Medicinal Chemistry Dissertations and Theses
https://hdl.handle.net/1808/14142
2024-03-29T05:49:05ZSTUDIES OF INTERACTIONS OF SMALL MOLECULES WITH MEMBRANES AND PROTEINS
https://hdl.handle.net/1808/30224
STUDIES OF INTERACTIONS OF SMALL MOLECULES WITH MEMBRANES AND PROTEINS
Phaniraj, Sahishna
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.
2019-12-31T00:00:00ZDiscovery of Novel Inhibitors of Cellular Efflux by High-Content Screening with a Fluorescent Mimic of Taxol
https://hdl.handle.net/1808/29848
Discovery of Novel Inhibitors of Cellular Efflux by High-Content Screening with a Fluorescent Mimic of Taxol
Smith, Tomas Joseph
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.
2019-08-31T00:00:00ZSynthesis and Evaluation of Fluorescent Tools for Studies of Cancer Biology
https://hdl.handle.net/1808/29842
Synthesis and Evaluation of Fluorescent Tools for Studies of Cancer Biology
Gao, Zhe
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.
2019-08-31T00:00:00ZStrategies for the Fluorine-Retentive Functionalization of Gem-Difluoroalkenes
https://hdl.handle.net/1808/29550
Strategies for the Fluorine-Retentive Functionalization of Gem-Difluoroalkenes
Orsi, Douglas
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.
2019-05-31T00:00:00Z