Human Metabolites of Bosentan Produced by Enzymes in Hypha’s PolyCYPs Cytochrome P450 Kit

Presented at: ISSX meeting, 2017, Providence, RI, USA

A cell-free kit of cytochrome P450 enzymes and ferredoxin / ferredoxin reductase redox partners, termed PolyCYPs, is under development for generating scalable quantities of oxidised metabolites. Cytochrome P450s in the kit have been derived from Hypha’s talented biotransforming actinomycetes and are capable of generating human and other mammalian metabolites of drug compounds. The catalytic abilities of two P450 enzymes in the kit, which have been cloned from two different actinomycete species into E. coli, are illustrated using bosentan.

Bosentan has one major active metabolite (Ro 48-5033), formed by hydroxylation at the t-butyl position and two other minor metabolites (Ro 47-8634 and Ro 64-1056) produced via O-demethylation of bosentan and Ro 48-5033. Recombinant enzymes PolyCYP 6.1 and PolyCYPTM14.1 were each able to produce one of the reported human metabolites. Furthermore, the third minor metabolite could be produced through use of a combination of PolyCYP 6.1 and PolyCYP 14.1. As well as utility for providing sufficient material for MetID, reactions can be scaled to produce milligram to gram quantities of metabolites and novel derivatives for further evaluation.