Remarkable and Unexpected Mechanism for (S)-3-Amino-4-(difluoromethylenyl)cyclohex-1-ene-1-carboxylic Acid as a Selective Inactivator of Human Ornithine Aminotransferase

Abstract

Human ornithine aminotransferase (hOAT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that was recently found to play an important role in the metabolic reprogramming of hepatocellular carcinoma (HCC) via the proline and glutamine metabolic pathways. The selective inhibition of hOAT by compound 10 exhibited potent in vivo antitumor activity. Inspired by the discovery of the aminotransferase inactivator (1S,3S)-3-amino-4-(difluoromethylene)­cyclopentane-1-carboxylic acid (5), we rationally designed, synthesized, and evaluated a series of six-membered-ring analogs. Among them, 14 was identified as a new selective hOAT inactivator, which demonstrated a potency 22× greater than that of 10. Three different types of protein mass spectrometry approaches and two crystallographic approaches were employed to identify the structure of hOAT-14 and the formation of a remarkable final adduct (32′) in the active site. These spectral studies reveal an enzyme complex heretofore not observed in a PLP-dependent enzyme, which has covalent bonds to two nearby residues. Crystal soaking experiments and molecular dynamics simulations were carried out to identify the structure of the active-site intermediate 27′ and elucidate the order of the two covalent bonds that formed, leading to 32′. The initial covalent reaction of the activated warhead occurs with *Thr322 from the second subunit, followed by a subsequent nucleophilic attack by the catalytic residue Lys292. The turnover mechanism of 14 by hOAT was supported by a mass spectrometric analysis of metabolites and fluoride ion release experiments. This novel mechanism for hOAT with 14 will contribute to the further rational design of selective inactivators and an understanding of potential inactivation mechanisms by aminotransferases.

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

“…The monofluorine analogue ( 6a ) inactivated GABA-AT via a similar pathway but resulted in the formation of a tight-binding aldehyde adduct, while the nonfluorine analogue ( 6b ) failed to serve as an MBI of GABA-AT (Figure ). The introduction of a double bond ( 7 , OV329 , Figure ) maintained the inactivation mechanism but greatly enhanced the potency, potentially as a result of the reduced acidity of the γ-proton. , Because of the structural similarity between these two aminotransferases, the aforementioned analogues also inactivated hOAT. , To improve the potency and selectivity toward hOAT over GABA-AT, six-membered ring analogue 8 (Figure ) was designed and synthesized, taking into account the relatively more flexible and larger active site of hOAT. Interestingly, analogue 8 was found to form a covalent adduct with attachments to both nearby Lys292 and *Thr322 (from the other subunit of a biological homodimer) in the catalytic pocket of hOAT, as indicated by cocrystal X-ray structures and protein mass spectra (MS) .…”

Determination of the pH dependence, substrate specificity, and turnovers of alternative substrates for human ornithine aminotransferase

“…Despite the fact that hOAT is mainly characterized as a δ-aminotransferase, several recent publications have shown that the enzyme is capable of reacting with cyclized ligands in which the amino group was located in γ position (28)(29)(30)(31). pH studies and alternative turnovers of hOAT Moreover, it was demonstrated that L-glutamate with a single α-amino group could also serve as a substrate in the first half-reaction (32).…”

Inactivators of Ornithine Aminotransferase for the Treatment of Hepatocellular Carcinoma

“…(B) X-ray cocrystal structure of double covalently bonded 23 ( 52 ) with Thr322 and Lys292 in OAT; PDB code: 7LON. Figure reproduced from ref . Copyright 2021 American Chemical Society.…”

“…A chimera of 22 and OV329 ( 25 ) resulted in 23 , which was expected, if it were an inactivator, to have an inactivation mechanism similar to that for 22 or 25 . Compound 23 is 22 times more efficient as an inactivator of OAT than 5 and comparable to 22 ; however, another completely different inactivation mechanism was revealed . On the basis of native mass spectrometry (to preserve noncovalent substrate binding and protein quaternary structure), intact protein mass spectrometry (to identify covalent adducts), top-down tandem mass spectrometry (to fragment the intact protein), protein crystallography, and metabolomics, the most plausible mechanisms for turnover and inactivation are shown in Scheme .…”

“…Compound 23 is 22 times more efficient as an inactivator of OAT than 5 and comparable to 22; however, another completely different inactivation mechanism was revealed. 63 On the basis of native mass spectrometry (to preserve noncovalent substrate binding and protein quaternary structure), intact protein mass spectrometry (to identify covalent adducts), top-down tandem mass spectrometry (to fragment the intact protein), protein crystallography, and metabolomics, the most plausible mechanisms for turnover and inactivation are shown in Scheme 6. The methods used to identify each of the intermediates and final adducts are given next to the structures.…”