BIO-ENGINEERING OF COMPOUNDS ACTIVE AGAINST MDR CANCER

描述（由申请人提供）：多重耐药性正在成为公共卫生的主要威胁。我们面临着对新的生物活性化合物和制造这些化合物的新方法的需求。链霉菌产生了大多数由细菌产生的抗生素，研究这种抗生素生物合成途径是开发抗生素生产新方法的基本前提。由灰色链霉菌产生的不寻常的聚酮，非肌动蛋白，是在多重耐药癌细胞中发现的p170糖蛋白外排泵的抑制剂。非肌动蛋白也是一种对革兰氏阳性细菌、分枝杆菌和真菌有活性的离子载体。非肌动蛋白是一种非手性化合物；由非乳酸前体的两个对映体组成的四聚体该生物具有两个镜像生物合成途径，每个镜像生物合成途径对应前体对映体。特别令人感兴趣的是非肌动蛋白生物合成的后期阶段，其中完整的大循环被组装起来，因为这些步骤可以被操纵来产生天然产物-合成非肌动蛋白的化合物杂化类似物。对非肌动蛋白生物合成系统的基础生物化学和应用生物化学将通过以下方式进行研究：(1)分析(+)-和(-)-非肌动蛋白合成酶，这两种酶催化相同的化学反应，但作用于底物的不同对映体。(2)酶NonL催化二聚体非乳酸前体生成非肌动蛋白的atp依赖性分析。该过程是一个独特的例子，一个生物偶联合成，其中一个非手性分子是由两个同手性前体缩合。(3)非肌动蛋白抗性基因的酶产物NonR分析。NonR可能是一种使非肌动蛋白失活的丝氨酸酯酶。这是一个基本的抗生素耐药机制的研究。我们假设NonR和第二种可能的酯酶NonD共同作用于非肌动蛋白的循环，从而控制非肌动蛋白的合成水平。(4)研究在体内和体外系统中衍生出非肌动蛋白生物合成的基础生化后期步骤，以产生一些天然产物-合成复合杂交抗生素。将对新化合物的生物学和化学性质进行评价。

DESCRIPTION (provided by applicant): Multiple drug resistance is becoming a major threat to public health. We are facing a need for both new biologically active compounds and for new methods of making such compounds. Streptomyces produce the majority of antibiotics that are made by bacteria and study of such antibiotic biosynthesis pathways is a basic prerequisite to developing new methods of antibiotic production. The unusual polyketide, nonactin, produced by Streptomyces griseus, is an inhibitor of the P 170-glycoprotein efflux pump found in multiple drug resistant cancer cells. Nonactin is also an ionophore active against Gram positive bacteria, mycobacteria, and fungi. Nonactin is an achiral compound; a tetramer made up from both enantiomers of a precursor nonactic acid. The organism has two mirror image biosynthesis pathways, one for each of the precursor enantiomers. Of particular interest are the late stages of nonactin biosynthesis wherein the complete macrocycle is assembled l as these steps can be manipulated to generate natural product- synthetic compound hybrid analogs of nonactin. The fundamental and applied biochemistry of the highly unusual nonactin biosynthesis system will be studied by: (1) Analysis of the (+)- and (-)-nonactate synthase enzymes, two enzymes that catalyze the same chemical reaction, yet upon different enantiomers of the substrate. (2) Analysis of the ATP-dependent formation of nonactin from dimeric nonactate precursors catalyzed by the enzyme NonL. The process is a unique example of a biological Coupe du Roi synthesis where an achiral molecule is made by the condensation of two homochiral precursors. (3) Analysis of NonR, the enzyme product of the nonactin resistance gene. NonR is likely a serine esterase that renders nonactin inactive. This is a study of a fundamental antibiotic resistance mechanism. We hypothesize that NonR, and a second likely esterase NonD, act together to recycle nonactin and thereby control nonactin synthesis levels. (4) Study of in vivo and in vitro systems derived from the basic biochemistry of the late steps of nonactin biosynthesis to produce a number of natural product-synthetic compound hybrid antibiotics. The biological and chemical properties of the new compounds will be evaluated.