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

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).

SUBMITTER: Shen S

PROVIDER: S-EPMC7549257 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Design and Mechanism of GABA Aminotransferase Inactivators. Treatments for Epilepsies and Addictions.

Project description:When the brain concentration of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) diminishes below a threshold level, the excess neuronal excitation can lead to convulsions. This imbalance in neurotransmission can be corrected by inhibition of the enzyme γ-aminobutyric acid aminotransferase (GABA-AT), which catalyzes the conversion of GABA to the excitatory neurotransmitter l-glutamic acid. It also has been found that raising GABA levels can antagonize the rapid elevation and release of dopamine in the nucleus accumbens, which is responsible for the reward response in addiction. Therefore, the design of new inhibitors of GABA-AT, which increases brain GABA levels, is an important approach to new treatments for epilepsy and addiction. This review summarizes findings over the last 40 or so years of mechanism-based inactivators (unreactive compounds that require the target enzyme to catalyze their conversion to the inactivating species, which inactivate the enzyme prior to their release) of GABA-AT with emphasis on their catalytic mechanisms of inactivation, presented according to organic chemical mechanism, with minimal pharmacology, except where important for activity in epilepsy and addiction. Patents, abstracts, and conference proceedings are not covered in this review. The inactivation mechanisms described here can be applied to the inactivations of a wide variety of unrelated enzymes.

| S-EPMC8459698 | biostudies-literature

Design and mechanism of tetrahydrothiophene-based ?-aminobutyric acid aminotransferase inactivators.

Project description:Low levels of ?-aminobutyric acid (GABA), one of two major neurotransmitters that regulate brain neuronal activity, are associated with many neurological disorders, such as epilepsy, Parkinson's disease, Alzheimer's disease, Huntington's disease, and cocaine addiction. One of the main methods to raise the GABA level in human brain is to use small molecules that cross the blood-brain barrier and inhibit the activity of ?-aminobutyric acid aminotransferase (GABA-AT), the enzyme that degrades GABA. We have designed a series of conformationally restricted tetrahydrothiophene-based GABA analogues with a properly positioned leaving group that could facilitate a ring-opening mechanism, leading to inactivation of GABA-AT. One compound in the series is 8 times more efficient an inactivator of GABA-AT than vigabatrin, the only FDA-approved inactivator of GABA-AT. Our mechanistic studies show that the compound inactivates GABA-AT by a new mechanism. The metabolite resulting from inactivation does not covalently bind to amino acid residues of GABA-AT but stays in the active site via H-bonding interactions with Arg-192, a ?-? interaction with Phe-189, and a weak nonbonded S···O?C interaction with Glu-270, thereby inactivating the enzyme.

| S-EPMC4390550 | biostudies-literature

Design, Synthesis, and In Vitro Evaluation of Novel Histone H3 Peptide-Based LSD1 Inactivators Incorporating ?,?-Disubstituted Amino Acids with ?-Turn-Inducing Structures.

Project description:Lysine-specific demethylase 1 (LSD1) mainly removes methyl groups of mono- or di-methylated lysine residues at the fourth position of histone H3 to epigenetically regulate the expression of genes associated with several diseases, such as cancer. Therefore, LSD1 inactivators are expected to be used as therapeutic agents. In this study, to identify novel peptide-based LSD1 inactivators, we focused on the X-ray structure of LSD1 complexed with a H3 peptide-based suicide substrate. It has been proposed that a methylated histone substrate forms three consecutive ?-turn structures in the active pocket of LSD1. Based on this, we designed and synthesized novel histone H3 peptide-based LSD1 inactivators 2a?c by incorporating various ?,?-disubstituted amino acids with ?-turn-inducing structures. Among synthetic peptides 2a?c, peptide 2b incorporating two 1-aminocyclohexanecarboxylic acids at both sides of a lysine residue bearing a trans-2-phenylcyclopropylamine (PCPA) moiety, which is a pharmacophore for LSD1 inactivation, was the most potent and selective LSD1 inactivator. These findings are useful for the further development of histone H3 peptide-based LSD1 inactivators.

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Ornithine aminotransferase versus GABA aminotransferase: implications for the design of new anticancer drugs.

Project description:Ornithine aminotransferase (OAT) and ?-aminobutyric acid aminotransferase (GABA-AT) are classified under the same evolutionary subgroup and share a large portion of structural, functional, and mechanistic features. Therefore, it is not surprising that many molecules that bind to GABA-AT also bind well to OAT. Unlike GABA-AT, OAT had not been viewed as a potential therapeutic target until recently; consequently, the number of therapeutically viable molecules that target OAT is very limited. In this review the two enzymes are compared with respect to their active-site structures, catalytic and inactivation mechanisms, and selective inhibitors. Insight is offered that could aid in the design and development of new selective inhibitors of OAT for the treatment of cancer.

| S-EPMC4415689 | biostudies-literature

Mechanism of Inactivation of GABA Aminotransferase by (E)- and (Z)-(1S,3S)-3-Amino-4-fluoromethylenyl-1-cyclopentanoic Acid.

Project description:When γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian central nervous system, falls below a threshold level, seizures occur. One approach to raise GABA concentrations is to inhibit GABA aminotransferase (GABA-AT), a pyridoxal 5'-phosphate-dependent enzyme that degrades GABA. We have previously developed (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115), which is 186 times more efficient in inactivating GABA-AT than vigabatrin, the only FDA-approved inactivator of GABA-AT. We also developed (E)- and (Z)-(1S,3S)-3-amino-4-fluoromethylenyl-1-cyclopentanoic acid (1 and 2, respectively), monofluorinated analogs of CPP-115, which are comparable to vigabatrin in inactivating GABA-AT. Here, we report the mechanism of inactivation of GABA-AT by 1 and 2. Both produce a metabolite that induces disruption of the Glu270-Arg445 salt bridge to accommodate interaction between the metabolite formyl group and Arg445. This is the second time that Arg445 has interacted with a ligand and is involved in GABA-AT inactivation, thereby confirming the importance of Arg445 in future inactivator design.

| S-EPMC4575653 | biostudies-literature

Computational Exploration of the Effects of Mutations on GABA Aminotransferase in GABA Aminotransferase Deficiency.

