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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F04A-T (20))
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Fabian, Miles
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California Institute of Technology
Engineering (All Types)
Schools of Engineering
United States
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Smith, Matthew A; Romero, Philip A; Wu, Timothy et al. (2013) Chimeragenesis of distantly-related proteins by noncontiguous recombination. Protein Sci 22:231-8
Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K B et al. (2013) General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH. Proc Natl Acad Sci U S A 110:10946-51
Coelho, Pedro S; Brustad, Eric M; Kannan, Arvind et al. (2013) Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes. Science 339:307-10
Coelho, Pedro S; Wang, Z Jane; Ener, Maraia E et al. (2013) A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo. Nat Chem Biol 9:485-7
Liu, Xiang; Bastian, Sabine; Snow, Christopher D et al. (2012) Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol. J Biotechnol 164:188-95
Brustad, Eric M; Lelyveld, Victor S; Snow, Christopher D et al. (2012) Structure-guided directed evolution of highly selective p450-based magnetic resonance imaging sensors for dopamine and serotonin. J Mol Biol 422:245-62
Brustad, Eric M; Arnold, Frances H (2011) Optimizing non-natural protein function with directed evolution. Curr Opin Chem Biol 15:201-10
Lelyveld, Victor S; Brustad, Eric; Arnold, Frances H et al. (2011) Metal-substituted protein MRI contrast agents engineered for enhanced relaxivity and ligand sensitivity. J Am Chem Soc 133:649-51