A Remarkable Difference That One Fluorine Atom Confers on the Mechanisms of Inactivation of Human Ornithine Aminotransferase by Two Cyclohexene Analogues of γ-Aminobutyric Acid

Abstract

Human ornithine aminotransferase (hOAT), a pyridoxal 5′-phosphate-dependent enzyme, plays a critical role in the progression of hepatocellular carcinoma (HCC). Pharmacological selective inhibition of hOAT has been shown to be a potential therapeutic approach for HCC. Inspired by the discovery of the nonselective aminotransferase inactivator (1R,3S,4S)-3-amino-4-fluoro cyclopentane-1-carboxylic acid (1), in this work, we rationally designed, synthesized, and evaluated a novel series of fluorine-substituted cyclohexene analogues, thereby identifying 8 and 9 as novel selective hOAT time-dependent inhibitors. Intact protein mass spectrometry and protein crystallography demonstrated 8 and 9 as covalent inhibitors of hOAT, which exhibit two distinct inactivation mechanisms resulting from the difference of a single fluorine atom. Interestingly, they share a similar turnover mechanism, according to the mass spectrometry-based analysis of metabolites and fluoride ion release experiments. Molecular dynamics (MD) simulations and electrostatic potential (ESP) charge calculations were conducted, which elucidated the significant influence of the one-fluorine difference on the corresponding intermediates, leading to two totally different inactivation pathways. The novel additionaromatization inactivation mechanism for 9 contributes to its significantly enhanced potency, along with excellent selectivity over other aminotransferases.

Turnover and Inactivation Mechanisms for (S)-3-Amino-4,4-difluorocyclopent-1-enecarboxylic Acid, a Selective Mechanism-Based Inactivator of Human Ornithine Aminotransferase

“…On the basis of our previous mechanistic studies of other related GABA-AT/ h OAT inactivators, , we propose three potential pathways (Scheme ). Initially, transimination with 6 would form the external aldimine M1 .…”

“…It should be noted that the α-amino group of 5-FMOrn forms a strong hydrogen bond with the phenol group of Tyr55 in the h OAT crystal complex (PDB entry 2OAT). Moreover, we also observed hydrogen bonds between the carboxylate groups of 1 (PDB entry 6OIA) and 2 (PDB entry 6V8C) and Tyr55 in h OAT crystal complexes. , Among the published h OAT inactivators, only 1 demonstrates (a) promising h OAT selectivity through potent irreversible inhibition of h OAT, (b) weak, reversible inhibition of GABA-AT ( K i = 4.2 mM), and (c) no inhibition of either aspartate aminotransferase (Asp-AT) or alanine aminotransferase (Ala-AT) (up to 4 mM) …”

“…Compound 1 has been demonstrated to be effective in vivo and is being investigated further in HCC patient-derived xenograft (PDX) models. On the basis of a similar strategy, we enlarged the ring system and further developed a cyclohexene-based analogue, WZ-2-051 ( 2 , Figure B), bearing a difluoro group . Compound 2 exhibited a 23-fold improvement in inactivation efficiency (defined by the k inact / K I ratio) against h OAT compared to 1 while showing 13.3-fold selectivity over GABA-AT.…”

“…Compound 2 exhibited a 23-fold improvement in inactivation efficiency (defined by the k inact / K I ratio) against h OAT compared to 1 while showing 13.3-fold selectivity over GABA-AT. The subsequent mechanistic studies disclosed that 2 undergoes a two-step fluoride ion elimination and inactivates h OAT through an addition-aromatization mechanism (Scheme B) . An additional example of a selective h OAT inactivator is 5-fluoromethylornithine (5-FMOrn, 3 ) inspired by the structure of h OAT substrate l -Orn and related to the structure of nonselective GABA-AT inactivator ( S )-4-amino-5-fluoropentanoic acid (AFPA) .…”

Enzymatic synthesis of fluorinated compounds

“…Hence, many fluorinated compounds have been developed as novel antitumor inhibitors, considering the problems of drug resistance, solubility, and toxicity. For example, diverse fluorinated cyclohexene derivatives were used to inhibit the activity of hOAT to treat HCC, because human ornithine aminotransferase (hOAT) plays an essential role in metabolic pathways of hepatocellular carcinoma (HCC) (Zhu et al 2020b ). Especially (1 R ,3 S ,4 S )-3-amino-4,4-difluoro cyclopentane-1-ene-1-carboxylate and (1 R ,3 S ,4 S )-3-amino-4-fluoro cyclopentane-1-ene-1-carboxylate, they showed the total different inactivation mechanisms from previous inhibitors.…”