Explorations of Drug Transport by P-Glycoprotein Using Molecular Dynamics Enabled by High Resolution Crystal Structures

The integral membrane efflux pump P-glycoprotein (P-gp, ABCB1) is a causative agent of multidrug resistance (MDR) to a wide variety of chemotherapeutics in human cancers. In many cancer subtypes, chemotherapy treatment can both induce and select for the overexpression of P-gp. When P-gp is overexpressed, the pump lowers the intracellular concentration of many drugs to sub-therapeutic levels, thereby conferring general MDR to the cancer, and rendering further chemotherapeutic treatment ineffective. Given the clinical importance of the enzyme, P-gp has long been a target for drug discovery and the focus of extensive mechanistic research. Until recently, the lack of known human P-gp structures has hindered efforts to understand the unusually polyspecific substrate profile and complex transport mechanism of the enzyme. Prior research by our laboratory has demonstrated the possibility of multiple transport pathways through the drug binding domains (DBDs) of the transporter, and of variable substrate binding sites using targeted molecular dynamics (TMD) simulations on homology models of human P-gp. Following the recent release of the cryo-EM structure of human P-gp, we performed novel simulations of the solved structure using four known, low energy conformations of P-gp homologues in various states along a putative ABC transporter catalytic efflux cycle. These simulations were done at long timescales using a combination of free and directed GPU-accelerated molecular dynamics with NAMD. These analyses included the new human P-gp structure and demonstrated the pumping of known P-gp substrates across the cell membrane. Parameterizations and simulations of molecules that are either suspected transport substrates or putative transport inhibitors of P-gp were performed. These computational studies support earlier observations, and explore the hypothetical mechanisms of P-gp-mediated substrate efflux and of P-gp transport inhibition. This work was supported by NIH NIGMS [R15GM094771-02].