In Silico and In Vitro Identification of P-Glycoprotein Inhibitors from a Library of 375 Phytochemicals

Abstract

Cancer therapy with clinically established anticancer drugs is frequently hampered by the development of drug resistance of tumors and severe side effects in normal organs and tissues. The demand for powerful, but less toxic, drugs is high. Phytochemicals represent an important reservoir for drug development and frequently exert less toxicity than synthetic drugs. Bioinformatics can accelerate and simplify the highly complex, time-consuming, and expensive drug development process. Here, we analyzed 375 phytochemicals using virtual screenings, molecular docking, and in silico toxicity predictions. Based on these in silico studies, six candidate compounds were further investigated in vitro. Resazurin assays were performed to determine the growth-inhibitory effects towards wild-type CCRF-CEM leukemia cells and their multidrug-resistant, P-glycoprotein (P-gp)-overexpressing subline, CEM/ADR5000. Flow cytometry was used to measure the potential to measure P-gp-mediated doxorubicin transport. Bidwillon A, neobavaisoflavone, coptisine, and z-guggulsterone all showed growth-inhibitory effects and moderate P-gp inhibition, whereas miltirone and chamazulene strongly inhibited tumor cell growth and strongly increased intracellular doxorubicin uptake. Bidwillon A and miltirone were selected for molecular docking to wildtype and mutated P-gp forms in closed and open conformations. The P-gp homology models harbored clinically relevant mutations, i.e., six single missense mutations (F336Y, A718C, Q725A, F728A, M949C, Y953C), three double mutations (Y310A-F728A; F343C-V982C; Y953A-F978A), or one quadruple mutation (Y307C-F728A-Y953A-F978A). The mutants did not show major differences in binding energies compared to wildtypes. Closed P-gp forms generally showed higher binding affinities than open ones. Closed conformations might stabilize the binding, thereby leading to higher binding affinities, while open conformations may favor the release of compounds into the extracellular space. In conclusion, this study described the capability of selected phytochemicals to overcome multidrug resistance.

A Bptf Inhibitor That Interferes with the Multidrug Resistance Pump to Sensitize Triple Negative Breast Cancer Cells to Chemotherapy

“…The basis for failure of these inhibitors in clinical trials has been varied, including the necessity of utilizing intolerably high doses, high toxicity, drug interactions, and limited efficacies ( 34,37 ). Nevertheless, efforts have continued to explore the use of natural and/or FDA-approved compounds which demonstrate P-gp inhibition activity for adjunctive therapeutic use with chemotherapy ( [38][39][40] ).…”

Natural Product-Derived Compounds for Targeting Multidrug Resistance in Cancer and Microorganisms

“…Schäfer et al [ 9 ] screened, using in silico studies, an in-house library of 375 phytochemicals bearing diverse scaffolds for their ability to overcome multidrug resistance in cancer, and selected six compounds for further in vitro investigation. The six selected compounds were two flavonoids, one alkaloid, and three terpenes.…”