Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor.

Semi-rational enzyme engineering is a powerful method to develop industrial biocatalysts. Profiting from advances in molecular biology and bioinformatics, semi-rational approaches can effectively accelerate enzyme engineering campaigns. Here, we present the optimization of a ketoreductase from Sporidiobolus salmonicolor for the chemo-enzymatic synthesis of ipatasertib, a potent protein kinase B inhibitor. Harnessing the power of mutational scanning and structure-guided rational design, we created a 10-amino acid substituted variant exhibiting a 64-fold higher apparent kcat and improved robustness under process conditions compared to the wild-type enzyme. In addition, the benefit of algorithm-aided enzyme engineering was studied to derive correlations in protein sequence-function data, and it was found that the applied Gaussian processes allowed us to reduce enzyme library size. The final scalable and high performing biocatalytic process yielded the alcohol intermediate with ≥ 98% conversion and a diastereomeric excess of 99.7% (R,R-trans) from 100 g L-1 ketone after 30 h. Modelling and kinetic studies shed light on the mechanistic factors governing the improved reaction outcome, with mutations T134V, A238K, M242W and Q245S exerting the most beneficial effect on reduction activity towards the target ketone.

Mass Balance of the Indoleamine 2,3-Dioxygenase Inhibitor Navoximod (GDC-0919) in Rats and Dogs: Unexpected Cyanide Release from Imidazo[5,1-a]isoindole and Species Differences in Glucuronidation.

Navoximod (GDC-0919) is a small molecule inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1) developed to reduce T cell immunosuppression associated with cancer. This study describes the absorption, metabolism, and excretion (AME) of navoximod in rats and dogs after a single oral dose of [14C]-navoximod. An unexpected thiocyanate metabolite M1 and a chiral inversion metabolite M51 were captured as the major circulating metabolites in rats, accounting for 30% and 18% of 0-24 hours exposure, respectively. These two metabolites combined had much lower systemic exposure in dogs and humans (<6% and <1%). The novel cyanide release is proposed to occur via 4,5-epoxidation on the fused imidazole ring, leading to ring opening and rearrangement along with the release of cyanide. The decyanated metabolites were identified and confirmed by synthetic standards, which supported the proposed mechanism. In dogs, glucuronidation to M19 was the major clearance mechanism, representing 59% of the dose in the bile of bile duct-cannulated (BDC) dogs and 19% of the dose in the urine of intact dogs. Additionally, M19 also represented 52% of drug related exposure in circulation in dogs. In comparison, in humans, navoximod was mainly cleared through glucuronidation to M28 and excreted in urine (60% of the dose). The differences in the metabolism and elimination observed in vivo were qualitatively recapitulated in vitro with liver microsomes, suspended hepatocytes, and cocultured primary hepatocytes. The striking species differences in regioselective glucuronidation is likely explained by the species differences in UGT1A9, which was mainly responsible for M28 formation in humans. SIGNIFICANCE STATEMENT: The results from this study demonstrated significant species differences in metabolism (especially glucuronidation) and elimination of navoximod among rats, dogs, and humans. The study also illustrated the mechanism of a novel cyanide release metabolism from the fused imidazo[5,1-a]isoindole ring. Such biotransformation should be kept in mind when working with imidazole-containing new chemical entities in drug discovery and development.