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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 STATEMENTThe 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.

Controlled enzymatic synthesis is an alluring alternative to solid‐phase synthesis and polymerase‐mediated incorporation of nucleotides for the crafting of chemically modified, therapeutic oligonucleotides. While this approach has met some success for the elaboration of long, unmodified DNA sequences, very little research efforts have been dedicated to xeno nucleic acids (XNAs). Here, we have evaluated the possibility of using various 3’‐O‐blocking groups for controlled synthesis of locked nucleic acids (LNA). LNA nucleosides were equipped with protecting groups used in synthetic organic chemistry and were evaluated for their stability. The most promising candidates, benzoyl‐ and pivaloyl‐protected nucleosides, were converted to the corresponding nucleotides. The resulting modified nucleotides were shown to be accepted by various polymerases. While single incorporation events were observed in high yields, strong esterase activity of polymerases represents a lasting hurdle that needs to be overcome. Overall, this article represents an additional step towards the controlled enzymatic synthesis of LNA‐containing oligonucleotides and could be extended to other sugar or nucleobase modified nucleotides. LNA nucleotidesequipped with different 3’‐O‐blocking groups were evaluated for their compatibility with controlled enzymatic synthesis. Benzoyl‐ and pivaloyl‐protected LNA nucleotides were readily accepted by polymerases.

The potential of biotechnology by means of biocatalysis or biosynthesis in organic synthesis is far from being fully exploited. For this reason a group of life science companies active in pharmaceuticals, flavour and fragrance, vitamin and fine chemicals businesses describe some examples of the use of enzymes in industrial organic synthesis and discuss why enzymes are still the exception rather than the rule in organic synthesis.

A 44-kDa c-type cytochrome was purified from membranes of Rhodobacter capsulatus. The cytochrome contained an ascorbate-reducible haem (α-max 556 nm) and a dithionite-reducible haem (α-max 552 nm) and had catalytic properties which indicated that it was a cytochrome c peroxidase. Increased levels of this cytochrome were observed when dimethylsulphoxide was present during phototrophic growth of cells but this was not observed when trimethylamine-N-oxide was present. The results indicate that the 44-kDa cytochrome is probably not directly involved in the dimethylsulphoxide/trimethylamine-N-oxide respiratory pathway of Rhodobacter capsulatus.

Arthrobacter globiformis amine oxidase produced by Escherichia coli cells grown in copper-depleted media was reported to undergo activation due to formation of its topaquinone cofactor in a copper-dependent autocatalytic reaction. Likewise, a mutated E. coli amine oxidase located in the cytoplasm was reported to form topaquinone autocatalytically in an EDTA-sensitive reaction. Here we show unequivocally that formation of an amine oxidase lacking topaquinone is primarily a consequence of the location of the enzyme in the cytoplasm rather than the level of copper in the growth medium. For E. coli, insertion of copper into apoamine oxidase and subsequent topaquinone formation occur after export of the apoenzyme into the periplasm.