-10-11T17:14:41Z
2016-10-11T17:14:41Z
2014-12-31
http://dissertations.umi.com/ku:13816
http://hdl.handle.net/1808/21649
Heat shock protein 90 KDa (Hsp90) belongs to family of proteins called molecular chaperone that are associated with protein folding and maturation. Hsp90 clients play a critical role in the pathogenesis of diseases such as cancer, neurodegeneration and infection. Currently, clinical trials are underway for various Hsp90 inhibitors, however, all of these inhibitors exhibit paninhibition of all four Hsp90 isoforms, which could be the cause of side effects observed with these inhibitors, including, hepatotoxicity, cardiotoxicity, and renal toxicity. Hence, the development of isoform selective Hsp90 inhibitor is needed to delineate the role each Hsp90 isoform plays towards the pathogenesis of these toxicities. One such isoform is the ER residing glucose regulated protein (Grp94), which is important for cellular communication and adhesion. Co-crystallization studies of radamide, an Hsp90 pan-inhibitor developed in our lab established that there exists a unique hydrophobic pocket found only in Grp94. To probe this pocket, two approaches have been investigated; 1) des-quinone analogs of radamide and 2) employing cis-amide isosteres. The co-crystal structure of cis-amide isostere compound BnIm bound to Gp94 and Hsp90 led to the discovery of a novel pocket in Grp94 due to ligand induced conformational change. This pocket has been probed by the modification of SNX 2112, a pan-inhibitor of Hsp90 that is currently undergoing clinical evaluation. These modifications have resulted in the identification of ACO1, which exhibits good potency and high selectivity towards Grp94. Rationale for the design of ACO analogs is discussed alongside their inhibition activities.
en
openAccess
Copyright held by the author.
Pharmaceutical sciences
Chemistry
Medicinal Chemistry
Structure-Based Design of Grp94-Selective Inhibitors
Thesis
oai:kuscholarworks.ku.edu:1808/216302020-10-16T14:28:24Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Yang, Ning
2016-10-11T15:41:47Z
2016-10-11T15:41:47Z
2013-12-31
http://dissertations.umi.com/ku:13060
http://hdl.handle.net/1808/21630
Tubulin-binding agents are an important class of therapeutics for cancer chemotherapy. However, their application can be limited by systemic toxicity, poor tissue specificity, and drug resistance. To address these issues, modification of tubulin-binding agents with drug delivery systems has shown significant promise. Our group has developed a novel technology for delivery of small molecules and macromolecules into mammalian cells. By mimicking small natural cell surface receptors, cargo linked to the membrane anchor N-alkyl-3beta-cholesterylamine can be internalized through a clathrin-mediated endocytic pathway and trapped in early/recycling endosomes. Co-administration with endosome-disruptive peptides enable 3beta-cholesterylamine-conjugated cargo to escape these early/recycling endosomes of living mammalian cells. This approach can be highly efficient as evidenced by fluorescence-based assays. To explore the potential of this system for delivery of tubulin-binding agents, we designed and synthesized a series of colchicine- and colchinol methyl ether-cholesterylamine conjugates. These disulfide-linked compounds were designed to deliver tubulin-binding agents into early/recycling endosomes to minimize their cytotoxic effect. Only upon the activation by an endosome disruptive peptide would these conjugates be released into the cytosol. Cleavage of a disulfide bond in a linker region by glutathione provides a mechanism of release. Cell viability assays demonstrated that carefully-designed conjugates to cholesterylamine can substantially reduce the toxicity of tubulin-binding agents by trapping the warhead in endosomes. After activation by endosome disruptive peptides, conjugates with a glutamic acid residue in the linker region proximal to colchinol methyl ether showed high potency against several cancer cell lines including Jurkat lymphocytes (T-cell leukemia) and PC3 prostate cancer cells.
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openAccess
Copyright held by the author.
Pharmaceutical sciences
cholesterylamine conjugates
delivery systems
endosome disruptive peptide
tubulin-binding agents
Synthetic delivery systems that control release of anticancer agents from early endosomes
Thesis
oai:kuscholarworks.ku.edu:1808/44382020-07-22T12:45:50Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Ranade, Adwait
2009-03-23T23:53:05Z
2009-03-23T23:53:05Z
2008-09-04
http://dissertations2.umi.com/ku:2728
http://hdl.handle.net/1808/4438
Novel Intramolecular Nucleophilic Addition Reactions: Formation of unusual N-O-Heterocyclic Enones Adwait R. Ranade, B. Tech. The University of Kansas, 2008 Cyclic enaminones, which possess vinylogous amide functionality in a closed ring structure, have been extensively studied and their versatility continues to spark interest in newer and more efficient methods for their enantiospecific synthesis and chemical modification. Due to their unique structural and chemical properties, enaminones are of interest in natural product and diversity-oriented synthesis (DOS). Many biologically active natural products and alkaloids belong to the indolizidine, pyrrolizidine, and quinolizidine classes of molecules. They can be synthesized via enaminones as intermediates. Therefore, the goal of this project was to develop a general methodology for the facile and enantiospecific synthesis of the enaminone scaffolds and then generate a library using this method. It was hypothesized that one-pot Boc deprotection/cyclization of β-hydroxylaminoynones would render N-oxy enaminones, which can be easily converted to enaminones by reductive cleavage of the N-O bond. Due to complications observed with such type of chemistry, the route was modified and a one-pot TBS deprotection/cyclization of β-hydroxylaminoynones was carried out. This approach yielded novel seven-membered N-O heterocyclic compounds. The mechanism is thought to be a 7-endo-dig cyclization to yield seven-membered 3,4-dihydro-1,2-oxazepin-5(2H)-ones. Reductive cleavage of the N-O bond in 3,4-dihydro-1,2-oxazepin-5(2H)-ones rendered 2,3-dihydropyridin-4-(1H)-ones (enaminones). Thus, the 7-membered novel oxazepinone scaffold and the known enaminone scaffold can be used for constructing diverse compound libraries. Additional chemical transformations can also be carried out on these scaffolds to obtain additional diverse chemotypes.
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openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Organic chemistry
Pharmaceutical chemistry
Novel Intramolecular Nucleophilic Addition Reactions: Formation of Unusual N-O-Heterocyclic Enones
Thesis
oai:kuscholarworks.ku.edu:1808/61832020-07-27T16:12:04Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Szostak, Michal
2010-05-03T01:03:07Z
2010-05-03T01:03:07Z
2009-10-08
http://dissertations.umi.com/ku:10586
http://hdl.handle.net/1808/6183
The research presented herein describes the development of synthetic methods to one-carbon bridged twisted amides and the study of properties of one-carbon bridged lactams. Initial investigations focused on electrostatic cation-pi and cation-n interactions as regiochemistry controlling feature of the intramolecular Schmidt reaction to provide access to one-carbon bridged amides. In cases where the reactive conformation of the azidohydrin intermediate is locked, the selectivity of the reaction depends on the electron density of an aromatic ring oriented in 1,3-diaxial relationship with regard to the diazonium cation. However, a placement of a heteroatom in the α-position to the ketone permits the synthesis of otherwise unsubstituted bridged amides from conformationally flexible ring systems. Also, described is the development of a general method of synthesis of one-carbon bridged amides relying on a transannular cyclization strategy. Next, experiments directed towards investigation of unusual properties of distorted amides are presented. One-carbon bridged lactams display superior to other bridged amides levels of hydrolytic stability. These lactams participate in a number of interesting and potentially useful reactions unknown to traditional amide bonds, including synthesis of remarkably stable tetrahedral intermediates and a direct conversion into bridged spiro-epoxyamines. The influence of the amide bond geometry on reactivity of distorted lactams is also discussed.
