Characterization of Cucurbitacin-Inspired Estrone Analogues as Novel Inhibitors of Human ATP- Binding Cassette Proteins (ABCB1 and ABCC1)

ATP-binding cassette (ABC) transporters are a large class of integral membrane proteins that contribute to key physiological functions in all organisms by utilizing ATP binding and hydrolysis to transport diverse substrates across membrane barriers. P-glycoprotein (P-gp/ ABCB1) and Multidrug Resistance protein 1 (MRP1/ABCC1) are widely reported ABC transporters associated with multidrug resistance in cancer. Multidrug resistance (MDR) mediated by P-gp and MRP1 is responsible for treatment failures of many metastatic cancers as a result of reduced accumulation, bioavailability and diminished potency of anticancer drugs. Currently, known P-gp and MRP1 inhibitors are limited due to toxicity, lack of selectivity and low therapeutic benefit which necessitates the investigation of novel compounds with improved potency and specificity. In this study, we investigated the inhibition potential of newly synthesized cucurbitacin-inspired estrone analogs (CIEAs) on P-gp and MRP1 mediated MDR. Utilizing a high content fluorescence-based transport activity assay initially developed in our lab, a library of 81 CIEAs were screened to identify inhibitors for P-gp and MRP1 proteins. The screening assay identified eight P-gp inhibitors using calcein-AM as a fluorescent reporter. A total of six MRP1 inhibitors were identified using doxorubicin and calcein red orange as fluorescent reporters. The inhibitory effect of the identified compounds on P-gp and MRP1 activity were confirmed using established cell-based functional assays. The identified inhibitors significantly increased the accumulation of P-gp substrate, calcein-AM and MRP1 substrates, doxorubicin and calcein red orange. Interestingly, the identified inhibitors demonstrated selective sensitization of P-gp and MRP1 overexpressing cancer cell lines towards different anticancer drugs. On the other hand, the identified inhibitors did not alter protein levels of both P-gp and MRP1. Furthermore, our in silico and FRET-based spectroscopic studies showed that the inhibitor compounds directly interact with P-gp and MRP1 proteins. Together, our findings demonstrated that CIEAs are promising drug candidates for further development into therapeutic agents against P-gp and MRP1-mediated multidrug resistance in cancer