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.

Figure 1.

Structures of analogs 9–15

Figure 2.

Deconvoluted intact protein mass spectra for hOAT samples. (A) Unmodified hOAT. (B) hOAT-14 analyzed at 25 °C (left); hOAT-14 analyzed at 55 °C (right). (C) The ratio of monomer and dimer in samples as a percent of the total protein signal. (D) Expanded view of unmodified hOAT (red) and hOAT-14 at 25 °C from 92–93 kDa. (E) Expanded view of unmodified hOAT (red) and hOAT-14 at 55 °C from 46–46.6 kDa.

Figure 3.

Localization of covalent modifications on hOAT. (A) Proposed structures and masses corresponding to hOAT adducts. HCD fragmentation maps for (B) unmodified hOAT, (C) hOAT-11b, and (D) hOAT-14, with associated scoring metric and modified residues colored for each adduct mass. Amino acid residues are numbered according to the full-length protein sequence (Uniprot accession: P04181) used in crystallography studies and not to the recombinantly expressed version of hOAT. For clarity, amino acid cleavage sites generating y199, y140, and y101 are shown in panel (B).

Figure 4.

Omit map (Fo-Fc at 2.5 σ) of the hOAT-14 cocrystal structure. Black dashed lines indicate hydrogen bonds.

Figure 5.

Final binding poses and average distances for the nucleophilic additions for 27 and 27` after 25 ns MD simulations. (A) MD simulation of 27 in the active site of hOAT with the intact salt bridge; (B) MD simulation of 27 in the active site of hOAT with the disrupted salt bridge; (C) MD simulation of 27` in the active site of hOAT with the disrupted salt bridge.

Figure 6.

Omit map (Fo-Fc at 2.5 σ) of intermediate 36 within the active site of hOAT. Black dashed lines indicate hydrogen bonds.

Figure 7.

Detection of metabolite 43 as the turnover product for hOAT-14. (A) Extracted ion chromatogram for 43 (+ESI, 170.04–170.08 m/z). (B) Confirmation of metabolite 43 using tandem mass spectrometry

Scheme 1.

Catalytic Mechanism of OAT

Scheme 2.

Inactivation mechanisms of GABA-AT by vigabatrin (1) and CPP-115 (5)

Scheme 3.

Syntheses of six-membered ring analogs 12–15 Reagents and conditions: (a) AgOC(O)CF₃, CH₃NO₂, 0 °C – r.t., 16h; then NH₃/MeOH, 1 h; (b) DMP, CH₃CN, NaHCO₃ (b) NaH, DMF; PMBCl, 0 °C – r.t., 16h; (c) CHF₂PO(OEt)₂, ^tBuLi (1.7 M in pentane), −100 °C – r.t., 2h; then reflux, 16h; (d) CAN, CH₃CN, H₂O, 0 °C - r.t., 10 h; (e) HCl (aq. 4 M), 75 °C, 5h. (e) 2-PySO₂CF₂H, ^tBuOK, DMF −60 °C, then −40 °C, 2h, NH₄Cl (saturated aq.), HCl (3M), overnight; (f) PhSeCl, KHMDS (1M in THF), −78 °C – r.t., 3h; (g) Boc₂O, DIPEA, DMAP, DCM, r.t., overnight; (h) K₂CO₃, EtOH, 0 °C - r.t. 3h; (i) m-CPBA, DCM, r.t., 3h; (j) HCl (aq. 4 M), AcOH, 80 °C, overnight.

Scheme 4.

Possible inactivation mechanisms for 14

Scheme 5.

Possible turnover mechanisms for 14 by hOAT

Scheme 6.

Plausible mechanism for 14 with hOAT