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openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Organic chemistry
Amide bonds
Cation-pi/cation-n interactions
Schmidt reaction
Tetrahedral intermediates
Transannular amidation
Twisted amides
Synthesis and Reactivity of Medium-Bridged Twisted Lactams
Dissertation
oai:kuscholarworks.ku.edu:1808/76422020-08-10T13:16:50Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Donnelly, Alison C.
2011-06-21T16:31:41Z
2011-06-21T16:31:41Z
2011-03-18
http://dissertations.umi.com/ku:11372
http://hdl.handle.net/1808/7642
Novobiocin, a known DNA gyrase inhibitor, binds to a nucleotide-binding site located on the Hsp90 C-terminus and induces degradation of Hsp90-dependent client proteins at ~700 &muM in breast cancer cells (SKBr3). Although many analogues of novobiocin have been synthesized in an attempt to improve upon this activity, it was only recently demonstrated that monomeric species can exhibit antiproliferative activity against various cancer cell lines. To further refine the essential elements of the coumarin core, a series of modified coumarin derivatives was synthesized and evaluated. Structure&ndashactivity relationships for novobiocin as an anti-cancer agent were elucidated through analogues that manifest low micromolar to nanomolar activity against several cancer cell lines. The compound that exhibited the best and most consistent activity has been further evaluated against a broader panel of cancers as well as taken into an in vivo model. Studies are ongoing to further refine the coumarin core, with the potential to replace it with a more suitable heterocyclic ring system. In addition to the coumarin portion, a noviose sugar and benzamide side chain are appended to the natural product. Because limited information exists regarding the role of the sugar appendage, a series of non-sugar derivatives was synthesized and evaluated to establish structure&ndashactivity relationships for the noviose region of novobiocin. These studies have produced simplified novobiocin analogues that manifest low micromolar activity against a panel of cancer cell lines. Likewise, studies have been executed to elucidate details concerning the benzamide side chain and its potential to make hydrophobic interactions with the binding pocket. The most promising compound from each of these series has demonstrated impressive activity against several cancer cell lines and have been evaluated in vivo. Efforts to understand the mechanism of action manifested by these diverse Hsp90 modulators are ongoing and have resulted in the existence of at least three distinct classes of Hsp90 C-terminal modulators. Moreover, collaborative studies with the NCI have revealed promising results with a compound that modulates Hsp90 through yet another disparate, but synergistic, mechanism. Through current studies, we hope to better solubilize the most potent compounds and advance novel Hsp90 modulators into clinical development.
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openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Pharmaceutical sciences
Organic chemistry
Oncology
Benzamide
Coumarin
C-terminus
Hsp90
Noviose
Novobiocin
Design, Synthesis and Evaluation of Non-Canonical Hsp90 Modulators
Dissertation
oai:kuscholarworks.ku.edu:1808/81482020-08-17T14:01:06Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Lovell, Kimberly M.
2011-10-09T01:41:08Z
2011-10-09T01:41:08Z
2011-08-31
http://dissertations.umi.com/ku:11672
http://hdl.handle.net/1808/8148
Kappa opioid (KOP) receptors have been shown to be involved in the control of several abuse related effects of central nervous system stimulants. KOP receptor agonists have been shown to modulate the activity of dopamine neurons and decrease self-administration of cocaine in a variety of species, while KOP receptor antagonists have the potential to be utilized as opioid abuse therapies and in the treatment of relapse. With this in mind, investigations were performed on the novel KOP receptor agonist neoclerodane diterpene salvinorin A. This natural product is the active component of the hallucinogenic mint plant Salvia divinorum and the first non-nitrogenous natural product having high affinity and efficacy at KOP receptors. Salvinorin A contains a furan ring, which in other furan containing natural products such as teucrin A and aflatoxin B1 has been identified to cause hepatotoxicity. In efforts to develop a more desirable pharmacological tool, structural modifications were made to salvinorin A in efforts explore the role of the furan ring in affinity and activity at KOP receptors and to reduce its potential for hepatotoxicity. Several ketone analogues were found to retain affinity at KOP receptors relative to salvinorin A and were versatile intermediates for the synthesis of other analogues. Surprisingly, benzisoxazole 187 was found to have increased affinity for MOP receptors. 2-Furanyl salvinorin A (185) was found to have similar efficacy and activity compared to salvinorin A. In addition, Captisol® was identified as a new vehicle for salvinorin A administration in pharmacological assays to eliminate some of the drawbacks of currently used vehicles. These studies assist with the identification of the pharmacophore of salvinorin A as well as the determination of structure-activity relationships, all of which will increase the potential for identification of novel opioid therapeutics.
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openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Pharmaceutical sciences
Organic chemistry
Furan
Kop receptors
Salvia divinorum
Salvinorin a
The Synthesis and Pharmacological Evaluation of Salvinorin A Analogues as Opioid Receptor Probes
Dissertation
oai:kuscholarworks.ku.edu:1808/43382018-01-31T20:08:15Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Lubbers, Donna Jeanne
2009-02-02T05:56:34Z
2009-02-02T05:56:34Z
2008-01-01
http://dissertations.umi.com/ku:10012
http://hdl.handle.net/1808/4338
As C−terminal inhibitors of a 90−kDa heat shock protein (Hsp90), novobiocin and its derivatives are a significant part of an emerging class of cancer chemotherapeutic agents. Previous studies have shown that analogues of the coumarin and benzamide moieties of novobiocin exhibit more than a 1000−fold improvement in activity over the parent compound. This thesis describes synthetic efforts toward the completion of noviose mimics to determine moieties that are critical for binding or can be altered for improved activity. Additionally, in the absence of a co−crystal structure for the Hsp90 C−terminus, there is a need to develop an accurate model to assist in efficient drug design. This work describes the use of molecular modeling and docking software to design new, potentially useful models of C−terminal interactions.
EN
openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Organic chemistry
Pharmaceutical chemistry
Chemistry
C-terminus
Computational
Hsp90
Novobiocin
Synthesis
Synthetic and computational efforts toward the understanding and development of novobiocin-derived inhibitors of Hsp90
Thesis
oai:kuscholarworks.ku.edu:1808/216362020-10-22T14:11:27Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Motiwala, Hashim F.
