SYNTHESIS OF MDR REVERSING POLYENES

多药耐药（MDR）是由于Pgp（P- 糖蛋白），通过MDR 1基因，一种ATP驱动的膜结合的多药耐药蛋白。 传送器。最近，我们开发了新的MDR的合成路线， 逆转剂，stipiamide，和更有效的，无毒的化合物6，7- 脱氢替匹酰胺（DHS，图1），可恢复 阿霉素对耐药人乳腺癌细胞（MCF-7adrR）的作用 低浓度（4 nM）。我们现在将研究新的化合物 在组合文库中具有非天然间隔区，和 二苯甲酮光标记。将生产非天然多烯， 确定MDR逆转的最佳结构要求， 用耐药癌细胞系和纯化的Pgp进行测定。多烯 将改变施替匹胺的区域，以最大限度地减少毒性， MDR逆转。与帕拉的双向交叉偶联反应 二取代苯将被研究以选择性地连接 两头将使用已知放射性标记的竞争研究， 验证直接Pgp绑定。每种化合物的毒性将是 使用正常癌细胞和具有添加的抗性细胞系测定 抗癌药抗1，2-羟甲基的替代途径将是 研究了手性助剂和催化羟醛缩合方法。的 官能度在两个末端和立体化学将被探测 使用组合库。耦合的条件将是 在1：1碘化物：乙炔化学计量下进行优化，并应用于 组合库化合物将作为混合样本池进行测定 使用MCF-7adrR MDR测定。Pgp上的特定残基将是 首次发现使用高效二苯甲酮- stipiamide光亲和标记剂。微测序将用于 识别结合位点中发现的特定残基。双功能 还将通过连接两个MDR逆转来制备双结构域化合物 化合物通过末端酰胺。使用这些试剂盒进行试剂盒测定 化合物将用于建立Pgp之间的距离约束 跨膜螺旋TM 6和TM 12已经暗示含有 药物和逆转剂结合位点。结果从图书馆， 双功能化合物和光标记将建立， 第一次，位置，绑定特性和距离 在Pgp的结合位点之间从这些实验中获得的数据 将直接影响Pgp的三维结构。这是一 重要的第一步，对独特的分子理解 识别特性，Pgp的转运机制，并将进一步 促进新的、更有效的逆转剂的设计。

Multi-drug resistance (MDR) is due to the expression of Pgp (P- glycoprotein), by the MDRl gene, an ATP-driven membrane-bound multi-drug transporter. Recently we have developed synthetic routes to new MDR reversal agents, stipiamide, and a more potent, non-toxic compound 6,7- dehydrostipiamide (DHS, fig.1) that restores the cytotoxicity of adriamycin to resistant human breast cancer cells (MCF-7adrR) at very low concentrations (4 nM). We will now investigate new compounds possessing non-natural spacers, in combinatorial libraries, and benzophenone photolabels. Non-natural polyenes will be produced to identify the optimal structural requirements for MDR reversal using assays with resistant cancer cell lines and purified Pgp. The polyene region of stipiamide will be changed to minimize toxicity and increase MDR reversal. Bi-directional cross coupling reactions with para- disubstituted benzenes will be investigated to selectively attach the two ends. Competition studies with known radiolabeles will be used to verify the direct Pgp binding. The toxicity of each compound will be determined using normal cancer cells and resistant cell lines with added cancer drug. Alternative routes to the anti-1,2-hydroxy methyl will be investigated with chiral auxiliaries and catalytic aldol approaches. The functionality at the two ends and the stereochemistry will be probed using combinatorial libraries. The conditions of the coupling will be optimized at 1:1 iodide:acetylene stoichiometry and applied to the combinatorial libraries. The compounds will be assayed as mixed pools using the MCF-7adrR MDR assay. Specific residues on Pgp will be identified, for the first time, using high-efficiency benzophenone- stipiamide photoaffinity labeler. Microsequencing will be used to identify specific residues found in the binding site. Bi-functional dual-domain compounds will also be made by linking two MDR reversal compounds through the terminal amide. Reversal assays with these compounds will be used to establish the distance constraints between Pgp transmembrane helices TM6 and TM12 that have been implicated to contain the drug and reversal agent binding sites. Results from the libraries, the bi-functional compounds, and the photolabels will establish, for the first time, the location, the binding characteristics, and the distances between the binding bites on Pgp. Data obtained from these experiments will have direct bearing on the 3D-structure of Pgp. This is an important first step toward a molecular understanding of the unique recognition properties, transport mechanism of Pgp, and will further facilitate the design of new, more potent reversal agents.