Potential of novel Heat Shock Protein 90 (HSP90) inhibitors for P-glycoprotein inhibition and cancer multidrug resistance reversal

Cancer chemotherapy is often compromised by development of multidrug resistance (MDR). Numerous strategies have been developed over recent decades to overcome cancer resistance but this issue remains unsolved in clinical practice. Dual-targeting by a single drug emerged as an unconventional approach to overcome incomplete efficacy of individual targeting agents. Heat Shock Protein 90 (HSP90) chaperone interacts with a broad range of client proteins involved in cancerogenesis and cancer progression. Its overexpression was found in several cancer types and thus it is considered a valuable target for anticancer treatment. However, HSP90 inhibitors were unsuccessful in clinical studies due to high toxicity, lack of selectivity against cancer cells and extrusion by membrane transporters such as P-glycoprotein (P-gp). P-gp is responsible for low efficacy of anticancer drugs in more than 50% of cancers. Recognizing the potential of new compounds to inhibit P-gp function and/or expression is essential in the search for effective anticancer agents. We have synthesized 11 novel HSP90 inhibitors containing an isoxazolonaphtoquinone core and identified candidates that inhibit P-gp and modulate MDR. HSP90 inhibitors were evaluated in MDR models comprised of sensitive and corresponding resistant cancer cells with P-gp overexpression (non-small cell lung carcinoma NCI-H460 and NCI-H460/R; colorectal adenocarcinoma DLD1 and DLD1-TxR) as well as human normal embryonic fibroblasts MRC-5. We have investigated the effect of HSP90 inhibitors on cell growth inhibition, P-gp function, and P-gp mRNA and protein expression. Additionally, optimization of HSP90 inhibitors\u2019 MDR modulation was performed by kinetics and dose response studies. Compounds 1 and 2 directly interacted with P-gp and inhibited its activity. Similar cytotoxicity of 1 and 2 in sensitive and MDR cancer cells indicated these compounds are not P-gp substrates. On contrary, the effect of compound 3 was significantly reduced in MDR cancer cells, indicating that this compound acts as P-gp substrate, exerting competitive inhibitory effect on P-gp. Inhibition of P-gp activity after 1, 2 and 3 treatment lasted 24 h. These compounds also showed good relative selectivity towards cancer cells. Compound 4 had no direct effect on P-gp activity but significantly suppressed P-gp expression after 72 h treatment