2016-10-11T16:09:29Z
2016-10-11T16:09:29Z
2014-05-31
http://dissertations.umi.com/ku:13294
http://hdl.handle.net/1808/21636
The research presented herein describes four separate projects, focusing on synthetic methodology development, discovery of novel cytotoxic agents, and natural product isolation. Catalysis of Intra- and Intermolecular Schmidt Reactions A method for carrying out the intramolecular Schmidt reaction of alkyl azides and ketones using a substoichiometric amount of catalyst is described. Following extensive screening, the use of the strong hydrogen-bond-donating solvent hexafluoro-2-propanol (HFIP) was found to be consistent with low catalyst loadings, which ranged from 2.5 mol % for favorable substrates to 25 mol % for more difficult cases. Reaction optimization, broad substrate scope, and preliminary mechanistic studies of this improved version of the reaction are discussed. The use of HFIP as the solvent also allowed for the extension of this methodology to intermolecular variants of Schmidt reaction favoring the development of mild, operationally simple, and more efficient protocols, requiring considerably less amounts of acid catalysts for these variants. Copper-Catalyzed Oxaziridine-Mediated C-(&ndash)H Bond Oxidation. The highly regioand chemoselective oxidation of an activated C−(&ndash)H bond via a copper-catalyzed reaction of oxaziridine is described. The oxidation proceeded with a variety of substrates, primarily comprising of allylic and benzylic examples, as well as one example of an otherwise unactivated tertiary C−(&ndash)H bond. The mechanism of the reaction is proposed to involve single-electron transfer (SET) to the oxaziridines to generate a copper-bound radical anion, followed by hydrogen atom abstraction and collapse to products, with regeneration of the catalyst by a final SET event. The involvement of allylic radical intermediates en route to the product was supported by a radical-trapping experiment with TEMPO. Synthesis and Cytotoxic Evaluation of Withalongolide A Analogues. The natural product withaferin A exhibits potent antitumor activity and other diverse pharmacological activities. The recently discovered withalongolide A, a C-19 hydroxylated congener of withaferin A, was reported to possess cytotoxic activity against head and neck squamous cell carcinoma (HNSCC). Interestingly, semisynthetic acetylated analogues of withalongolide A were shown to be considerably more cytotoxic than the parent compound. To further explore the structure-(&ndash)activity relationship (SAR), 20 new semisynthetic analogues of this highly oxygenated withalongolide A were designed, synthesized, and evaluated for cytotoxic activity against four different cancer cell lines. A number of derivatives were found to be more potent than the parent compound and withaferin A. Isolation of Withalongolide O from Physalis longifolia. The SAR analysis of reported bioactive withanolides revealed certain crucial structural requisites for possessing a potent cytotoxic activity. The semisynthesis of a putative unnatural withanolide incorporating all the basic and essential structural features to boost the antiproliferative activity was contemplated. Withaferin A was considered as an appropriate starting material for this purpose. Although the semisynthetic efforts met with failure, it was during the isolation of withaferin A from the crude plant extract that we discovered a novel withanolide, withalongolide O. The structure of withalongolide O was determined using various spectroscopic techniques and subsequently confirmed by X-ray crystallographic analysis. Both withalongolide O and its diacetate exhibited potent cytotoxicity against four different cancer cell lines.
en
openAccess
Copyright held by the author.
Pharmaceutical sciences
Organic chemistry
HFIP
oxaziridine
Schmidt reaction
withalongolide
I. Catalysis of Intra- and Intermolecular Schmidt Reactions. II. Copper-Catalyzed Oxaziridine-Mediated C-H Bond Oxidation. III. Synthesis and Cytotoxic Evaluation of Withalongolide A Analogues.
Dissertation
oai:kuscholarworks.ku.edu:1808/61782020-07-28T14:52:57Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Khownium, Kriangsak
2010-05-03T00:45:44Z
2010-05-03T00:45:44Z
2009-12-15
http://dissertations.umi.com/ku:10667
http://hdl.handle.net/1808/6178
Part I. The natural products oximidine I and II are potent and selective anticancer agents at nanomolar concentrations. Their structures and potent cytotoxicity are similar to the recently discovered natural products salicylihalamides, apicularens, and lobatamides, which are members of the benzolactone enamide family. However, it is believed that the enamide, a crucial moiety for anticancer activity, will be labile under physiological conditions. This has led to our hypothesis that the replacement of the enamide moiety with a stable warhead will allow the discovery of novel anticancer agents with improved pharmacokinetics. We selected three different warheads that are likely to be stable under physiological conditions and that could undergo nucleophilic addition like the enamide. We prepared by total synthesis oximidine analogues that carry a vinyl sulfone, a boronic acid and an α -keto oxadiazole instead of the enamide side chain. Based on CoMSIA/QSAR analysis, we have designed, prepared and evaluated new analogs of oximidine II. Part II. Lipopolyamines bind to the lipid A moiety of lipopolysaccharide, a constituent of Gram-negative bacterial membranes, and neutralize its toxicity in animal models of endotoxic shock. In an effort to identify non-polyamine scaffolds with similar endotoxin-recognizing features, we had observed an unusually high frequency of hits containing guanylhydrazone scaffolds in high-throughput screens. We now describe the syntheses and preliminary structure-activity relationships in a homologous series of bis-guanylhydrazone compounds decorated with hydrophobic functionalities. These first-generation compounds bind and neutralize lipopolysaccharide with a potency comparable to that of polymyxin B, a peptide antibiotic known to sequester LPS.
EN
openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Organic chemistry
Anti-cancer
Comsia
Novel
Oximidine
Sepsis
V-atpase
Synthesis and evaluation of modified oximidine analogues as anticancer agents and of terephthalaldehyde-bis-guanylhydrazones as endotoxin sequestering agents
Dissertation
oai:kuscholarworks.ku.edu:1808/216332020-10-22T14:22:52Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Garg, Gaurav
2016-10-11T16:00:32Z
2016-10-11T16:00:32Z
2014-05-31
http://dissertations.umi.com/ku:13323
http://hdl.handle.net/1808/21633
Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that plays a pivotal role in protein homeostasis in responses to cellular stress. Hsp90 regulates the conformational maturation, activation, and integrity of a wide array of client proteins, including oncogenic proteins (Her2, Raf1, Akt, CDK4 etc.) associated with all six hallmarks of cancer. Consequently, Hsp90 inhibition offers a unique opportunity for the simultaneous degradation of multiple anti-cancer targets and hence, for the development of cancer chemotherapeutics. Hsp90 exists as a homodimer with each monomer consisting of a druggable domain; the N-terminal domain, the middle domain, and the C-terminus. The majority of research has focused on development of Hsp90 N-terminal inhibitors. In fact, all Hsp90 inhibitors in clinical trials belong to this class. One of the major drawbacks associated with N-terminal inhibitors is the concomitant induction of the pro-survival response, which results in an upregulation of Hsp's and affects the dosing schedule. As a result, alternative strategies are sought for the development of future Hsp90 inhibitors. Over the last decade, Hsp90 C-terminal inhibitors have emerged an attractive alternative for Hsp90 modulation. These inhibitors exhibit similar inhibitory activity to N-terminal inhibitors, but do not induce the pro-survival response and could potentially circumvent the clinical limitations imposed on N-terminal Inhibitors. Presented herein are the design, synthesis and biological evaluation of ring-constrained novobiocin analogues that provide new insights into the Hsp90 C-terminal binding pocket and SAR's that can be used for future analog development. In addition, identification of a novel class of Hsp90 inhibitors is discussed. These new agents provide a platform upon which future Hsp90 inhibitors can be built upon.
en
openAccess
Copyright held by the author.
