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

pubs.acs.org/acsmedchemlett Letter Mechanism-Based Design of 3‑Amino-4Halocyclopentenecarboxylic Acids as Inactivators of GABA Aminotransferase Sida Shen, Peter F. Doubleday, Pathum M. Weerawarna, Wei Zhu, Neil L. Kelleher, and Richard B. Silverman* Cite This: ACS Med. Chem. Lett. 2020, 11, 1949−1955 ACCESS Metrics & More Read Online sı Supporting Information * Article Recommendations ABSTRACT: Aminotransferases are pyridoxal 5′-phosphate-dependent enzymes that catalyze reversible transamination reactions between an amino acid and an α-keto acid, playing a critical role in cellular nitrogen metabolism. It is evident that γ-aminobutyric acid aminotransferase (GABA-AT), which balances the levels of inhibitory and excitatory neurotransmitters, has emerged as a promising therapeutic target for epilepsy and cocaine addiction based on mechanism-based inactivators (MBIs). In this work, we established an integrated approach using computational simulation, organic synthesis, biochemical evaluation, and mass spectrometry to facilitate our design and mechanistic studies of MBIs, which led to the identification of a new cyclopentene-based analogue (6a), 25-times more efficient as an inactivator of GABA-AT compared to the parent compound (1R,3S,4S)-3-amino-4-fluorocyclopentane carboxylic acid (FCP, 4). KEYWORDS: GABA aminotransferase, cyclopentene, deprotonation, rate constant, inactivation efficiency A suggesting that modulation of the deficient level of GABA in the CNS might produce an anticonvulsant effect. Among various approaches to increase the brain concentrations of GABA (e.g., GABA prodrugs and glutamic acid decarboxylase (GAD) activators),4 mechanism-based inactivators (MBIs) of GABA-AT are attractive because of their unique inactivation mechanisms and their successful advancement into preclinical/ clinical stages.4 Unlike other irreversible inhibitors, MBIs are unreactive prior to conversion into an active species in the catalytic site of the target enzyme, thus minimizing unwanted off-target effects.6 Vigabatrin (1, Sabril, Figure 1B), a MBI of GABA-AT, exhibits anticonvulsant activity and was approved by the FDA in 2009 as an adjunctive therapy for refractory partial seizures.7 Mechanistic studies revealed that it irreversibly inhibits GABAAT by covalent modification through two different mechanisms, a Michael addition pathway (70%) and an enamine pathway (30%), leading to its anticonvulsant effect.8,9 It was minotransferases (ATs) are essential enzymes that catalyze two coupled transamination reactions between an amino acid and an α-keto acid, thus playing an important role in nitrogen metabolism in cells. All ATs require pyridoxal 5′-phosphate (PLP) as a cofactor, which is linked to a basic lysine residue in the catalytic pocket through a Schiff base, to convert an amino acid into the corresponding carbonyl compound with concomitant conversion of PLP into pyridoxamine 5′-phosphate (PMP) in the first half-reaction. In the second half-reaction, ATs catalyze the reaction of PMP with an acceptor α-keto acid to perform another transfer of an amino group, thereby converting PMP back to PLP.1,2 Recent findings have demonstrated that pharmacological inhibition of certain ATs (e.g., γ-aminobutyric acid AT and ornithine AT) is a therapeutic strategy aimed to treat neurological disorders and cancers, respectively.3,4 γ-Aminobutyric acid aminotransferase (GABA-AT, E.C. 2.6.1.19) catalyzes the degradation of the prime inhibitory neurotransmitter GABA to succinic semialdehyde (SSA) with the generation of the major excitatory neurotransmitter Lglutamate (L-Glu) from α-ketoglutarate (α-KG) (Figure 1A).4 Normal functioning of the central nervous system (CNS) requires well-balanced levels of inhibitory and excitatory neurotransmitters; a reduction in the level of GABA has been implicated in the symptoms associated with epilepsy,5 © 2020 American Chemical Society Special Issue: Medicinal Chemistry: From Targets to Therapies Received: December 30, 2019 Accepted: February 13, 2020 Published: February 18, 2020 1949 https://dx.doi.org/10.1021/acsmedchemlett.9b00672 ACS Med. Chem. Lett. 2020, 11, 1949−1955 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett Letter (Arg192 and Arg445).14,15 CPP-115 has been investigated in a Phase I clinical trial,10 as a compassionate use medication and as a treatment for infantile spasms,16 while OV329 suppressed the release of brain dopamine at a dose of 0.1 mg/kg in a rat model of cocaine addiction.14 Therefore, mechanism-based inactivation of GABA-AT has served as an effective approach to discover novel therapeutic treatments for different neurological disorders. In 2000, (1R,3S,4S)-3-amino-4-fluorocyclopentane carboxylic acid (FCP, 4, Figure 1B) was reported as an inactivator of GABA-AT.17 In 2004, crystallography with GABA-AT revealed that FCP covalently modifies the Lys329-PLP linkage by forming imine adduct M5 (Scheme 1) derived from an enamine inactivation mechanism.18 The proposed inactivation mechanism of FCP is initiated by FCP acting as a substrate to form Schiff base M1 with PLP, followed by deprotonation (M2) and subsequent elimination of fluoride ion to afford the imine intermediate (M3). Subsequent Lys329 attack at the imine moiety of M3 releases the enamine metabolite (M4) and PLP is returned to Lys329. M4 covalently modifies the Lys329−PLP complex to generate adduct M5 via an enamine mechanism. However, except for the cocrystal structure, its mechanism was not well supported. Therefore, at the beginning of this work, we resynthesized FCP (Scheme S1) and further elucidated its mechanisms of inactivation and alternative turnover using mass spectrometry with the intent of using this as a basis for new inactivator design. In the present work, the kinetic constants for FCP against GABA-AT (Table 1) indicate that FCP had a greater binding Figure 1. Coupled transamination reactions of GABA-AT (A) and structures of MBI representatives 1−5 (B). also found to prevent cocaine addiction at a dose of 300 mg/kg in a rat model.10 However, there are considerable concerns regarding the permanent visual damage associated with longterm vigabatrin administration, which results because of its low inactivation efficiency and poor blood-brain barrier (BBB) permeability, which demand high daily doses (1−3 g per day) that eventually impair its clinical profile.11,12 Recent findings identified cyclopentane-based analogue CPP-115 (2, Figure 1B) and cyclopentene-based analogue OV329 (3, Figure 1B), which exhibit several hundred-fold improved inactivation efficiency relative to vigabatrin.13,14 GABA-AT crystal structures in complex with CPP-115/OV329 demonstrated that their difluoromethylenyl groups are converted into a carboxylate group in the binding site, and both compounds inactivate the enzyme via tight electrostatic interactions between the two carboxylate groups and two arginine residues Table 1. Kinetic Constants of Analogues FCP and 6a−6c with GABA-ATa cmpd FCP (...truncated)