Project description:Gamma-aminobutyric acid (GABA) transaminase-also called GABA aminotransferase (GABA-AT)-deficiency is a rare autosomal recessive disorder characterized by a severe neonatal-infantile epileptic encephalopathy with symptoms such as seizures, hypotonia, hyperreflexia, developmental delay, and growth acceleration. GABA transaminase deficiency is caused by mutations in GABA-AT, the enzyme responsible for the catabolism of GABA. Mutations in multiple locations on GABA-AT have been reported and their locations have been shown to influence the onset of the disease and the severity of symptoms. We examined how GABA-AT mutations influence the structural stability of the enzyme and GABA-binding affinity using computational methodologies such as molecular dynamics simulation and binding free energy calculation to understand the underlying mechanism through which GABA-AT mutations cause GABA-AT deficiency. GABA-AT 3D model depiction was carried out together with seven individual mutated models of GABA-AT. The structural stability of all the predicted models was analyzed using several tools and web servers. All models were evaluated based on their phytochemical values. Additionally, 100 ns MD simulation was carried out and the mutated models were evaluated using RMSD, RMSF, Rg, and SASA. gmxMMPBSA free energy calculation was carried out. Moreover, RMSD and free energy calculations were also compared with those obtained using online web servers. Our study demonstrates that P152S, Q296H, and R92Q play a more critical role in the structural instability of GABA-AT compared with the other mutated models: G465R, L211F, L478P, and R220K.

| S-EPMC10342115 | biostudies-literature

Rational Design, Synthesis, and Mechanism of (3<i>S</i>,4<i>R</i>)-3-Amino-4-(difluoromethyl)cyclopent-1-ene-1-carboxylic Acid: Employing a Second-Deprotonation Strategy for Selectivity of Human Ornithine Aminotransferase over GABA Aminotransferase.

Project description:Human ornithine aminotransferase (hOAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that contains a similar active site to that of γ-aminobutyric acid aminotransferase (GABA-AT). Recently, pharmacological inhibition of hOAT was recognized as a potential therapeutic approach for hepatocellular carcinoma. In this work, we first studied the inactivation mechanisms of hOAT by two well-known GABA-AT inactivators (CPP-115 and OV329). Inspired by the inactivation mechanistic difference between these two aminotransferases, a series of analogues were designed and synthesized, leading to the discovery of analogue 10b as a highly selective and potent hOAT inhibitor. Intact protein mass spectrometry, protein crystallography, and dialysis experiments indicated that 10b was converted to an irreversible tight-binding adduct (34) in the active site of hOAT, as was the unsaturated analogue (11). The comparison of kinetic studies between 10b and 11 suggested that the active intermediate (17b) was only generated in hOAT and not in GABA-AT. Molecular docking studies and pKa computational calculations highlighted the importance of chirality and the endocyclic double bond for inhibitory activity. The turnover mechanism of 10b was supported by mass spectrometric analysis of dissociable products and fluoride ion release experiments. Notably, the stopped-flow experiments were highly consistent with the proposed mechanism, suggesting a relatively slow hydrolysis rate for hOAT. The novel second-deprotonation mechanism of 10b contributes to its high potency and significantly enhanced selectivity for hOAT inhibition.

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INTERACTION OF ASPARTATE AMINOTRANSFERASE WITH AMINO ACIDS.

Project description:1. The capacity of various amino acids to convert the pyridoxal form of aspartate aminotransferase into the pyridoxamine form has been investigated. 2. Glutamate has the highest converting capacity; aspartate, alpha-aminopimelate, alpha-aminoadipate and other amino acids follow. 3. The converting capacity of the various amino acids assayed is connected with their structural features. 4. A possible role of amino acids as secondary substrates of aspartate aminotransferase is suggested.

| S-EPMC1206790 | biostudies-other

Design and synthesis of potential mechanism-based inhibitors of the aminotransferase BioA involved in biotin biosynthesis.

Project description:BioA, a pyridoxal 5'-phosphate (PLP) dependent aminotransferase, catalyzes the second step of biotin biosynthesis, converting 7-keto-8-aminopelargonic acid (KAPA) into 7,8-diaminopelargonic acid (DAPA). Amiclenomycin (ACM) isolated from cultures of different Streptomyces strains is a potent mechanism-based inhibitor of BioA that operates via an aromatization mechanism, irreversibly labeling the PLP cofactor. However, ACM is plagued by inherent chemical stability. Herein we describe the synthesis of four inhibitors, inspired by ACM but containing an allylic amine as the chemical warhead, designed to both improve stability and operate via a complementary Michael addition-pathway upon enzymatic oxidation of the allylic amine substrate to an enimine. Acyclic analogue M-1 contains a terminal olefin as the pro-Michael acceptor. The synthesis of M-1 features an alkyne-zipper reaction and the Overman rearrangement as key synthetic operations. The cyclic analogues M-2/3/4 contain either an endocyclic or exocyclic olefin as the pro-Michael acceptor. These were all prepared using a common strategy employing DIBAL reduction of a precursor bicyclic lactam, followed by in situ Horner-Wadsworth-Emmons (HWE) olefination as the key synthetic steps.

| S-EPMC3448953 | biostudies-literature

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