Evolution of cytochrome p450s for the diversification of drug scaffolds

描述（申请人提供）：选择性碳氢化合物活化仍然是合成有机化学的巨大挑战之一。非活化的烷基和芳基C-H键的特定功能化将是巨大的好处，不仅在一般的合成方法，特别是在药物发现的铅多样化方面。在本建议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提供的非凡特异性。具体目标：1)将利用定向进化技术对来自巨芽孢杆菌的细菌细胞色素p450 BM-3进行工程改造，以产生一组催化剂，该催化剂可选择性地对三种特殊药物支架进行羟基化：苯二氮卓类药物、芳基吡啶类药物和二氢吡啶类药物。然后，这些进化的酶将在每个支架家族中筛选混杂活性，以检查它们作为类似结构化合物多样化的多面手催化剂的能力，从而促进新药的开发。2)该提案还将引入新的方法，将p450芳基羟化酶活性的进化与可选择标记的表达结合起来，从而使可筛选的突变蛋白数量大幅增加（从103-104增加到> - 109）。该方法引入了一种用于检测蛋白质活性的产原体策略。转录的小分子诱导剂将通过一个连接体连接到感兴趣的p450底物，该连接体在芳基羟基化反应中被选择性地切割。然后释放的诱导剂分子可以激活抗生素抗性基因的表达，这样可以通过在选择性培养基上生长来选择活性p450突变体。这种方法将被用于开发酶羟基化芳基环的特权药物支架。公共卫生相关性：药物样结构的化学多样化在寻找治疗兴趣分子的持续努力中至关重要。本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。这些催化剂将减少步骤的数量，以及创造具有潜在有趣生物学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.