Pharmaceutical sciences
Organic chemistry
Design, Synthesis and Biological Evaluation of Ring-constrained and Biphenyl Derivatives as Hsp90 C-terminal Inhibitors
Thesis
oai:kuscholarworks.ku.edu:1808/43372020-07-22T13:02:46Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Han, Huijong
2009-02-02T05:54:53Z
2009-02-02T05:54:53Z
2008-10-23
http://dissertations2.umi.com/ku:2753
http://hdl.handle.net/1808/4337
Infectious diseases and cancers are the second and third largest causes of death worldwide. UDP-N-acetyl-glucosamine (UNAG) enolpyruvyl transferase (MurA)and Cyclin-dependent kinases (CDKs) are proven as antibiotic and anticancer targets, respectively. MurA belongs to the enolpyruvyl transferase family of enzymes. MurA catalyzes the first committed step in the biosynthesis of cell wall peptidoglycan, and is the target of fosfomycin, a naturally occurring broad-spectrum antibiotic. Ever increasing resistance of bacteria to fosfomycin has placed an emphasis on the identification and characterization of novel MurA inhibitors. Knowledge of the detailed enzymatic mechanism is essential for the discovery of potent MurA inhibitors. The studies on the mutant MurA enzymes Arg91Lys, Asp123Ala, Arg120Ala, and Cys115Asp, revealed key catalytic residues and residues important for the conformational changes in the enzymatic reaction. Several new inhibitors of MurA were identified by High-Throughput Screening (HTS), and kinetically characterized. It appears that most of these compounds bind to the open conformation of MurA, and thus the inhibitor binding site is largely solvent exposed. These results suggest that MurA inhibitors need to be designed to induce the open-closed transformation of the enzyme, like the natural substrate UNAG. Such inhibitors should be much more potent than the inhibitors discovered in this work. CDK2 plays a critical role in the G1- to S-phase checkpoint of the cell cycle. Only a few drugs targeting CDK2 are in clinical trials, thus there is a need for the discovery of novel CDK2 inhibitors. Six CDK2 inhibitor scaffolds were identified by HTS, and the molecular modes of action of four of them were thoroughly characterized by steady-state kinetics and crystallography. Structure-Activity Relationship (SAR) analysis of the four scaffolds gave rise to the design of analogs with excellent potency. In addition, computational studies were performed, and novel CDK2 inhibitor scaffolds were designed. The selectivities and cytotoxic properties of these inhibitors are not known yet.
EN
openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Chemistry
Biochemistry
Structure-based drug design
Mura
Cdk2
X-ray crystallography
Structure-activity relationships
The Application of X-ray Crystallography towards the Design of Novel Inhibitors of MurA and CDK2
Dissertation
oai:kuscholarworks.ku.edu:1808/261542018-04-19T20:13:59Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Johnson, Stephanie
2018-03-09T22:34:13Z
2018-03-09T22:34:13Z
2017-05-31
http://dissertations.umi.com/ku:15233
http://hdl.handle.net/1808/26154
The ability of ligands to differentially regulate the activity of signaling pathways coupled to a receptor potentially enables researchers to optimize therapeutically relevant efficacies, while minimizing activity at pathways that lead to adverse effects. Recent studies have demonstrated the functional selectivity of kappa opioid receptor (KOR) ligands acting at KOR expressed by rat peripheral pain sensing neurons. In addition, KOR signaling leading to antinociception and dysphoria occur via different pathways. Based on this information, it can be hypothesized that a functionally selective KOR agonist would allow researchers to optimize signaling pathways leading to antinociception while simultaneously minimizing activity towards pathways that result in dysphoria. In this study, our goal was to alter the structure of U50,488 such that efficacy was maintained for signaling pathways important for antinociception (inhibition of cAMP accumulation) and minimized for signaling pathways that reduce antinociception. Thus, several compounds based on the U50,488 scaffold were designed, synthesized, and evaluated at KORs. Selected analogues were further evaluated for inhibition of cAMP accumulation, activation of extracellular signal-regulated kinase (ERK), and inhibition of calcitonin gene-related peptide release (CGRP). The data obtained demonstrates that modification of the structure of U50,488 changed the signaling pathway regulation. Specifically, we identified three functionally selective KOR ligands (4b, 9u, and 9ac) that inhibit cAMP accumulation, similar to U50,488, but, unlike U50,488, do not activate ERK. In addition, the ability to inhibit CGRP release showed monotonic concentration-response curves, indicating that a pathway leading to nociception is not activated. These data suggest that the efficacy for specific signaling pathways can be finely tuned by structural modifications to a given ligand.
en
openAccess
Copyright held by the author.
Pharmaceutical sciences
Organic chemistry
Chemistry
functional
kappa
opioid
selectivity
U50
488
Design and Synthesis of Functionally Selective Kappa Opioid Receptor Ligands
Thesis
oai:kuscholarworks.ku.edu:1808/52352020-07-22T12:47:27Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Fang, Weijie
2009-05-31T21:57:18Z
2009-05-31T21:57:18Z
2008-09-04
http://dissertations2.umi.com/ku:2725
http://hdl.handle.net/1808/5235
Our research focuses on the development of potent and highly selective peptide ligands for kappa (κ) opioid receptors and examination of the structure-activity relationships (SAR) for activity at these receptors. Both κ opioid receptor agonists and antagonists have potential therapeutic applications for a variety of diseases. Dynorphin (Dyn) A is an endogenous heptadecapeptide agonist at κ opioid receptors. Dyn A-based peptide antagonists could be useful pharmacological tools for studying κ opioid receptors. Arodyn is an acetylated Dyn A analog identified in our laboratory that is a potent and highly selective κ opioid receptor antagonist. One main purpose of this dissertation research was to evaluate the role of the N-terminal 'message' sequence (especially Phe1 and Phe3) of arodyn in κ opioid receptor affinity, selectivity and efficacy. These two positions were substituted with other aromatic or nonaromatic residues. The other main purpose of this research was to develop conformationally restricted analogs of arodyn and Dyn A with high affinity and/or selectivity that can be used to explore the structural and conformational requirements for interaction of these ligands with κ opioid receptors, and to explore the SAR for agonist vs. antagonist activity at κ receptors. Different cyclization strategies were explored for the synthesis of cyclic arodyn and Dyn A analogs, including cyclization through lactam bond formation or ring-closing metathesis (RCM).
