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Publication STUDIES OF INTERACTIONS OF SMALL MOLECULES WITH MEMBRANES AND PROTEINS(University of Kansas, 2019-12-31) Phaniraj, SahishnaThe 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.Publication Discovery of Novel Inhibitors of Cellular Efflux by High-Content Screening with a Fluorescent Mimic of Taxol(University of Kansas, 2019-08-31) Smith, Tomas JosephFluorescence-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.Publication Synthesis and Evaluation of Fluorescent Tools for Studies of Cancer Biology(University of Kansas, 2019-08-31) Gao, ZheA 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.Publication Strategies for the Fluorine-Retentive Functionalization of Gem-Difluoroalkenes(University of Kansas, 2019-05-31) Orsi, DouglasFluorination 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.Publication Studies of novel targeted drug delivery systems and molecular probes of cancer biology(University of Kansas, 2019-05-31) Knewtson, Kelsey ErinCancer 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.Publication Investigation of the Phenyl Ring of Imidazoquinolines(University of Kansas, 2018-05-31) Hunt, Jordan RobertThe 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.Publication A New Generation of Isoform Selective Hsp90 Inhibitors: Targeting the Cytosolic Hsp90 Isoforms(University of Kansas, 2018-05-31) Mishra, SanketHeat 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.Publication The Development of Organelle-localized Hsp90 Isoform-selective Inhibitors(University of Kansas, 2017-12-31) Crowley, Vincent MatthewMolecular 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.Publication Exploration of Salvinorin A for the Development of Pain and Addiction Therapies(University of Kansas, 2017-08-31) Crowley, Rachel S.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.Publication 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.(University of Kansas, 2017-05-31) Ma, HuiyongDevelopment 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.Publication Design and synthesis of cyclic analogs of the kappa opioid receptor antagonist arodyn(University of Kansas, 2018-05-31) Gisemba, SolomonOpioid 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.Publication Advances in Heterocyclic Synthesis through Ring Expansions and Flow Chemistry(University of Kansas, 2018-05-31) Charaschanya, ManwikaThis 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.Publication Exploration of the Mechanisms of Adjuvanticity for Toll-like Receptor Agonists(University of Kansas, 2017-08-31) Salyer, Alex Christopher DeanSmall-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.Publication The Development of Small Molecules that Modulate Molecular Chaperones Hsp90 and Hsp70(University of Kansas, 2017-12-31) Forsberg, Leah KathleenThe 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.Publication Synthetic Strategies to Access Biologically Important Fluorinated Motifs: Fluoroalkenes and Difluoroketones(University of Kansas, 2017-05-31) Yang, Ming-HsiuFluorine 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.Publication Design and Synthesis of Functionally Selective Kappa Opioid Receptor Ligands(University of Kansas, 2017-05-31) Johnson, StephanieThe 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.Publication Copper-Catalyzed Decarboxylative Trifluoromethylation Reactions(University of Kansas, 2017-05-31) Ambler, Brett R.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.Publication An Improved Synthesis of the Pacific Blue Fluorophore and Fluorescence-based Studies of Receptor-Ligand Interactions(University of Kansas, 2016-12-31) Lee, Molly MarieEarly-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.Publication Unfolding the Hsp90 Foldasome: Structure-Activity Relationship Studies on EGCG and Development of Isoform-Selective Inhibitors(University of Kansas, 2016-08-31) Khandelwal, AnujThe 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.Publication SYNTHESIS AND PHENOTYPIC DISCOVERY OF MOLECULAR PROBES OF BIOLOGICAL SYSTEMS(University of Kansas, 2015-12-31) Meinig, James MatthewThe 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.