Identification of Novel Inhibitors of P‐glycoprotein Mediated Multidrug Resistance

Abstract

Multidrug resistance (MDR) in cancers due to the overexpression of multidrug transporter proteins such as P‐glycoprotein (P‐gp) is a leading cause of chemotherapy failure. P‐gp uses ATP binding and hydrolysis to export a broad range of structurally dissimilar compounds, including many of the most commonly used chemotherapeutics. While this mechanism helps to protect tissues from toxins, the overexpression of P‐gp presents a major obstacle to cancer pharmacotherapy by preventing chemotherapeutics from reaching effective concentrations in the cancer cells. Reversing MDR by inhibiting P‐gp mediated efflux has been attempted for many years. The search for MDR reversing agents has resulted in a number of hit and lead compounds, many of which competitively inhibited chemotherapeutic transport by P‐gp, but to date none have succeeded in becoming FDA‐approved drugs. By searching for compounds that interact preferentially with the ATP binding domains of P‐gp, instead of the drug binding domain, we aim to identify inhibitors of P‐gp with novel mechanisms of action. We predict that these properties will be more suitable for future development of co‐therapeutics to reverse MDR.In in silico studies using targeted molecular dynamics and massively parallel drug docking of millions of drug‐like molecules, we have identified hundreds of potential inhibitors predicted to have high affinity for the ATP binding domain of P‐gp but low affinity for its drug binding domain. We previously reported that four of these compounds inhibited P‐gp catalyzed ATP hydrolysis (Brewer FK, Follit CA, Vogel PD, Wise JG (2014) Molecular Pharmacology, 86(6), 716–726) and three of those reversed the P‐gp mediated MDR phenotype of prostate cancer cells in culture (Follit CA, Brewer FK, Wise JG, Vogel PD (2015) Pharmacology Research & Perspectives 3(5): e00170). Here, we describe our continued search for potent P‐gp inhibitors using a cell‐based assay for initial screening of predicted P‐gp inhibitors that reports on the metabolic activity of the cells. “Hits” that were found to reverse MDR in a prostate cancer cell line after assaying ~100 computationally identified compounds, were further evaluated by live cell imaging. The majority of inhibitors identified in the metabolic activity assays also demonstrated inhibition of P‐gp‐catalyzed efflux of P‐gp substrates in the cell imaging experiments, supporting the usefulness of the metabolic activity assay as a medium‐throughput primary screen.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM09477102] to JGW/PDV, SMU Dissertation Fellowship, SMU University Research Council, SMU Center for Drug Discovery, Design and Delivery, The Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.