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Pharmaceutical chemistry
Design and Synthesis of Novel Linear and Cyclic Peptide Ligands for Kappa Opioid Receptors
Dissertation
oai:kuscholarworks.ku.edu:1808/261582018-04-19T20:19:05Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Yang, Ming-Hsiu
2018-03-09T22:41:58Z
2018-03-09T22:41:58Z
2017-05-31
http://dissertations.umi.com/ku:15287
http://hdl.handle.net/1808/26158
Fluorine plays an important role in drug design, because of some unique features imparted by fluorine. The incorporation of fluorine into small molecules can modulate molecular physicochemical properties, metabolic stability, lipophilicity, and binding affinity to the target proteins. However, few fluorinated molecules are biosynthesized by enzymes. This means incorporating fluorine into the molecules relies on synthetic methods. Thus, efficient synthetic strategies to access the molecules bearing a variety of privileged fluorinated moieties are important for drug discovery. Fluoroalkenes are an isopolar and isosteric mimic of an amide bond with distinct biophysical properties, including decreased H-bond donating and accepting abilities, increased lipophilicity, and metabolic stability. Moreover, fluoroalkenes can also serve as probes for conducting conformational analyses of amides. These potential applications require the development of efficient methods to access fluoroalkenes. In chapter 2, a Shapiro fluorination strategy to access peptidomimetic fluoroalkenes is demonstrated. The Shapiro fluorination reactions convert a ketone into a fluoroalkene in one or two steps. Moreover, this method uses inexpensive and readily available reagents, and no transition metals are involved in the reactions. Thus, it provides an operation-simple alternative to access fluoroalkenes in medicinal chemistry. a,a-difluoroketones represent a privileged substructure in medicinal chemistry, and serves as inhibitors to many hydrolytic enzymes, such as serine and aspartyl proteases. From chapters 3 to 5, palladium-catalyzed decarboxylative methods are developed for accessing a-alkyl- and a-aryl-a,a-difluoroketones. This decarboxylative strategy overcomes two major challenges associated with alkylation reactions of a,a-difluoroketone enolates. Chapter 3 demonstrates that decarboxylation regioselectively generates a,a-difluoroketone enolates, which are difficult to access by base deprotonation. Moreover, palladium catalysis enables the coupling of the a,a-difluoroketone enolate with benzylic electrophiles to form a key C(a)–C(sp3) bond. In chapter 4, an orthogonal catalytic system is developed for accessing linear and branched a-allyl-a,a-difluoroketones. Two distinct mechanisms are involved in the formation of the regioisomers. Chapter 5 describes a base-mediated selective para-C–H difluoroalkylation of arenes, which represents a different strategy for para-C–H functionalization of arenes compared to the known methods. These decarboxylative coupling reactions provide structurally diverse a,a-difluoroketone derivatives, and should be useful for accessing potential biological probes and therapeutics.
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Organic chemistry
Chemistry
Synthetic Strategies to Access Biologically Important Fluorinated Motifs: Fluoroalkenes and Difluoroketones
Dissertation
oai:kuscholarworks.ku.edu:1808/254342018-01-31T20:07:48Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Meinig, James Matthew
2017-11-17T22:37:18Z
2017-11-17T22:37:18Z
2015-12-31
http://dissertations.umi.com/ku:14290
http://hdl.handle.net/1808/25434
The Peterson laboratory has had a long-standing interest in fluorescent probes of biological systems. My research in the Peterson group has focused on the design, synthesis, and biological evaluation of fluorescent small molecules that exhibit specific patterns of subcellular localization and studies of their downstream biological effects. The relationship between this approach and the strategy of phenotypic drug discovery is described in Chapter 1. Chapter 2 describes the discovery of the intrinsic blue fluorescence of the potent anti- cancer/anti-viral compound AKT inhibitor-IV (AKTIV), and how we used this property to discover that its mechanism of biological action involves accumulation in mitochondria and associated effects on mitochondrial morphology and cellular bioenergetics. Chapter 3 describes the synthesis of a novel class of hydrophobic fluorinated rhodol fluorophores that selectively accumulate in the endoplasmic reticulum. These fluorophores were shown to enable delivery of linked small- molecules to control a specific biological pathway in this organelle. Building on these studies, Chapter 4 describes screening of a variety of fluorescent probes against the vertebrate model organism zebrafish (Danio rerio). These studies led to the discoveries that hydrophobic rhodamines can be used to target zebrafish mitochondria, and acid-activated fluorophores can accumulate in acidic tissues of the embryonic yolk. Chapter 5 describes another project involving the synthesis of novel cholesteryl dimers and analysis of the in vitro stability of liposomes that incorporate these compounds.
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Copyright held by the author.
Biology
Organic chemistry
Phenotypic Discovery
Subcellular Targeting
SYNTHESIS AND PHENOTYPIC DISCOVERY OF MOLECULAR PROBES OF BIOLOGICAL SYSTEMS
Dissertation
oai:kuscholarworks.ku.edu:1808/302242022-02-24T15:51:12Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Phaniraj, Sahishna
2020-03-29T17:30:58Z
2020-03-29T17:30:58Z
2019-12-31
http://dissertations.umi.com/ku:16904
http://hdl.handle.net/1808/30224
The modern molecular understanding of biological systems relies on integration of principles, practices, and techniques from diverse fields such as molecular biology, biochemistry, and biophysics. This approach has provided great insights into the molecular complexity of living cells. However, studies of some biological assemblies, such as the dynamic collection of lipids, proteins, and other biomolecules that make up cellular membranes, remains a challenge. To simplify these systems, and enable investigations of their structure and function, a wide range of membrane-mimetic models have been developed. Chapter 1 of this dissertation reviews some of the most important methods for studies of these types of biological systems, including recently developed lipidic nanodiscs. In Chapter 2 of this dissertation, I describe the design and construction of a new class of nanodiscs that are stabilized by covalent crosslinking of a membrane scaffold protein. These nanodiscs, termed SpyDiscs, uniquely enable imaging of pore formation by membrane-disruptive peptides. Another class of molecules that interacts with biological membranes is described in Chapter 3 of this dissertation. In this chapter, we report the synthesis of a new class of hydrophobic fluorescent probes that can be used to visualize the endoplasmic reticulum of living cells. The final chapter of this dissertation describes a fluorescence polarization assay developed to study proteinprotein interactions involved in iron homeostasis in the pathogenic bacterium Pseudomonas aeruginosa. These binding studies along with other observations offer a promising target for inhibition of this pathogen as a strategy to overcome multidrug resistance observed with this superbug.
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Copyright held by the author.
