Search for new MDR modulator and analysis of its target molecule

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1) In the course of our study of bioactive substances from majine organisms, we focused on a search for reversing substances of multidrug-resistance (MDR) in tumor cells and isolated agosterol A from a marine sponge Spongia sp. We examined the action of agosterol A for both the P-gp-or MRP1-mediated MDR cells and clalified that agosterol A inhibited the drug transportation through P-gp and/or MRP1 by affecting those drug efflux pumps directly.2) From the structure-activity relationship study, each of the 3,4,6-acetoxyl groups and 11,22-hydroxyl groups was elucidated to be crucial for reversing MDR in tumor cells. Furthermore, we synthesized the 125I-labeled photoaffinity probe of agosterol A.3) We succeeded in photolabeling bf MRP1 using the synthtic photoaffinity probe of agosterol A.Interestingly, the probe showed the affinity to MRP1 only in the presence of glutathione. We found that the probe photolabeled the C-proximal molecule of MRP1 (C932-1531; TMD2). Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and mutated MRP1s, we found that LO is the site on MRP1 which interacts with GSH. Furthermore, we demonstrated that the GSH-dependent binding site of the probe lies between amino acid 1223 and 1295, a region of MRP1 which includes'TM helix 17. Furthermore, our findings suggest that the charged amino acid Arg1249 is indispensable for MRP1 substrates interaction whereae the C terminal region (C1295-1531).