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Design and Mechanism of (S)-3-Amino-4-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic Acid, a Highly Potent ?-Aminobutyric Acid Aminotransferase Inactivator for the Treatment of Addiction.

ABSTRACT: ?-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.

SUBMITTER: Juncosa JI

PROVIDER: S-EPMC5812813 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Publications

Design and Mechanism of (S)-3-Amino-4-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic Acid, a Highly Potent γ-Aminobutyric Acid Aminotransferase Inactivator for the Treatment of Addiction.

Juncosa Jose I JI Takaya Kenji K Le Hoang V HV Moschitto Matthew J MJ Weerawarna Pathum M PM Mascarenhas Romila R Liu Dali D Dewey Stephen L SL Silverman Richard B RB

Journal of the American Chemical Society 20180130 6

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 challeng ...[more]

PMID: 29381352

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Project description:Gamma-aminobutyric acid aminotransferase (GABA-AT), a pyridoxal 5'-phosphate dependent enzyme, catalyzes the degradation of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) to succinic semialdehyde with concomitant conversion of pyridoxal 5'-phosphate (PLP) to pyridoxamine 5'-phosphate (PMP). The enzyme then catalyzes the conversion of alpha-ketoglutarate to the excitatory neurotransmitter L-glutamate. Racemic 4-amino-3-fluorobutanoic acid (3-F-GABA) was shown previously to act as a substrate for GABA-AT, not for transamination, but for HF elimination. Here we report studies of the reaction catalyzed by GABA-AT on (R)- and (S)-3-F-GABA. Neither enantiomer is a substrate for transamination. Very little elimination from the (S)-enantiomer was detected using a coupled enzyme assay; The rate of elimination of HF from the (R)-enantiomer is at least 10 times greater than that for the (S)-enantiomer. The (R)-enantiomer is about 20 times more efficient as a substrate for GABA-AT catalyzed HF elimination than GABA is a substrate for transamination. The (R)-enantiomer also inhibits the transamination of GABA 10 times more effectively than the (S)-enantiomer. Using a combination of computer modeling and the knowledge that vicinal C-F and C-NH3+ bonds have a strong preference to align gauche rather than anti to each other, it is concluded that on binding of free 3-F-GABA to GABA-AT the optimal conformation places the C-NH3+ and C-F bonds gauche in the (R)-enantiomer but anti in the (S)-enantiomer. Furthermore, the dynamic binding process and the bioactive conformation of GABA bound to GABA-AT have been inferred on the basis of the different biological behavior of the two enantiomers of 3-F-GABA when they bind to the enzyme. The present study suggests that the C-F bond can be utilized as a conformational probe to explore the dynamic binding process and provide insight into the bioactive conformation of substrates, which cannot be easily determined by other biophysical approaches.

Dataset Information

Design and Mechanism of (S)-3-Amino-4-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic Acid, a Highly Potent ?-Aminobutyric Acid Aminotransferase Inactivator for the Treatment of Addiction.

Publications

Design and Mechanism of (S)-3-Amino-4-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic Acid, a Highly Potent γ-Aminobutyric Acid Aminotransferase Inactivator for the Treatment of Addiction.

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