Pharmaceutical sciences
Endoplasmic reticulum probes
Membrane-active peptides
Nanodiscs
Pseudomonas aeruginosa
SpyTag/SpyCatcher system
STUDIES OF INTERACTIONS OF SMALL MOLECULES WITH MEMBRANES AND PROTEINS
Dissertation
oai:kuscholarworks.ku.edu:1808/295472021-10-28T19:35:58Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Knewtson, Kelsey Erin
2019-09-06T19:30:25Z
2019-09-06T19:30:25Z
2019-05-31
http://dissertations.umi.com/ku:16576
http://hdl.handle.net/1808/29547
https://orcid.org/0000-0002-3346-304X
Cancer is a complex family of diseases. As our understanding of cancer biology has improved, so has our ability to treat the pathology associated with this condition. Traditional anticancer therapeutics lack selectivity and cause many side effects. These side effects can reduce the quality of life of the patient and limit the doses of drugs that need to be used to fully eradicate cancer cells. In recent years, a deeper understanding of cancer has led to the development of targeted therapies that exploit molecular differences between cancer cells and healthy cells. These newer targeted therapies often have reduced side-effects compared with traditional drugs, to the great benefit of patients. Sections of this dissertation focus on a specific class of targeted anticancer therapeutics called antibody drug conjugates (ADCs). ADCs combine the targeting power of antibodies with the cell killing mechanisms of potent toxins. These therapies can overcome some of the resistance that can emerge against therapeutic antibodies, and the low therapeutic index of associated toxins, but there is still room for improvement. Many patients treated with ADCs experience severe side effects. Additionally, most ADCs are generated by attaching the toxins to the antibody in a random manner, generating very heterogenous mixtures of therapeutics. In this dissertation, a system that combines ADCs with endosome disruptive peptides is explored as a possible method to improve the therapeutic index of ADCs. This system employs ADCs composed of poorly membrane permeable toxins whose toxicity is triggered by endosome disruptive peptides that allow these toxins to reach the cytoplasm. A novel approach to generate more homogenous ADCs is also described. This method takes advantage of the binding of Protein A from Staphylococcus aureus to human antibodies to direct the labeling of the antibody with small molecules. This dissertation also describes basic cancer biology research related to the cellular production of reactive oxygen/nitrogen species. Control of the levels of oxidants and reductants is very important for the normal function of cells and imbalances are linked to many disease states including cancer. We describe the development and use of novel fluorescent sensors of the important biological oxidant peroxynitrite. These sensors are able to detect endogenous production of peroxynitrite by macrophages upon phagocytosis of opsonized beads, a process that previously reported sensors are unable to detect. Better sensors of peroxynitrite such as those discussed here could aid the study of this oxidant and its role in cancer and other diseases.
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Copyright held by the author.
Chemistry
Biology
antibody drug conjugate
endosome disruption
fluorescence
peroxynitrite
Studies of novel targeted drug delivery systems and molecular probes of cancer biology
Dissertation
oai:kuscholarworks.ku.edu:1808/61872020-07-23T13:58:52Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Schroeder, Chad E.
2010-05-03T01:21:49Z
2010-05-03T01:21:49Z
2009-04-29
http://dissertations.umi.com/ku:10282
http://hdl.handle.net/1808/6187
Sakurai and Mukaiyama aldol additions were carried out with 3-Azido-hexanal under chelation and non-chelation conditions. The reactions were generally found to be diastereofacially selective in favor of the anti stereoisomer and showed simple diastereoselectivity in favor syn substitution. The relative stereochemistry of the addition products were deduced from NOE experiments on cyclic amines that were produced from intramolecular Schmidt, Staudinger/aza-Wittig, and catalytic hydrogenation reactions. The findings indicate that substituted N-heterocycles can be made diastereoselectively in a couple of steps from simple azido aldehydes by carefully selecting the reaction conditions.
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Organic chemistry
Nucleophilic Additions to 3-Azido-hexanal
Thesis
oai:kuscholarworks.ku.edu:1808/80382020-08-13T13:10:36Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Axtman, Matthew
2011-09-22T00:29:13Z
2011-09-22T00:29:13Z
2011-06-13
http://dissertations.umi.com/ku:11614
http://hdl.handle.net/1808/8038
The heat shock protein 90 (Hsp90) family of molecular chaperones is responsible for the conformational maturation of nascent polypeptides and refolding of denatured proteins. Hsp90 is known to play an important role in the regulation of cell signaling, survival and proliferation, transforming it into a promising target for the treatment of several diseases, including cancer. Proteins associated with all six hallmarks of cancer, as described by Weinberg, are Hsp90 clients. Since these proteins depend upon Hsp90, its inhibition has the potential to simultaneously disrupt all six and halt the malignant progression using a single small molecule. Hsp90 is abundantly expressed in the cell and accounts for 1-2% of the total cellular protein, making itone of the most prevalent proteins in eukaryotic cells. The Hsp90 isoforms are responsible for the conformational maintenance of greater than 150 proteins. There are four distinct isoforms, including Hsp90á, Hsp90â, GRP94 and TRAP1. Hsp90á and â are located in the cytoplasm; Hsp90á is the major inducible form, while Hsp90â is the constitutively active form. In contrast, GRP94 is found in the endoplasmic reticulum and TRAP1 is located in the mitochondrial matrix. Hsp90 exists as a homodimer consisting of an N- and C-terminal domain, connected by a middle domain. The Hsp90 N-terminus contains an ATP binding site, responsible for the ATPase activity of the chaperone associated with folding of clients. Small molecules that preferentially bind to this nucleotide binding site inhibit the ability of Hsp90 to properly fold polypeptides, ultimately tagging them for degradation through the ubiquitin-proteasome pathway. Natural product inhibitors of the N-terminal ATP binding site include the ansamycin antibiotic geldanamycin (GDA) and the macrocyclic lactone radicicol (RDC). Blagg and co-workers have previously reported the chimeric N-terminal inhibitor radamide, which combines the resorcinol portion of RDC and the quinone portion of GDA through a flexible linker. Radamide manifests a slightly greater binding affinity for GRP94 (Kd = 0.52 ìM) versus cytosolic Hsp90 (Kd = 0.87 ìM). This phenomenon is elucidated by examining the unique binding conformations adopted by radamide when crystal structures of were obtained using each isoform. When bound to Hsp90, radamide exhibits a linear conformation, while when bound to GRP94 the quinone portion is bent towards a pocket that is not accessible in Hsp90á or â. By employing conformational constraint, it was proposed that the quinone portion of radamide could exhibit the bent conformation seen in the natural products and result in increased affinity for Hsp90 as well as selective binding to the cytosolic isoforms. In contrast to radamide, a recent paper by Gasiewicz and co-workers examined the effects of flavones and (-)-Epigallocatechin-3-Gallate (EGCG), the major polyphenolic catechin found in green tea, on the aryl hydrocarbon receptor (AHR). Due to its diverse medicinal properties, EGCG has been proposed as a potential treatment for several diseases. The study by Gasiewicz revealed that EGCG functions through a different mechanism of action than the known flavone antagonists, interacting with the Hsp90 C-terminus rather than the AHR. It was confirmed that EGCG binds specifically to the C-terminal nucleotide binding site, either within or proximal to the site where the natural product novobiocin is thought to bind. Previous studies have determined that EGCG manifests an IC50 of ~150 ìM against MCF-7 cells. Upon examination of an overlay of EGCG and novobiocin, the structural similarities of the coumarin and the catechin cores become apparent. It was proposed that design of molecules based on the structure-activity relationships established for novobiocin may result in Hsp90-specific inhibitors based upon the EGCG scaffold.
