GSH facilitates the binding and inhibitory activity of novel multidrug resistance protein 1 (MRP1) modulators

MRP1 ( ABCC1 ) is a membrane transporter that confers multidrug resistance in cancer cells by exporting chemotherapeutic agents, often in a reduced glutathione (GSH)‐dependent manner. This transport activity can be altered by compounds (modulators) that block drug transport while simultaneously stimulating GSH efflux by MRP1. In MRP1‐expressing cells, modulator‐stimulated GSH efflux can be sufficient to deplete GSH and increase sensitivity to chemotherapy, enhancing cancer cell death. Further development of clinically useful MRP1 modulators requires a better mechanistic understanding of modulator binding and its relationship to GSH binding and transport. Here, we explore the mechanism of two MRP1 small molecule modulators, 5681014 and 7914321, in relation to a bipartite substrate‐binding cavity of MRP1. Binding of these modulators to MRP1 was dependent on the presence of GSH but not its reducing capacity. Accordingly, the modulators poorly inhibited organic anion transport by K332L‐mutant MRP1, where GSH binding and transport is limited. However, the inhibitory activity of the modulators was also diminished by mutations that limit E 2 17βG but spare GSH‐conjugate binding and transport (W553A, M1093A, W1246A), suggesting overlap between the E 2 17βG and modulator binding sites. Immunoblots of limited trypsin digests of MRP1 suggest that binding of GSH, but not the modulators, induces a conformation change in MRP1. Together, these findings support the model, in which GSH binding induces a conformation change that facilitates binding of MRP1 modulators, possibly in a proposed hydrophobic binding pocket of MRP1. This study may facilitate the structure‐guided design of more potent and selective MRP1 modulators.