Evolution of cytochrome p450s for the diversification of drug scaffolds

描述（由申请人提供）：选择性C-H活化仍然是合成有机化学中的巨大挑战之一。未活化的烷基和芳基C-H键的特定官能化将具有巨大的益处，不仅在一般的合成方法方面，而且特别是在药物发现的多样化方面。本提案中提出的实验旨在利用酶系统对具有治疗意义的分子的选择性氧化提供的非凡特异性。具体目标：1）来自巨大芽孢杆菌的细菌细胞色素p450 BM-3将使用定向进化进行工程改造，以产生一组催化剂，其位点选择性地羟基化三种特权药物支架：苯并二氮杂卓，芳基吲哚和二氢吡啶。然后，这些进化的酶将在每个支架家族中筛选混杂活性，以检查它们作为具有相似结构的化合物多样化的通用催化剂的能力，从而促进新药的开发。2)该提议还将引入新的方法来将p450芳基-羟化酶活性的进化与选择标记的表达偶联，从而允许可以筛选的突变蛋白的数量显著增加（从103-104增加到>109）。该方法引入了用于检查蛋白质活性的前诱导剂策略。小分子转录诱导剂将通过响应芳基羟基化而选择性裂解的接头连接至感兴趣的p450底物。然后释放的诱导物分子可以激活抗生素抗性基因的表达，使得可以通过在选择性培养基上生长来选择活性p450突变体。这种方法将被用来开发酶，羟基化的特权药物支架的芳基环。公共卫生相关性：药物样结构的化学多样化在寻找治疗感兴趣的分子的持续努力中是必不可少的。该提案旨在通过开发促进新药合成的催化剂来提高这一过程的效率。这些催化剂应该减少步骤的数量以及产生具有潜在感兴趣的生物特性的新分子所需的成本。

DESCRIPTION (provided by applicant): Selective C-H activation remains one of the great challenges in synthetic organic chemistry. The specific functionalization of unactivated alkyl and aryl C-H bonds would be of enormous benefit, not only in terms of general synthetic methodology but particularly with regard to lead diversification in drug discovery. The experiments presented in this proposal are designed to exploit the extraordinary specificity provided by enzymatic systems towards the selective oxidation of molecules with therapeutic interest. Specific Aims: 1) The bacterial cytochrome p450 BM-3 from Bacillus megaterum will be engineered using directed evolution to generate a panel of catalysts that site-selectively hydroxylate three privileged drug scaffolds: the benzodiazepines, the arylindoles, and the dihydropyridines. These evolved enzymes will then be screened for promiscuous activity within each scaffold family to examine their ability to act as generalist catalysts for the diversification of compounds of similar structure and thus facilitate the development of new drugs. 2) This proposal will also introduce novel methodology to couple the evolution of p450 aryl-hydroxylase activity to the expression of a selectable marker allowing the number of mutant proteins that can be screened to be increased substantially (from 103-104 to >109). This methodology introduces a proinducer strategy for examining protein activity. A small molecule inducer of transcription will be linked to a p450 substrate of interest through a linker that is selectively cleaved in response to aryl hydroxylation. The released inducer molecule can then activate the expression of an antibiotic resistance gene such that active p450 mutants can be selected for by growth on selective media. This methodology will be exploited to develop enzymes that hydroxylate aryl rings of privileged drug scaffolds. PUBLIC HEALTH RELEVANCE: The chemical diversification of drug like structures is essential in the ongoing effort to find molecules of therapeutic interest. This proposal aims to improve the efficiency of this process by developing catalysts that facilitate the synthesis of new drugs. These catalysts should decrease the number of steps as well as the cost required to create new molecules with potentially interesting biological properties.