Search for new MDR modulator and analysis of its target molecule

1)在主要生物活性物质的研究过程中，我们致力于寻找肿瘤细胞多药耐药(MDR)的逆转物质和从海绵Spongia sp.中分离出agosterol A。我们检测了Aagosterol A对P-gp或MRP1介导的MDR细胞的作用，证实Aagosterol A通过直接影响P-gp和/或MRP1的药物外排泵来抑制药物的转运。2)从构效关系研究来看，3，4，6-乙酰氧基和11，22-羟基在逆转肿瘤细胞MDR中起关键作用。此外，我们还合成了~(125)I标记的银杏叶提取物A的光亲和探针。3)我们用合成的银杏甾醇A的光亲和探针成功地标记了MRP1。有趣的是，该探针仅在谷胱甘肽存在的情况下才与MRP1有亲和力。我们发现该探针光标记了MRP1(C932-1531；TMD2)的C-近端分子。利用表达各种截短、共表达和突变的MRP1的膜小泡在GSH存在和不存在的情况下进行光标记法研究，我们发现LO是MRP1上与GSH相互作用的位点。此外，我们还证明了该探针的GSH依赖的结合部位位于MRP1的氨基酸1223和1295之间，该区域包括TM螺旋17。此外，我们的发现表明，荷电的氨基酸Arg1249是MRP1底物相互作用所必需的，其中C末端(C1295-1531)是必不可少的。

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).