γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. Inhibition of GABA aminotransferase (GABA-AT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, has been established as a possible strategy for the treatment of substance abuse. The raised GABA levels that occur as a consequence of this inhibition have been found to antagonize the rapid release of dopamine in the ventral striatum (nucleus accumbens) that follows an acute challenge by an addictive substance. In addition, increased GABA levels are also known to elicit an anticonvulsant effect in patients with epilepsy. We previously designed the mechanism-based inactivator (1S,3S)-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid (2), now called CPP-115, that is 186 times more efficient in inactivating GABA-AT than vigabatrin, the only FDA-approved drug that is an inactivator of GABA-AT. CPP-115 was found to have high therapeutic potential for the treatment of cocaine addiction and for a variety of epilepsies, has successfully completed a Phase I safety clinical trial, and was found to be effective in the treatment of infantile spasms (West syndrome). Herein we report the design, using molecular dynamics simulations, synthesis, and biological evaluation of a new mechanism-based inactivator, (S)-3-amino-4-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic acid (5), which was found to be almost 10 times more efficient as an inactivator of GABA-AT than CPP-115. We also present the unexpected crystal structure of 5 bound to GABA-AT, as well as computational analyses used to assist the structure elucidation process. Furthermore, 5 was found to have favorable pharmacokinetic properties and low off-target activities. In vivo studies in freely moving rats showed that 5 was dramatically superior to CPP-115 in suppressing the release of dopamine in the corpus striatum, which occurs subsequent to either an acute cocaine or nicotine challenge. Compound 5 also attenuated increased metabolic demands (neuronal glucose metabolism) in the hippocampus, a brain region that encodes spatial information concerning the environment in which an animal receives a reinforcing or aversive drug. This multidisciplinary computational design to preclinical efficacy approach should be applicable to the design and improvement of mechanism-based inhibitors of other enzymes whose crystal structures and inactivation mechanisms are known.

γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concentration of GABA falls below a threshold level, convulsions can occur. Inhibition of GABA-AT raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurological disorders, including drug addiction. Among the analogues that we previously developed, (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115) showed 187 times greater potency than that of vigabatrin, a known inactivator of GABA-AT and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently, CPP-115 was shown to have no adverse effects in a Phase I clinical trial. Here we report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes GABA-AT-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of two fluoride ions and coenzyme conversion to pyridoxamine 5′-phosphate (PMP). The partition ratio for CPP-115 with GABA-AT is about 2000, releasing cyclopentanone-2,4-dicarboxylate (22) and two other precursors of this compound (20 and 21). Time-dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP (20), which disrupts an electrostatic interaction between Glu270 and Arg445 to form an electrostatic interaction between Arg445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. This represents a novel mechanism for inactivation of GABA-AT and a new approach for the design of mechanism-based inactivators in general.

Ornithine aminotransferase (OAT) and γ-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.

Based on the structure of the superpotent 5-HT 2A agonist 2-(4-bromo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine, which consists of a ringsubstituted phenethylamine skeleton modified with an N-benzyl group, we designed and synthesized a small library of constrained analogues to identify the optimal arrangement of the pharmacophoric elements of the ligand. Structures consisted of diversely substituted tetrahydroisoquinolines, piperidines, and one benzazepine. Based on the structure of (S,S)-9b, which showed the highest affinity of the series, we propose an optimal binding conformation. (S,S)-9b also displayed 124-fold selectivity for the 5-HT 2A over the 5-HT 2C receptor, making it the most selective 5-HT 2A receptor agonist ligand currently known.

The protein regulator of the operon and its own gene (GabR) is a transcriptional activator that regulates transcription of γ-aminobutyric acid aminotransferase (GABA-AT; GabT) upon interactions with pyridoxal-5'-phosphate (PLP) and GABA, and thereby promotes the biosynthesis of glutamate from GABA. We show here that the external aldimine formed between PLP and GABA is apparently responsible for triggering the GabR-mediated transcription activation. Details of the "active site" in the structure of the GabR effector-binding/oligomerization (Eb/O) domain suggest that binding a monocarboxylic γ-amino acid such as GABA should be preferred over dicarboxylic acid ligands. A reactive GABA analog, ()-4-amino-5-fluoropentanoic acid (AFPA), was used as a molecular probe to examine the reactivity of PLP in both GabR and a homologous aspartate aminotransferase (Asp-AT) from as a control. A comparison between the structures of the Eb/O-PLP-AFPA complex and Asp-AT-PLP-AFPA complex revealed that GabR is incapable of facilitating further steps of the transamination reaction after the formation of the external aldimine. Results of in vitro and in vivo assays using full-length GabR support the conclusion that AFPA is an agonistic ligand capable of triggering GabR-mediated transcription activation via formation of an external aldimine with PLP.

A novel class of isochroman dopamine analogues, 1, originally reported by Abbott Laboratories, had greater than 100-fold selectivity for D1-like vs. D2-like receptors. We synthesized a parallel series of chroman compounds, 2, and showed that repositioning the oxygen in the heterocyclic ring reduced potency and conferred D2-like receptor selectivity to these compounds. In silico modeling supported the hypothesis that the altered pharmacology for 2 was due to potential intramolecular hydrogen bonding between the oxygen in the chroman ring and the meta-hydroxyl of the catechol moiety. This interaction realigns the catechol hydroxyl groups and disrupts key interactions between these ligands and critical serine residues in TM5 of the D1-like receptors. This hypothesis was tested by the synthesis and pharmacological evaluation of a parallel series of carbocyclic compounds, 3. Our results suggest that when the potential for intramolecular hydrogen bonding is removed, D1-like receptor potency and selectivity is restored.

We have developed two new continuous, coupled assays for ornithine-δ-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multi-well plates for convenience and high throughput. The first assay is based on the reduction of Δ1-pyrroline-5-carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1 (PYCR1), which results in the concomitant oxidation of NADH to NAD+. This procedure was found to be three times more sensitive than previous methods, and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of L-glutamate, produced during the regeneration of the cofactor PLP of OAT by an unamplified modification of the commercially available Amplex® Red L-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT; measuring the transformation of L-ornithine at high concentrations by this assay is complicated by the fact that it also acts as a substrate for the L-glutamate oxidase (GluOx) reporter enzyme.

We have synthesized three analogues of 4-amino-5-fluorohexanoic acids as potential inactivators of γ-aminobutyric acid aminotransferase (GABA-AT), which were designed to combine the potency of their shorter chain analogue, 4-amino-5-fluoropentanoic acid (AFPA), with the greater enzyme selectivity of the antiepileptic vigabatrin (Sabril®). Unexpectedly, these compounds failed to inactivate or inhibit the enzyme, even at high concentrations. On the basis of molecular modeling studies, we propose that the GABA-AT active site has an accessory binding pocket that accommodates the vinyl group of vigabatrin and the fluoromethyl group of AFPA, but is too narrow to support the extra width of one distal methyl group in the synthesized analogues.