Scientific posters

PolyCYP6, a functional bacterial Class I cytochrome P450, was successfully mined from one of Hypha’s actinomycete strains and converts the non-steroidal anti-inflammatory drug diclofenac into its hydroxylated human metabolites. Improvement of the PolyCYP cytochrome P450 cell factory for efficient whole-cell biotransformation was achieved through co-expression with specific redox partners and changing components of the T7 expression plasmid. The highest yielding recombinant strain expressed PolyCYP6 with the P. putida reductase system. An ampicillin resistant marker & high copy vector also further improved yields of hydroxylated products.

The first-in-class JAK inhibitor, ruxolitinib (trade names Jakafi® and Jakavi®), is primarily metabolized to a complex mixture of stereoisomeric cyclopentyl hydroxyl and keto metabolites. Application of Hypha’s microbial-based biocatalytic C-H bond activation to ruxolitinib resulted in the production of an array of hydroxylated and further oxidized keto metabolites, many of which corresponded to circulating human metabolites. All possible oxidized isomers of the aliphatic cyclopentyl moiety were derived from a variety of microbial species which were readily scaled up, enabling efficient production of stereoisomer metabolite standards for structural characterization and bioanalytical monitoring.

Metabolism of drugs often results in the formation of major circulating metabolites derived from mixed clearance pathways, and can include both primary and secondary metabolites. Human metabolism of Incyte’s investigational new drug epacadostat (EPA) forms 3 major circulating plasma metabolites (Boer et al., 2016). Glucuronidation of EPA to form M9 is the dominant metabolic pathway, in conjunction with formation of an amidine M11 and an N-dealkylated metabolite, M12. Boer and co-workers showed that reductive metabolism by gut microbiota results in M11, which is then absorbed and further modified by CYP enzymes to form the secondary metabolite M12.

Introducing chemical diversity into a drug candidate late in the optimisation process has several applications including exploration of SAR (structure-activity relationships). Biocatalysis can provide access to chemical space in a complementary manner to chemical synthesis, thereby broadening coverage of the SAR map to better understand how small changes in the molecular structure affect biological potency.

A cell-free kit of cytochrome P450 enzymes and ferredoxin/ferredoxin reductase redox partners, termed PolyCYPs^®, is being developed for generating scalable quantities of oxidised metabolites. P450 cytochromes in the kit have been derived from some of Hypha’s most talented biotransforming bacteria and are capable of generating human and other mammalian metabolites of drug compounds.

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.