This dissertation focuses upon dynamic agents of multidrug resistance (MDR). We used a combination of in silico and in vitro techniques to investigate two membrane transporters that confer MDR – P-glycoprotein, which confers MDR in human cancers, and MtrD, which confers MDR in Neisseria gonorrhoeae. Inhibitors targeting both proteins have tremendous potential for use as co-therapeutics in the treatment of multidrug resistant cancers, or of multidrug resistant infections. However, previously identified inhibitors of P-gp have failed clinical trials due to off-target effects and associated toxicities. Furthermore, the molecular mechanism of antibiotic transport by MtrD is poorly understood, and this dearth of knowledge obfuscates efforts to target MtrD in drug discovery screens. For these reasons, inhibitors targeting either P-gp or MtrD are not available for clinical use.
In this work, we used rigorous and robust computational techniques to investigate P-gp and MtrD. Our studies employed free and biased all atom Molecular Dynamics simulations to study the conformational and transport dynamics of these MDR-critical proteins. We then used massively parallel molecular docking experiments to screen millions of compounds prior to purchasing them for testing in the laboratory. Finally, to test our computationally-derived hypotheses, we employed a suite of cell-based and biophysical assays; these included MTT cell viability assays, Confocal Microscopy, Fluorescence Accumulation Assays, and LC-MS/MS accumulation assays.
In this work, we contributed to the understanding of how MtrD effluxes its antibiotic substrates, thereby rendering them ineffective as treatments for N. gonorrhoeae. Our data identify new areas of MtrD to target in drug discovery efforts. Furthermore, we used a massively parallel virtual drug screening program to select compounds for testing as P-gp inhibitors and tested those compounds against five different human cell lines in vitro. We report a 13% hit rate for P-gp inhibitors, a massive improvement over other virtual-assisted screens. As our last project, we used MD simulations and cell-based assays to demonstrate that P-gp can efflux the Alzheimer’s associated Aβ peptides. This project significantly expands the substrate profile of this already promiscuous transporter.
John G. Wise
Biochemistry, Cell Biology, Life Sciences, Molecular Biology
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Ammerman, Lauren, "Dynamic Mechanisms Of Multidrug Resistance In Human Cancers And Gram Negative Pathogens" (2021). Biological Sciences Theses and Dissertations. 11.
Biochemistry, Biophysics, and Structural Biology Commons, Bioinformatics Commons, Biology Commons