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This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Chemistry
Egcg
Hsp90 inhibitors
Radamide
Design, Synthesis, and Evaluation of Site-Specific Hsp90 Inhibitors
Thesis
oai:kuscholarworks.ku.edu:1808/80402020-08-07T17:07:51Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Bailey, Kyle
2011-09-22T00:36:04Z
2011-09-22T00:36:04Z
2011-05-27
http://dissertations.umi.com/ku:11605
http://hdl.handle.net/1808/8040
High-throughput screening and rational design can be used to create bioactive compounds with high affinity for selected therapeutic targets. However, a significant challenge in preclinical drug development is the identification of off-target proteins that contribute to phenotypic effects. This limits the understanding of the molecular basis of such effects, thus subverting rational drug design and hindering the identification of new therapeutic targets. Most previous strategies for proteome-wide target identification (target ID) have involved incubating cell lysates with compound-conjugated affinity resins. Despite their simplicity, such approaches can subject the proteome to conditions that prevent the detection of small molecule-protein interactions. The yeast three-hybrid system is an attractive alternative that uses genetic tools to screen for protein-small molecule interactions in cellulo. This thesis describes efforts to improve the utility of the yeast three-hybrid system to screen for drug targets. The proposed improvements utilize 1) the native fluorescence of green fluorescent protein (GFP) to identify interactions by flow-cytometry and fluorescence-activated cell sorting (FACS) and 2) the extreme affinity of streptavidin to search the mammalian proteome with biotinylated probes. The first objective required improvements to the sensitivity and dynamic range of a reporter vector encoding a popular GFP spectral variant. A new reporter vector was constructed and shown to exhibit better fluorescent properties compared to an existing reporter in a yeast one-hybrid assay. This reporter was also used to detect ligand dependent dimerization of the estrogen receptor β and progesterone receptor proteins. The second goal involved efforts to create a reduced valency streptavidin to enhance sensitivity for detection of biotinylated molecules in yeast three-hybrid systems. Circular permutations of wild-type and a low affinity mutant of streptavidin were constructed and fused to create dimeric streptavidins with variable valency. These constructs were tested with yeast three-hybrid assays using a GFP variant reporter, and shown to have altered profiles in fluorescence-based assays.
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This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Molecular biology
Pharmacology
Drug
Gfp
Target
Three-hybrid
Yeast
Improving the Yeast Three-Hybrid System for High-Throughput Target Discovery
Thesis
oai:kuscholarworks.ku.edu:1808/216612018-01-31T20:07:48Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Singh, Gurpreet
2016-10-11T19:12:43Z
2016-10-11T19:12:43Z
2015-12-31
http://dissertations.umi.com/ku:14373
http://hdl.handle.net/1808/21661
Abstract This work describes the development of methods to access synthetically useful chiral diols in enantiomerically pure form. First chapter describes the development of a stereodivergent approach for enantioenriched synthesis of 2-cyclopentene-1,3-diol that was later converted to 4-hydroxy-2-cyclopentenones (4-HCPs), which are highly privileged synthetic building blocks with numerous applications in natural product syntheses and pharmaceuticals. The present approach enables the gram scale synthesis of 4-HCPs with chemically diverse protecting groups, in a stereodivergent manner. In chapter 2, we describe the development of a unified strategy for the stereodivergent synthesis of enantioenriched 1,3-dihydroxy substituted six-membered carbo- and heterocyclic rings. The previously known approaches for accessing these compounds involve multiple synthetic steps and one or more enzymatic steps. We developed a purely synthetic approach to synthesize enantioenriched carbo- and heterocyclic six-membered 1,3-diols from a common, highly economical commercial available starting material. In Chapter 3, we described the development of a small-molecule library of stereochemically diverse compounds by integrating enantioenriched carbo- and heterocyclic 1,3-diols, and natural α-amino acids.
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openAccess
Copyright held by the author.
Organic chemistry
Chemistry
3-cyclohexanediols
3-dihydroxypiperidines
cyclopentenones
piperidines
prostaglandin-inspired libraries
small-molecule libraries
Stereodivergent Synthesis of Enantioenriched Five- and Six-Membered Cyclic-1,3-diols and Applications Toward Library Synthesis
Dissertation
oai:kuscholarworks.ku.edu:1808/275792019-01-16T19:36:28Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Gisemba, Solomon
2019-01-01T20:18:52Z
2019-01-01T20:18:52Z
2018-05-31
http://dissertations.umi.com/ku:16069
http://hdl.handle.net/1808/27579
Opioid receptors are important therapeutic targets for mood disorders and pain. Kappa opioid receptor (KOR) antagonists have recently shown potential for treating drug addiction and depression. Arodyn (Ac[Phe1,2,3,Arg4,D-Ala8]Dyn A(1-11)-NH2), an acetylated dynorphin A (Dyn A) analog, has demonstrated potent and selective KOR antagonism, but can be rapidly metabolized by proteases. Cyclization of arodyn could enhance metabolic stability and potentially stabilize the bioactive conformation to give potent and selective analogs. Accordingly, novel cyclization strategies utilizing ring closing metathesis (RCM) were pursued. However, side reactions involving olefin isomerization of O-allyl groups limited the scope of the RCM reactions, and their use to explore structure-activity relationships of aromatic residues. Here we developed synthetic methodology in a model dipeptide study to facilitate RCM involving Tyr(All) residues. Optimized conditions that included microwave heating and the use of isomerization suppressants were applied to the synthesis of cyclic arodyn analogs. Initial pharmacological data indicates the constraints involving aromatic residues were generally well tolerated at KOR with most of the analogs exhibiting affinities within 3- to 4-fold that of arodyn. RCM was also used in the synthesis of head to side chain cyclized arodyn analogs. Attempted cyclizations involving Tyr(All) residues proceeded in low yields, in contrast to cyclizations involving AllGly residues. However, ring contraction products as a result of olefin isomerization were also observed during the latter cyclizations. The resulting head to side chain cyclized arodyn analogs exhibited a 5-fold decrease in KOR affinity compared to arodyn. We further explored synthesis of arodyn analogs cyclized in both the N-terminal and C-terminal segments resulting in bicyclic arodyn analogs. Here, we present the synthesis of two initial bicyclic peptide KOR ligands with different topologies. The RCM-based bicyclic arodyn analog exhibited KOR affinity within 3-fold that of arodyn, whereas the lactam-based bicyclic analog displayed a substantial loss in affinity for KOR. There are currently no reports of bicyclic opioid peptide ligands and such bicyclic arodyn analogs could be useful as pharmacological tools.
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Copyright held by the author.
Chemistry
Pharmacology
Arodyn
Dynorphin A analogs
Kappa opioid receptor antagonists
Peptide cyclization
Ring-closing metathesis
Tyrosine(allyl)
Design and synthesis of cyclic analogs of the kappa opioid receptor antagonist arodyn
Dissertation
oai:kuscholarworks.ku.edu:1808/55822020-07-27T14:50:57Zcom_1808_100com_1808_1260col_1808_14142col_1808_1951
Amolins, Michael Wayne
2009-11-02T20:51:18Z
2009-11-02T20:51:18Z
2009-07-09
http://dissertations.umi.com/ku:10470
http://hdl.handle.net/1808/5582
This thesis represents a discussion of the advancements made in the area of Hsp90 therapeutics, and the development of a novel Hsp90 inhibitory scaffold of natural product origin. Through utilization of a recently reported firefly luciferase assay, our group was able to conduct a high-throughput screen that resulted in the identification of several Hsp90 inhibitors that contained a 1,4-naphthoquinone scaffold. From this, a library of naphthoquinones was synthesized. To further probe structure-activity relationships, a comparative molecular field analysis was performed, and a second generation of 1,4-naphthoquinones was developed. Although the results were promising, concerns arose regarding the nature of the scaffold itself. It is well documented that quinone-based scaffolds exert cytotoxicity through Michael reaction chemistry or redox activity. To circumvent this concern, the core scaffold was changed from a 1,4-naphthoquinone core to a structurally similar flavone. This was completed through utilization of molecular modeling and Western Blot analyses.
