(±)-(1S,2R,5S)-5-Amino-2-fluorocyclohex-3-enecarboxylic Acid. A Potent GABA Aminotransferase Inactivator that Irreversibly Inhibits via an Elimination−Aromatization Pathway

Abstract

Inhibition of γ-aminobutyric acid aminotransferase (GABA-AT) raises the concentration of GABA, an inhibitory neurotransmitter in human brain, which could have therapeutic applications for a variety of neurological diseases including epilepsy. Based on studies of several previously synthesized conformationally-restricted GABA-AT inhibitors, (±)-(1S,2R,5S)-5-amino-2-fluorocyclohex-3-ene carboxylic acid (12) was designed as a mechanismbased inactivator. This compound was shown to irreversibly inhibit GABA-AT; substrate protects the enzyme from inactivation. Mechanistic experiments demonstrated the loss of one fluoride ion per active site during inactivation and the formation of N-m-carboxyphenylpyridoxamine 5′-phosphate (26), the same product generated by inactivation of GABA-AT by gabaculine (8). An elimination-aromatization mechanism is proposed to account for these results.γ-Aminobutyric acid aminotransferase (GABA-AT, E.C. 2.6.1.19) is the enzyme responsible for the degradation of γ-Aminobutyric acid (GABA), one of the major inhibitory neurotransmitters in the mammalian central nervous system, 1 to succinic semialdehyde. Inhibition of this enzyme results in an increased concentration of GABA in the brain and could have therapeutic applications in neurological disorders including epilepsy, 2 Parkinson's disease, 3 Huntington's chorea, 4 and Alzheimer' disease. 5 In fact, vigabatrin (1 , Figure 1), an irreversible inactivator of GABA-AT, is a drug for the treatment of epilepsy. 6 It has also been found that an increase in the availability of GABA blocks the effects of drug addiction. 7We recently reported several fluorine-containing conformationally-restricted analogues of GABA ( Figure 1, 2-4) as potential meschanism-based inactivators 8 of GABA-AT, but they turned out to have only minimal reversible inhibitory activity; the non-fluorinated parent compound also was devoid of substrate or inhibitory activity. 9 However, the corresponding † The authors are grateful to the National Institutes of Health (GM66132) for financial support of this research. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript cyclopentane analogue of 2, namely 5, was shown to be a time-dependent inactivator of GABA-AT; 10 it inactivates the enzyme by an enamine mechanism. 11 Likewise, (1R,4S)-(+)-4-amino-2-cyclopentene-1-carboxylic acid (6) is a substrate and inhibitor of GABA-AT but not an inactivator, 12 whereas, the corresponding cyclohexene analogue (7) is a time-dependent inactivator of GABA-AT, which also was demonstrated to inactivate the enzyme by an enamine mechanism, in this case leading to a ternary complex between the enzyme, the PLP cofactor, and 7. 13 It was reasoned that the flexible chair conformation of the cyclohexane ring is responsible for the inability of these compounds to inactivate the enzyme, and that the more rigid structures of the corresponding cyclopentane or cyclohexene analogues are more effective inactivators. This also is consistent with the observation that the natural produ...

A Remarkable Difference That One Fluorine Atom Confers on the Mechanisms of Inactivation of Human Ornithine Aminotransferase by Two Cyclohexene Analogues of γ-Aminobutyric Acid

“…Mechanistic pathway b is a direct aromatization of 14a/14b to yield a tight binding adduct 20a/20b, which is similar to the inactivation mechanism of another fluorinated cyclohexene analogue. 34 Mechanistic pathway c involves nucleophilic addition of Lys292 to conjugated olefin 14a/14b, yielding adducts 21a/ 21b.…”

Putative Irreversible Inhibitors of the Human Sodium-Dependent Bile Acid Transporter (hASBT; SLC10A2) Support the Role of Transmembrane Domain 7 in Substrate Binding/Translocation

“…Whereas, the application of substrate analogues as irreversible inhibitors has been applied previously in the enzyme research field, this approach has found limited application as a tool in transporter research. For example, Wang and colleagues (17) synthesized a series of fluorinated GABA analogs which functioned as irreversible inhibitors of GABA aminotransferase. In the present study, we selected for chloro- and mesyl- groups since they are better leaving groups compared to fluoride.…”

Design, Docking Simulations, Synthesis, and in vitro and in vivo Behavioral Assessment of m‐Aminobenzoic Acid Analogues as GABA‐AT Inhibitors

“…Additionally, molecules unable to cross the BBB, must rely on various endogenous transporters. [13] Some GABA analogues that are unsaturated between the amino and carboxylic group, including cis-amino crotonic acid, trans-amino crotonic acid, and aminotetrolic acid, are characterized by an increased specificity to recognize GABAergic receptors; [14][15][16][17] similarly, cyclic systems, such as gabaculine, a naturally occurring neurotoxin, analog of m-aminobenzoic acid (MABA) and GABA-AT inhibitor, are able to cross the BBB (Figure 1) [18][19][20] and these structures are closely related with our proposed compounds in the Table 1.…”