Evolution of cytochrome p450s for the diversification of drug scaffolds

描述(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键的特定官能化不仅在一般合成方法方面，而且在药物发现中的领导多样化方面，都将是巨大的好处。本提案中提出的实验旨在利用酶系统对具有治疗兴趣的分子的选择性氧化所提供的非凡特异性。具体目标：1)来自巨大芽孢杆菌的细菌细胞色素P450 BM-3将通过定向进化来产生一组催化剂，这些催化剂可以选择性地对三种有特权的药物支架进行羟基化：苯二氮卓、芳基吲哚和二氢吡啶。然后，将对这些进化的酶在每个支架家族中的混杂活性进行筛选，以检查它们作为通用催化剂的能力，以使结构相似的化合物多样化，从而促进新药的开发。2)这项建议还将引入新的方法，将P450芳基羟基酶活性的进化与可选择标记的表达相结合，从而使可筛选的突变蛋白的数量大幅增加(从103-104增加到109)。这种方法引入了一种检测蛋白质活性的诱导剂策略。一个小分子转录诱导剂将通过一个连接体连接到感兴趣的P450底物上，该连接体在芳基羟化反应中被选择性地切割。释放的诱导剂分子然后可以激活抗生素耐药性基因的表达，从而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羟基化药物支架的芳香环的酶。公共卫生相关性：类药物结构的化学多样化对于正在进行的寻找治疗感兴趣的分子的努力至关重要。这项提议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。这些催化剂应该会减少产生具有潜在生物学特性的新分子所需的步骤数量和成本。

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.