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This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Pharmaceutical chemistry
Chemistry
Biochemistry
Organic chemistry
Cancer
Flavone
Heat shock proteins
Molecular modeling
Naphthoquinone
Neuroprotection
Natural Product Inhibitors of Hsp90: Potential Leads for Drug Discovery
Thesis
oai:kuscholarworks.ku.edu:1808/216432018-01-31T20:07:51Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Hymel, David
2016-10-11T16:54:37Z
2016-10-11T16:54:37Z
2014-08-31
http://dissertations.umi.com/ku:13612
http://hdl.handle.net/1808/21643
Over the past several years, the Peterson group has developed synthetic mimics of cholesterol that rapidly incorporate into the plasma membrane of mammalian cells. These N-alkyl-3&beta-cholesterylamines cycle between the plasma membrane and early/recycling endosomes similar to natural cell surface receptors. Because of this unique activity, cholesterylamines have been linked to protein-binding and other motifs to generate artificial cell surface receptors. More recently, they have been conjugated to membrane disruptive peptides to generate synthetic agents that selectively permeabilize early endosomes and deliver cell-impermeable small molecules to the cytosol. Although this pioneering work has significant potential as a system to deliver cell-impermeable small molecule drugs and/or therapeutic agents, additional research is needed to understand the mechanism of action and develop therapeutic applications. To further evaluate this system, we first investigated the structure-activity relationships of cholesterylamines using fluorescent analogues designed to probe the mechanism of cellular uptake. This study produced probes with robust activity that suggests an unprecedented mechanism of cholesterol uptake on mammalian cell surfaces. Second, we investigated novel endosome disruptive peptides that provided insights into their mechanism of action. Additionally, we applied this knowledge to obtain proof-of-concept with antibody conjugates and endosome disruptive peptides as a new strategy to selectively deliver small molecules to tumor cells that overexpress specific cell surface receptors. Another project involves the development of a fluorescence-based method to detect protein-protein interactions in complex biological systems. This method utilizes a novel fluorinated fluorophore that undergoes proximity-driven exchange between lysine residues at the interface of a protein complex. Transfer of this fluorophore from donor to acceptor lysine residues produces a fluorescent protein partner that can be detected by gel electrophoresis or proteomics methods. Since lysine is prevalent at the interface of numerous protein complexes, this method may be useful to identify novel protein-protein interactions and/or factors that affect these interactions.
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Pharmaceutical sciences
Organic chemistry
Biochemistry
Antibody Conjugates
Cholesterol
Drug Delivery
Endocytosis
Pore-Forming Peptides
Protein-Protein Interactions
Synthetic Molecular Probes of Endocytosis, Escape from Endosomes, and Protein-Protein Interactions
Dissertation
oai:kuscholarworks.ku.edu:1808/54662020-07-23T14:59:59Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Leighty, Matthew
2009-08-31T03:51:36Z
2009-08-31T03:51:36Z
2009-05-04
http://dissertations.umi.com/ku:10394
http://hdl.handle.net/1808/5466
The first total synthesis of boehmeriasin A was achieved in seven steps from readily available materials with an overall yield of 29%. The absolute stereochemistry of the natural product was determined to be of the (R)-configuration. (-)-(R)-Boehmeriasin A demonstrated potent cytotoxicity in several cancer cell lines including drug resistant cancer cells where paclitaxel is inactive. An efficient and mild method for the construction if chromones was developed that requires only a single purification step at the end of the sequence and results in moderate to good yields of the isolated chromones. This method was applied toward the synthesis of 5-hydroxy-2-(2-phenylethyl)chromone, a neuroprotective chromone. The reduction of tertiary amides using Cp2Zr(H)Cl to the corresponding aldehydes on a preparatory scale results in good isolated yields of the products. Through a modified workup procedure, issues not previously observed were overcome to afford good isolated yields of the corresponding aldehydes.
EN
openAccess
This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
Organic chemistry
Boehmeriasin a
Flavone
Natural product synthesis
Vinylogous amide
Vinylogous ester
Vinylogous Esters and Amides: Useful Synthons for Diversity-Oriented and Natural Product Synthesis
Dissertation
oai:kuscholarworks.ku.edu:1808/61822020-07-28T12:35:26Zcom_1808_1260com_1808_100col_1808_1952col_1808_14142
Hirt, Erin Elaine
2010-05-03T01:00:57Z
2010-05-03T01:00:57Z
2009-09-29
http://dissertations.umi.com/ku:10583
http://hdl.handle.net/1808/6182
This thesis describes three new advances in the synthesis of nitrogen containing heterocycles. The first two chapters discuss the development of two different domino reaction sequences: a Diels-Alder/Schmidt and a Diels-Alder/acylation sequence. The third chapter then explores the development of a first-generation catalytic Schmidt reaction. The domino Diels-Alder/Schmidt reaction exploits two modes of reactivity. The first method reacted the azide and ketone groups on separate molecules, since it had been established in previous work that intermolecular Schmidt reactions only occur under special circumstances. An initial Diels¬-Alder reaction followed by a subsequent intramolecular Schmidt reaction was observed. This mode of reactivity was used to synthesize several interesting alkaloid-like skeleta. The second method was to deactivate the ketone for intramolecular azide attack by converting it into an enone. The Schmidt reaction did not occur until the enone had participated in a Diels-Alder reaction, thus providing control of this domino reaction. The enone deactivation method was used to form trans-hexahydroindoles and homopyrrolo[2.1-j]quinolin-5-ones. Also described herein is the development of a one-pot domino Diels-Alder/acylation strategy to form octahydroisoquinolinone scaffolds. The reaction exploits the reactivity of maleic anhydride toward variously substituted amino dienes providing exclusively the endo product. This work resulted in a scalable synthetic sequence tolerant of a wide range of substitution. The products also contain olefin and carboxylic acid groups suitable for further functionalization. Finally, studies towards the development of a catalytic, intramolecular Schmidt reaction of ketones and azides are described. Building on an initial positive result employing 50 mol% Sc(OTf)3 in water, conditions were explored for promoting this reaction sequence. Reaction surveys exploring solvents, Lewis and protic acids, and reaction conditions were completed. These studies led to the identification of conditions using a phase transfer catalyst (n-Bu4NOH or n-Bu4NCl) and microwave irradiation that accelerate the reaction with a broader range of substrates.
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Organic chemistry
Azides
Diels-Alder reaction
Methodology
Schmidt reaction
Development of Three Reaction Methodologies En Route to Nitrogen Containing Heterocycles: a Diels-Alder/Schmidt, a DIels-Alder/Acylation and a Catalytic Intramolecular Schmidt
Dissertation