Use of engineered cytochromes P450 for accelerating drug discovery and development

Abstract

Numerous steps in drug development, including the generation of authentic metabolites and late-stage functionalization of candidates, necessitate the modification of often complex molecules, such as natural products. While it can be challenging to make the required regio- and stereoselective alterations to a molecule using purely chemical catalysis, enzymes can introduce changes to complex molecules with a high degree of stereo- and regioselectivity. Cytochrome P450 enzymes are biocatalysts of unequalled versatility, capable of regio- and stereoselective functionalization of unactivated CH bonds by monooxygenation. Collectively they catalyze over 60 different biotransformations on structurally and functionally diverse organic molecules, including natural products, drugs, steroids, organic acids and other lipophilic molecules. This catalytic versatility and substrate range makes them likely candidates for application as potential biocatalysts for industrial chemistry. However, several aspects of the P450 catalytic cycle and other characteristics have limited their implementation to date in industry, including: their lability at elevated temperature, in the presence of solvents, and over lengthy incubation times; the typically low efficiency with which they metabolize non-natural substrates; and their lack of specificity for a single metabolic pathway. Protein engineering by rational design or directed evolution provides a way to engineer P450s for industrial use. Here we review the progress made to date toward engineering the properties of P450s, especially eukaryotic forms, for industrial application, and including the recent expansion of their catalytic repertoire to include non-natural reactions.

Keywords: Biocatalysis; CH functionalization; Drug development; Drug metabolism; Late-stage functionalization; Monooxygenase; Thermostable.