Mechanism of Inactivation of Ornithine Aminotransferase by (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)cyclopentane-1-carboxylic Acid

Abstract

The inhibition of ornithine aminotransferase (OAT), a pyridoxal 5′-phosphate-dependent enzyme, has been implicated as a treatment for hepatocellular carcinoma (HCC), the most common form of liver cancer, for which there is no effective treatment. From a previous evaluation of our aminotransferase inhibitors, (1S,3S)-3-amino-4-(perfluoropropan-2-ylidene)cyclopentane-1carboxylic acid hydrochloride (1) was found to be a selective and potent inactivator of human OAT (hOAT), which inhibited the growth of HCC in athymic mice implanted with human-derived HCC, even at a dose of 0.1 mg/kg. Currently, investigational new drug (IND)-enabling studies with 1 are underway. The inactivation mechanism of 1, however, has proved to be elusive. Here we propose three possible mechanisms, based on mechanisms of known aminotransferase inactivators: Michael addition, enamine addition, and fluoride ion elimination followed by conjugate addition. On the basis of crystallography and intact protein mass spectrometry, it was determined that 1 inactivates hOAT through fluoride ion elimination to an activated 1,1′-difluoroolefin, followed by conjugate addition and hydrolysis. This result was confirmed with additional studies, including the detection of the cofactor structure by mass spectrometry and through the identification of turnover metabolites. On the basis of this inactivation mechanism and to provide further evidence for the mechanism, analogues of 1 (19, 20) were designed, synthesized, and demonstrated to have the predicted selective inactivation mechanism. These analogues highlight the importance of the trifluoromethyl group and provide a basis for future inactivator design.

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

“…The crystal structure (PDB entry 7LK1) shown in Figure A and Figure S3 indicates that PLP is covalently linked to 6 , and a covalent bond between Lys292 and 6 tethers the compound to the enzyme. The covalent bond between Lys292 and 6 represents a stable gem -diamine species that has not been observed in any previous h OAT/ligand crystals. ,,, This observation further validated the gem -diamine precursors ( M12 or M13 , Scheme ) of enamine intermediate M8 . Moreover, two alternate conformations of this intermediate were observed, which differ in the position of the carboxylate group derived from 6 .…”

“…IC 50 values were obtained using nonlinear regression analysis in GraphPad Prism 8 of a 9-point enzymatic assay with a 2-fold serial dilution against GABA-AT. The partition ratios were determined under conditions in the presence of α-KG, while fluoride ion release results were determined in the absence of α-KG. b Data were extracted from ref . c Enzyme activity remaining was measured as a function of the number of equivalents of 4 – 6 relative to enzyme concentration. Linear regression analysis was used on the linear portion of the curves to obtain the x-intercept, which was the turnover number (partition ratio = turnover number – 1).…”

“…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) …”

“…A cyclopentane-based analogue, termed BisCF 3 ( 1 , Figure B), bearing a bis­(trifluoromethyl) group as its warhead, was identified to be a selective MBI of h OAT while only showing millimolar reversible inhibition of GABA-AT. Recent mechanistic studies have revealed that one of its trifluoromethyl groups undergoes fluoride ion elimination, leading the ligand to covalently modify the catalytic Lys292 residue by conjugate addition (Scheme A). , It is considered that the sterically bulky bis­(trifluoromethyl) group may not access the more narrow pocket of GABA-AT as readily, influencing the initial binding pose between the ligand and the enzyme, which may be responsible for its reversible inhibition of this enzyme. Compound 1 has been demonstrated to be effective in vivo and is being investigated further in HCC patient-derived xenograft (PDX) models.…”

Chemical Aspects of Human and Environmental Overload with Fluorine

“…This reactive intermediate then either binds strongly to the target enzyme or forms a covalent bond with it. 77 Fluorination can be used to create mechanism-based inhibitors for two reasons. On one hand, fluorine and hydrogen are isosteric, so after the H → F exchange the new compound usually remains a substrate of the target enzyme.…”