Rational Design, Synthesis, and Mechanism of (3S,4R)-3-Amino-4-(difluoromethyl)cyclopent-1-ene-1-carboxylic Acid: Employing a Second-Deprotonation Strategy for Selectivity of Human Ornithine Aminotransferase over GABA Aminotransferase

Abstract

Human ornithine aminotransferase (hOAT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that contains a similar active site to that of γ-aminobutyric acid aminotransferase (GABA-AT). Recently, pharmacological inhibition of hOAT was recognized as a potential therapeutic approach for hepatocellular carcinoma. In this work, we first studied the inactivation mechanisms of hOAT by two well-known GABA-AT inactivators (CPP-115 and OV329). Inspired by the inactivation mechanistic difference between these two aminotransferases, a series of analogues were designed and synthesized, leading to the discovery of analogue 10b as a highly selective and potent hOAT inhibitor. Intact protein mass spectrometry, protein crystallography, and dialysis experiments indicated that 10b was converted to an irreversible tight-binding adduct (34) in the active site of hOAT, as was the unsaturated analogue (11). The comparison of kinetic studies between 10b and 11 suggested that the active intermediate (17b) was only generated in hOAT and not in GABA-AT. Molecular docking studies and pK a computational calculations highlighted the importance of chirality and the endocyclic double bond for inhibitory activity. The turnover mechanism of 10b was supported by mass spectrometric analysis of dissociable products and fluoride ion release experiments. Notably, the stopped-flow experiments were highly consistent with the proposed mechanism, suggesting a relatively slow hydrolysis rate for hOAT. The novel second-deprotonation mechanism of 10b contributes to its high potency and significantly enhanced selectivity for hOAT inhibition.

Structural and Mechanistic Basis for the Inactivation of Human Ornithine Aminotransferase by (3S,4S)-3-Amino-4-fluorocyclopentenecarboxylic Acid

“…The incorporation of a double bond into a cyclopentane system has significantly improved the inactivation efficiency of our recent MBIs by enhancing the reactivity of the key C γ hydrogen and providing conformational changes [ 26 , 43 , 44 , 45 ]. In this work, we carried out an integrated mechanistic study with h OAT and 5 , demonstrating that the addition of a double bond also influences the inactivation mechanism pathways.…”

Ornithine aminotransferase and carbamoyl phosphate synthetase 1 involved in ammonia metabolism serve as novel targets for early stages of gastric cancer

“…Silverman et al 14 have recently synthesized OAT inactivators to inhibit the activity of OAT and lead to downregulation of l ‐Gln and growth inhibition of hepatocellular carcinoma through targeting the Wnt/β‐catenin pathway, which suggests that OAT was an important chemotherapy target for treatment of cancer. Zhu et al 15 recently showed that both human OAT and γ‐aminobutyric acid aminotransferase have a similar active site. They used two inhibitors of γ‐aminobutyric acid aminotransferase, CPP‐115 and OV329, to inhibit the activity of OAT by synthesizing a series of analogues.…”

Horner–Wadsworth–Emmons reaction as an excellent tool in the synthesis of fluoro-containing biologically important compounds

“…Among others, the HWE reaction between lithiated fluoro(phenylsulfonyl)methylphosphonate 9′ and a ketone 184 in THF was used in the first step of synthesis to afford 185 -( Z ) isomer exclusively (Scheme 35). 124…”

“…Among others, the HWE reaction between lithiated fluoro ( phenylsulfonyl)methylphosphonate 9′ and a ketone 184 in THF was used in the first step of synthesis to afford 185-(Z) isomer exclusively (Scheme 35). 124 The resulting building block 185 was then transformed into derivatives 186 and 187. The authors obtained also other fluorinated analogues 188, 189 (Fig.…”

“…This compound proved to be very good tight-binding, irreversible inhibitor of hOAT (K i = 0.022 ± 0.004 mM) with weak reversible inhibitory activity against other human aminotransferases such as: Asp-AT, Ala-AT, and GABA-AT. 124 Xu obtained a series of compounds containing vinyl sulfone or sulfoxide moieties as anti-tubulin polymerization inhibitors targeting the colchicine binding site on tubulin. The major modification involved replacement of the α,β-unsaturated ketone moiety of well-known anti-tubulin chalcones 125 with a vinyl sulfone system.…”