Structure-ATPase Activity Relationship of Rhodamine Derivatives as Potent Inhibitors of P-Glycoprotein CmABCB1.

Understanding the transport and inhibition mechanisms of substrates by P-glycoprotein (P-gp) is one of the important approaches in addressing multidrug resistance (MDR). In this study, we evaluated a variety of rhodamine derivatives as potential P-gp inhibitors targeting CmABCB1, a P-gp homologue, with a focus on their ATPase activity. Notably, a Q-rhodamine derivative with an o,o'-dimethoxybenzyl ester moiety (RhQ-DMB) demonstrated superior affinity and inhibitory activity, which was further confirmed by a drug susceptibility assay in yeast strains expressing CmABCB1. Results from a tryptophan fluorescence quenching experiment using a CmABCB1 mutant suggested that RhQ-DMB effectively enters and binds to the inner chamber of CmABCB1. These findings underscore the promising potential of RhQ-DMB as a tool for future studies aimed at elucidating the substrate-bound state of CmABCB1.

Crystal structure of CmABCB1 multi-drug exporter in lipidic mesophase revealed by LCP-SFX.

CmABCB1 is a Cyanidioschyzon merolae homolog of human ABCB1, a well known ATP-binding cassette (ABC) transporter responsible for multi-drug resistance in various cancers. Three-dimensional structures of ABCB1 homologs have revealed the snapshots of inward- and outward-facing states of the transporters in action. However, sufficient information to establish the sequential movements of the open-close cycles of the alternating-access model is still lacking. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has proven its worth in determining novel structures and recording sequential conformational changes of proteins at room temperature, especially for medically important membrane proteins, but it has never been applied to ABC transporters. In this study, 7.7 mono-acyl-glycerol with cholesterol as the host lipid was used and obtained well diffracting microcrystals of the 130 kDa CmABCB1 dimer. Successful SFX experiments were performed by adjusting the viscosity of the crystal suspension of the sponge phase with hy-droxy-propyl methyl-cellulose and using the high-viscosity sample injector for data collection at the SACLA beamline. An outward-facing structure of CmABCB1 at a maximum resolution of 2.22 Šis reported, determined by SFX experiments with crystals formed in the lipidic cubic phase (LCP-SFX), which has never been applied to ABC transporters. In the type I crystal, CmABCB1 dimers interact with adjacent molecules via not only the nucleotide-binding domains but also the transmembrane domains (TMDs); such an interaction was not observed in the previous type II crystal. Although most parts of the structure are similar to those in the previous type II structure, the substrate-exit region of the TMD adopts a different configuration in the type I structure. This difference between the two types of structures reflects the flexibility of the substrate-exit region of CmABCB1, which might be essential for the smooth release of various substrates from the transporter.