Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5′-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover

Abstract

Potent mechanism-based inactivators can be rationally designed against pyridoxal 5′-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-amino-butyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.

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 .…”

“…The covalent bond between Lys292 and 6 represents a stable gem-diamine species that has not been observed in any previous hOAT/ligand crystals. 9,12,13,24 This observation further validated the gemdiamine precursors (M12 or M13, Scheme 5) 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.…”

“…Moreover, in the published crystal structure of the native enzyme (holo-hOAT; PDB entry 1OAT), Arg413 typically forms a salt bridge with Glu235, which was observed for several co-crystals of hOAT with different inactivators. 16,24,27 In the hOAT/6 soaking structure (Figure 5A), the salt bridge of Arg413-Glu235 is broken because of the formation of an alternative salt bridge between the carboxylate of one intermediate and Arg413. Interestingly, the Arg413−Glu235 salt bridge is also found to be broken in the hOAT/6 co-crystal structure, while no direct interaction is observed between Arg413 and the ligand.…”

Mechanism-Based Design of 3-Amino-4-Halocyclopentenecarboxylic Acids as Inactivators of GABA Aminotransferase

“…According to the literature, FCP shows a different turnover mechanism when incubated with aspartate aminotransferase, affording a ketone metabolite without releasing fluoride ion ( S11 in Scheme S2). In the current study with GABA-AT, we did not detect this metabolite. Intact protein mass spectrometry of GABA-AT inactivated by FCP produced a mass shift caused by covalent modification, which corresponded to PLP-bound ketone M8 (Scheme ; theoretical, 357.06 Da; observed, 357.27 Da; shown in Table ).…”

“…To prepare two other cyclopentene analogues (6b and 6c), bearing chlorine and bromine atoms, respectively, the synthetic routes were initiated from tosylation (17) and PMB deprotection of 10 to afford 18 (Scheme 3). After Boc protection (19), a one-pot hydrolysis and elimination was carried out with K 2 CO 3 /MeOH to obtain the desired cyclopentene (20). Intermediate 20 was successfully converted to the desired monochloro-(21a) or monobromo-(21b) substituted compound using NCS or NBS, respectively; the sole diastereomers formed may have been facilitated by neighboring group participation in the halogenation step.…”