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 E2 17βG but spare GSH-conjugate binding and transport (W553A, M1093A, W1246A), suggesting overlap between the E2 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.