SYNTHESIS OF MDR REVERSING POLYENES

MDR逆转多烯的合成

• 批准号: 6180509
• 负责人: MERRITT B ANDRUS
• 金额: $ 17.31万
• 依托单位: BRIGHAM YOUNG UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180509
• 关键词: P glycoprotein; affinity labeling; antibiotics; binding sites; cell line; combinatorial chemistry; doxorubicin; drug design /synthesis /production; multidrug resistance; polyenes; protein structure function; stereochemistry

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.

多重耐药性（MDR）是由于 Pgp（P- 糖蛋白），由 MDR1 基因产生，一种 ATP 驱动的膜结合多药 运输者。最近我们开发了新 MDR 的合成路线 逆转剂、斯蒂吡酰胺和一种更有效、无毒的化合物 6,7- 脱氢斯蒂酰胺（DHS，图 1）可恢复细胞毒性 阿霉素对耐药人乳腺癌细胞 (MCF-7adrR) 的作用非常强 低浓度（4 nM）。我们现在将研究新化合物 在组合文库中拥有非天然间隔区，以及 二苯甲酮光标签。将生产非天然多烯 确定 MDR 逆转的最佳结构要求 使用耐药癌细胞系和纯化的 Pgp 进行测定。多烯 斯蒂吡酰胺的区域将被改变以最小化毒性并增加 多药耐药逆转。与对位的双向交叉偶联反应 将研究二取代苯以选择性地连接 两端。已知放射性标记的竞争研究将用于 验证直接 Pgp 绑定。每种化合物的毒性为 使用正常癌细胞和耐药细胞系并添加 癌症药物。抗 1,2-羟甲基的替代途径将是 用手性助剂和催化羟醛方法进行了研究。这 将探测两端的功能性和立体化学 使用组合库。耦合条件为 按 1:1 碘化物:乙炔化学计量优化并应用于 组合库。这些化合物将作为混合池进行分析 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- 斯蒂吡酰胺光亲和标记仪。微测序将用于 识别结合位点中发现的特定残基。双功能 双结构域化合物也将通过连接两个 MDR 逆转来制备 通过末端酰胺形成化合物。用这些进行逆转测定 化合物将用于建立 Pgp 之间的距离约束 跨膜螺旋 TM6 和 TM12 被认为含有 药物和逆转剂的结合位点。来自图书馆的结果， 双功能化合物和光标签将建立 第一次、位置、绑定特征和距离 Pgp 上的结合位点之间。从这些实验中获得的数据 将直接影响 Pgp 的 3D 结构。这是一个 迈向分子理解独特的重要的第一步 Pgp的识别特性、传输机制，并将进一步 促进新的、更有效的逆转剂的设计。