BIO-ENGINEERING OF COMPOUNDS ACTIVE AGAINST MDR CANCER

描述：非肌动蛋白是170-kDa-P-糖蛋白的抑制物 4‘-O-四氢吡喃拉霉素在多药耐药中的介导外排 红白血病K562细胞。非肌动蛋白及其大四内酯同系物是 ALS在体外对癌细胞具有活性，并显示出肿瘤抑制作用 在小鼠体内研究中的活性。此外，大四内酯类化合物的作用是 典型的离子载体抗生素对革兰氏细菌、真菌、 和分枝杆菌。非肌动蛋白不是目前的候选药物，因为它 毒性相对较高。然而，自然产生的同源物有 比非肌动蛋白更具活性：从QSAR分析中，我们提出了一系列 NE类化合物具有更高的潜在活性。 化学合成大四内酯同系物并不是一件容易的事，因为它 需要分别合成非乳酸的两个对映体； 选择性地将非乳酸单元组装成四聚中间体；以及 最后进行大内酯化反应，形成大四内酯环。 相比之下，灰色链霉菌菌株ETH A7796可以很容易地转换 发酵过程中添加1gl-1的非肌动蛋白。我们建议将最好的方面结合起来 化学合成和生物合成，以制造新的大环内酯类化合物。 化学合成将被用来制造外消旋非乳酸类似物，通过 已建立的高效路线。基因改造后的生物合成 一株灰链霉菌将实现立体特异的生物转化 非乳酸类似于新的大环内酯类化合物。 这项建议的具体目标是： 1.灰链霉菌A7796的纯化及其关键酶性质的研究 非肌动蛋白生物合成，非乳酸合成酶，催化 一种转化为非乳酸的无环前体。 2.分离和鉴定非肌动蛋白生物合成簇基因。 3.使用非肌动蛋白生物合成受阻的突变菌株，制造四个 典型的大环内酯类似物，并研究它们的物理和 生物属性。

DESCRIPTION: Nonactin is an inhibitor of the 170-kDa-P-glycoprotein mediated efflux of 4'-O-tetrahydropyranyladriamycin in multi-drug resistant erythroleukemia K562 cells. Nonactin and its macrotetrolide homologues are als active against cancer cell lines in vitro and have shown tumor reducing activity in in vivo studies with mice. Further, the macrotetrolides act as typical ionophore antibiotics being effective against Gram+ bacteria, fungi, and mycobacteria. Nonactin is not a current drug candidate because of its relatively high toxicity. The naturally occurring homologues, however, have greater activity than nonactin: from a QSAR analysis we propose a series of ne compounds which have potentially much higher activity. Chemical synthesis of macrotetrolide homologues is not trivial as it requires the separate synthesis of both enantiomers of nonactic acid; selective assembl of nonactic acid units into a tetrameric intermediate; and finally a macrolactonization reaction to form the macrotetrolide ring. Streptomyces griseus strain ETH A7796, in comparison, can readily make over 1 gL-1 of nonactin in fermentation. We propose to combine the best aspects of chemical synthesis and 'biosynthesis' to make new macrotetrolides. Chemical synthesis will be used to make racemic nonactic acid analogs via established, efficient routes. 'Biosynthesis' in a genetically altered strain of S. griseus will achieve the stereospecific biotransformation of the nonactic acid analogs into new macrotetrolides. The specific aims of this proposal are: 1. To purify from S. griseus ETH A7796 and characterize the key enzyme in nonactin biosynthesis, nonactate synthase, which catalyzes the conversion of a acyclic precursor into nonactic acid. 2. To isolate and characterize genes of the nonactin biosynthesis cluster. 3. To use a mutant strain, disrupted in nonactin biosynthesis, to make four prototypical macrotetrolide analogs, and to study their physical and biologica properties.