SYNTHESIS OF MDR REVERSING POLYENES

多药耐药(MDR)与Pgp的表达有关(P<0.05)。 糖蛋白)，由mdr1基因，一种三磷酸腺苷驱动的膜结合多药 传送器。最近，我们开发了合成路线来合成新的MDR 反转剂，司匹胺，和一种更有效的无毒化合物6，7- 脱氢己二胺(DHS，图1)，恢复其细胞毒性 阿霉素对耐药人乳腺癌细胞(MCF-7adrR)的体外耐药作用 低浓度(4 NM)。我们现在要研究新的化合物 在组合文库中具有非自然间隔区，以及 二苯甲酮类光标记。非天然聚烯将被生产来 确定MDR逆转的最佳结构要求 用耐药癌细胞株和纯化的Pgp进行检测。多烯 将改变己二胺的区域，以将毒性降至最低 MDR逆转。对位偶联反应的双向交叉偶联反应 将对双取代苯进行研究，以选择性地将 两端。已知无线电标签的竞争研究将用于 检验直接PGP绑定。每种化合物的毒性将是 用正常癌细胞和耐药细胞系测定 抗癌药物。反1，2-羟甲基的替代路线将是 用手性助剂和催化醇方法进行了研究。这个 两端的官能度和立体化学将被探索 使用组合库。耦合的条件将是 在1：1碘：乙炔化学计量比下进行优化，并应用于 组合库。这些化合物将被检测为混合池 采用MCF-7adrR多药耐药实验。PGP上的特定残留物将是 首次使用高效二苯甲酮进行鉴定- 己二胺光亲和标记剂。微测序将用于 确定在结合部位发现的特定残基。双功能 双结构域化合物也将通过连接两个MDR逆转而制成 通过末端酰胺的化合物。用这些方法进行逆转分析 将使用化合物来建立PGP之间的距离约束 跨膜螺旋TM6和TM12被认为含有 药物与逆转剂的结合部位。来自图书馆的结果， 双功能化合物和光标记将为 第一次、位置、绑定特征和距离 在Pgp上的结合片段之间。从这些实验中获得的数据 将直接关系到PGP的3D结构。这是一个 迈向独特的分子理解的重要第一步 Pgp的识别特性、转运机制，并将进一步 促进设计新的、更有效的逆转剂。

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.