Efficient Screening of Cytochrome P450 BM3 Mutants for Their Metabolic Activity and Diversity toward a Wide Set of Drug-Like Molecules in Chemical Space

Abstract

ABSTRACT:In the present study, the diversity of a library of drug-metabolizing bacterial cytochrome P450 (P450) BM3 mutants was evaluated by a liquid chromatography-mass spectrometry (LC-MS)-based screening method. A strategy was designed to identify a minimal set of BM3 mutants that displays differences in regio-and stereoselectivities and is suitable to metabolize a large fraction of drug chemistry space. We first screened the activities of six structurally diverse BM3 mutants toward a library of 43 marketed drugs (encompassing a wide range of human P450 phenotypes, cLogP values, charges, and molecular weights) using a rapid LC-MS method with an automated method development and data-processing system. Significant differences in metabolic activity were found for the mutants tested and based on this drug library screen; nine structurally diverse probe drugs were selected that were subsequently used to study the metabolism of a library of 14 BM3 mutants in more detail. Using this alternative screening strategy, we were able to select a minimal set of BM3 mutants with high metabolic activities and diversity with respect to substrate specificity and regiospecificity that could produce both human relevant and BM3 unique drug metabolites. This panel of four mutants (M02, MT35, MT38, and MT43) was capable of producing P450-mediated metabolites for 41 of the 43 drugs tested while metabolizing 77% of the drugs by more than 20%. We observed this as the first step in our approach to use of bacterial P450 enzymes as general reagents for lead diversification in the drug development process and the biosynthesis of drug(-like) metabolites.

CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

“…This progress can directly be of use for efficient and timesaving production of human drug metabolites. High yields and conversion rates can already be achieved by using corresponding human (Rushmore et al, 2000;Vail et al, 2005;Schroer et al, 2010;Geier et al, 2012;Schiffer et al, 2015) or suitable nonhuman (Taylor et al, 1999;Otey et al, 2006;Sawayama et al, 2009;Reinen et al, 2011;Di Nardo and Gilardi, 2012;Kiss et al, 2015;Ren et al, 2015) P450 enzymes in a whole-cell system to produce respective metabolites. The majority of published bacterial P450 enzymes used for the conversion of drugs are mutants of CYP102A1 (BM3) from Bacillus megaterium.…”

Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs

“…Positions which were selected for mutagenesis were residues 72, 74, 82, 87 and 437 which appear to be key active site residues and have profound influence on regio-and stereoselectivity of P450 BM3 mutants. 20,22,28,29 …”

“…18 Recent studies have reported the ability of cytochrome P450 BM3 mutants to produce human relevant drug metabolites of several drugs including NSAIDs. [19][20][21] Engineered P450 BM3 mediated hydroxylation of NSAIDs like diclofenac, ibuprofen and naproxen have been reported. [22][23][24][25] In the present study, fenamic acid NSAIDs (Figure 1) have been used as examples to demonstrate the ability of P450 BM3 mutants to produce human relevant metabolites.…”

Subtle structural changes in the Asp251Gly/Gln307His P450 BM3 mutant responsible for new activity toward diclofenac, tolbutamide and ibuprofen

“…The direct use of human cytochrome P450 is limited by the need of a redox partner and the poor stability and activity. However, bacterial counterparts such as cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium, can be directly used [3] or engineered to metabolise drugs and to produce the same metabolites as the human enzymes [4][5][6][7][8][9][10][11][12][13][14]. This protein is a selfsufficient fatty acids monoxygenase containing a NADPH-dependent reductase (BMR) and a P450 catalytic domain (BMP) fused in a single polypeptidic chain [15,16].…”