Abstract

Multidrug resistance (MDR) is a major cause of chemotherapy failure. Overexpression of ATP-binding cassette (ABC) transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two well-studied drug transporters which are associated with MDR. These two transporters also act as a major functional unit of the blood brain barrier to protect the brain from xenobiotics and toxins. Lack of clinically approved P-gp and BCRP inhibitors renders chemotherapy treatments of many MDR cancers ineffective and obstructs drug uptake into the brain.

Using computational methods, we have identified new compounds that inhibit P-gp (Brewer et al., Mol. Pharmacol. 2014). Several of these compounds show successful MDR reversal in the drug resistant DU145TXR prostate cancer cell line (Follit et al., Pharmacol. Res. Perspect. 2015). Here, we further analyze these P-gp inhibitors 29, 34 and 45 in a P-gp over-expressing ovarian cancer cell line, A2780ADR, and a P-gp over-expressing prostate cancer cell line, DU145TXR. Treatment of chemotherapeutics with the in silico identified inhibitors led to a higher mortality in MDR cancer cells in tests conducted with both conventional 2D and 3D-spheroid cell-based assays. Using a novel assay based on cellular accumulation of compounds in a P-gp overexpressing cell line, we report that 29, 34 and 45 do not function as P-gp transport substrates. It was found that the efficacy of chemotherapy can be enhanced by initial cotreatment of chemotherapeutics with P-gp inhibitor 29 followed by an extended treatment using only inhibitor 29 without having chemotherapeutics in the media. Further, we describe the effects of chemical variants of the P-gp inhibitor 29 in experiments aimed at improving the pharmacological characteristics of the inhibitor. The variants were generated using computational approaches or by structure-based design in attempts to improve P-gp inhibition. Multiple variants showed an improved efficacy in reversing paclitaxel resistance in the P-gp over-expressing DU145 TXR prostate cancer cell line. We achieved a 100% success rate using computational lead optimization in obtaining variants of P-gp inhibitor 29 which do not function as transport substrates of P-gp.

In related work, we developed a breast cancer cell line that overexpresses the BCRP transporter for the purpose of identifying inhibitors of the BCRP drug pump. Three of the variants of inhibitor 29 could reverse the BCRP-mediated MDR in this BCRP overexpressing breast cancer cell line. Further, we identified several other compounds that interfered with BCRP mediated transport in vitro screened from an inhouse library of small molecules.

The experimental inhibitors of P-gp and BCRP in this study appear to be promising candidates for further development into co-therapeutics to treat MDR cancers and modulate the blood brain barrier.

Degree Date

Spring 5-18-2019

Document Type

Dissertation

Degree Name

Ph.D.

Department

Biology

Advisor

John G. Wise

Subject Area

Biological Sciences, General, Cell Biology, Life Sciences, Pharmacology

Number of Pages

198

Format

.pdf

Creative Commons License

Creative Commons Attribution-Noncommercial 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License

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