Theoretical and Mechanistic Validation of Global Kinetic Parameters of the Inactivation of GABA Aminotransferase by OV329 and CPP-115

Abstract

((S)-3-Amino-(difluoromethylenyl)­cyclopent-1-ene-1-carboxylic acid (OV329) is a recently discovered inactivator of γ-aminobutyric acid aminotransferase (GABA-AT), which has 10 times better inactivation efficiency than its predecessor, CPP-115, despite the only structural difference being an endocyclic double bond in OV329. Both compounds are mechanism-based enzyme inactivators (MBEIs), which inactivate GABA-AT by a similar mechanism. Here, a combination of a variety of computational chemistry tools and experimental methods, including quantum mechanical (QM) calculations, molecular dynamic simulations, progress curve analysis, and deuterium kinetic isotope effect (KIE) experiments, are utilized to comprehensively study the mechanism of inactivation of GABA-AT by CPP-115 and OV329 and account for their experimentally obtained global kinetic parameters k inact and K I. Our first key finding is that the rate-limiting step of the inactivation mechanism is the deprotonation step, and according to QM calculations and the KIE experiments, k inact accurately represents the enhancement of the rate-limiting step for the given mechanism. Second, the present study shows that the widely used simple QM models do not accurately represent the geometric criteria that are present in the enzyme for the deprotonation step. In contrast, QM cluster models successfully represent both the ground state destabilization and the transition state stabilization, as revealed by natural bond orbital analysis. Furthermore, the globally derived K I values for both of the inactivators represent the inhibitor constants for the initial binding complexes (K d) and indicate the inactivator competition with the substrate according to progress curve analysis and the observed binding isotope effect. The configurational entropy loss accounts for the difference in K I values between the inactivators. The approach we describe in this work can be employed to determine the validity of globally derived parameters in the process of MBEI optimization for given inactivation mechanisms.

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) .…”

“…25 The introduction of a double bond (7, OV329, Figure 2) maintained the inactivation mechanism but greatly enhanced the potency, potentially as a result of the reduced acidity of the γ-proton. 27,29 Because of the structural similarity between these two aminotransferases, the aforementioned analogues also inactivated hOAT. 20,27 To improve the potency and selectivity toward hOAT over GABA-AT, sixmembered ring analogue 8 30 (Figure 2) was designed and synthesized, taking into account the relatively more flexible and larger active site of hOAT.…”

Structural and Mechanistic Basis for the Inactivation of Human Ornithine Aminotransferase by (3S,4S)-3-Amino-4-fluorocyclopentenecarboxylic Acid

“…The incorporation of a double bond into a cyclopentane system has significantly improved the inactivation efficiency of our recent MBIs by enhancing the reactivity of the key C γ hydrogen and providing conformational changes [ 26 , 43 , 44 , 45 ]. In this work, we carried out an integrated mechanistic study with h OAT and 5 , demonstrating that the addition of a double bond also influences the inactivation mechanism pathways.…”

OV329, a novel highly potent γ‐aminobutyric acid aminotransferase inactivator, induces pronounced anticonvulsant effects in the pentylenetetrazole seizure threshold test and in amygdala‐kindled rats

“…During this drug‐testing phase, the ADT determination performed by an ascending staircase procedure (refer to Supplement) always started three steps below the previous vehicle ADT. Because OV329 is expected to induce an irreversible inactivation of GABA‐AT requiring resynthesis of the enzyme, 40 the vehicle postcontrol ADT determination was made not earlier than 1 week following the previous drug trial (equally to the ivPTZ‐ST protocol). This was not necessary after vehicle injections, which is why the testing interval could be shorter after vehicle injections.…”