Evolution of cytochrome p450s for the diversification of drug scaffolds

细胞色素 p450 的进化使药物支架多样化

基本信息

批准号：
8214361
负责人：
Eric Michael Brustad
金额：
$ 2.57万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-02-01 至 2011-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8214361
关键词：
Antibiotic Resistance Arabinose Bacillus (bacterium)Benzodiazepines Beryllium Binding Biological Cell Survival Chemicals Cleaved cell Couples Cytochrome P450 Cytochromes Development Dihydropyridines Disadvantaged Dissociation Elements Engineering Enzymes Evolution Family General Practitioners Genes Genetic Transcription Growth Health Hydrogen Bonding Hydroxylation Lead Libraries Ligands Link Location Methodology Methods Mixed Function Oxygenases Molecular Nature Organic Chemistry Pharmaceutical Preparations Procedures Process Property Proteins Protocols documentation Reaction Resolution Screening procedure Site Solutions Specificity Structure System Techniques Therapeutic Time Transition Elements catalyst cost design dihydropyridine directed evolution drug discovery high throughput screening improved interest molecular recognition mutant novel oxidation promoter research study response scaffold small molecule

项目摘要

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.

描述（由申请人提供）：选择性 C-H 活化仍然是有机合成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，不仅在一般合成方法方面，而且特别是在药物发现中的先导多样化方面。本提案中提出的实验旨在利用酶系统提供的非凡特异性来选择性氧化具有治疗意义的分子。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设计，以产生一组催化剂，这些催化剂可对三种特殊药物支架进行位点选择性羟基化：苯二氮卓类、芳基吲哚类和二氢吡啶类。然后将筛选这些进化的酶在每个支架家族中的混杂活性，以检查它们作为相似结构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) 该提案还将引入新的方法，将 p450 芳基羟化酶活性的进化与选择性标记的表达结合起来，从而使可以筛选的突变蛋白的数量大幅增加（从 103-104 到 >109）。该方法引入了用于检查蛋白质活性的生产者策略。转录的小分子诱导剂将通过接头连接到感兴趣的 p450 底物，该接头响应芳基羟基化而选择性裂解。然后释放的诱导分子可以激活抗生素抗性基因的表达，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。该方法将用于开发使特权药物支架的芳环羟基化的酶。公共卫生相关性：药物结构的化学多样化对于不断寻找具有治疗意义的分子至关重要。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数量以及创造具有潜在有趣的生物特性的新分子所需的成本。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Optimizing non-natural protein function with directed evolution.

通过定向进化优化非天然蛋白质功能。

DOI：
10.1016/j.cbpa.2010.11.020
发表时间：
2011-04
期刊：
Current opinion in chemical biology
影响因子：
7.8
作者：
Brustad EM;Arnold FH
通讯作者：
Arnold FH

Chimeragenesis of distantly-related proteins by noncontiguous recombination.

通过非连续重组实现远缘相关蛋白质的嵌合。

DOI：
发表时间：
2013-02
期刊：
影响因子：
0
作者：
Smith, Matthew A;Romero, Philip A;Wu, Timothy;Brustad, Eric M;Arnold, Frances H
通讯作者：
Arnold, Frances H

General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH.

将酮醇酸还原异构酶辅因子依赖性从 NADPH 逆转为 NADH 的一般方法。

DOI：
发表时间：
2013-07-02
期刊：
影响因子：
11.1
作者：
Brinkmann;Flock, Tilman;Cahn, Jackson K B;Snow, Christopher D;Brustad, Eric M;McIntosh, John A;Meinhold, Peter;Zhang, Liang;Arnold, Frances H
通讯作者：
Arnold, Frances H

Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol.

乳酸乳球菌乙醇脱氢酶 LlAdhA 的结构引导工程可提高异丁醛向异丁醇的转化。

DOI：
10.1016/j.jbiotec.2012.08.008
发表时间：
2012-12-15
期刊：
Journal of biotechnology
影响因子：
4.1
作者：
Liu, Xiang;Bastian, Sabine;Snow, Christopher D.;Brustad, Eric M.;Saleski, Tatyana E.;Xu, Jian-He;Meinhold, Peter;Arnold, Frances H.
通讯作者：
Arnold, Frances H.

A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo.

丝氨酸取代的 P450 在体内高效催化卡宾转移至烯烃。

DOI：
发表时间：
2013-08
期刊：
影响因子：
14.8
作者：
Coelho, Pedro S;Wang, Z Jane;Ener, Maraia E;Baril, Stefanie A;Kannan, Arvind;Arnold, Frances H;Brustad, Eric M
通讯作者：
Brustad, Eric M