P34 HUMAN METABOLITES OF BOSENTAN PRODUCED BY ENZYMES IN HYPHA’S POLYCYPSTM CYTOCHROME P450 KIT.

Jonathan Steele , Hypha Discovery Ltd, Uxbridge, United Kingdom
Antonio de Riso , Hypha Discovery Ltd, Uxbridge, United Kingdom
Headley Williams , Hypha Discovery Ltd, Uxbridge, United Kingdom
Richard Phipps , Hypha Discovery Ltd, Uxbridge, United Kingdom
Silvia Bardoni , Hypha Discovery Ltd, Uxbridge, United Kingdom
Chris Drake , Hypha Discovery Ltd, Uxbridge, United Kingdom
Stephen Wrigley , Hypha Discovery Ltd, Uxbridge, United Kingdom
Julia Shanu-Wilson , Hypha Discovery Ltd, Uxbridge, United Kingdom
Frank Scheffler , Hypha Discovery Ltd, Uxbridge, United Kingdom
Sebastian Schulz , University College London, London, United Kingdom
John Ward , University College London, London, United Kingdom

 

A cell-free kit of cytochrome P450 enzymes and ferredoxin / ferredoxin reductase redox partners, termed PolyCYPsTM, is under development for generating scalable quantities of oxidised metabolites. Cytochrome P450s in the kit have been derived from some of 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, is illustrated using bosentan. Bosentan is an endothelin receptor antagonist used for treatment of pulmonary artery hypertension. It has one major active metabolite (Ro 48-5033) which is formed by hydroxylation at the t-butyl position1, and which contributes 10-20% of total activity of the parent2. Two other minor metabolites (Ro 47-8634 and Ro 64-1056) are produced via O-demethylation of bosentan and Ro 48-50331. Recombinant enzymes PolyCYPTM6.1 and PolyCYPTM14.1 were each able to produce one of the reported human metabolites. Furthermore, the third minor metabolite was produced through use of a combination of PolyCYPTM6.1 and PolyCYPTM14.1. 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.

 

1Weber et al., 1999. Absorption, excretion and metabolism of the endothelin receptor antagonist bosentan in healthy male subjects. DMD 27(7), 810-815.

 

2Tracleer® (bosentan) product information. Actelion Pharmaceuticals US, Inc. October 2016.

http://www.tracleer.com/assets/PDFs/Tracleer_Full_Prescribing_Information.pdf