Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (1S,3S)-3-Amino-4-difluoromethylene-1-cyclopentanoic Acid (CPP-115)

Abstract

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.

A case report on the efficacy of vigabatrin analogue (1 S , 3 S )-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid (CPP-115) in a patient with infantile spasms

“…(1 S , 3 S )-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid (CPP-115) is a high-affinity vigabatrin analogue. CPP-115 was developed to produce greater therapeutic potency than vigabatrin without retinal toxicity [10] , [11] . The molecular structure prevents formation of the reactive metabolite implicated in retinal toxicity [10] .…”

Rational Optimization of Mechanism-Based Inhibitors through Determination of the Microscopic Rate Constants of Inactivation

“…As a result, MBIs are widely used in medicine, accounting for over 50 marketed drugs 6 with numerous development efforts ongoing. 7,8 …”

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

“…The partition ratio for CPP-115 with GABA-AT was reported to be about 2000, releasing cyclopentanone-2,4-dicarboxylate and two other precursors of this compound. 17 …”

“…17 Here, the salt bridge between Arg445 and Glu270 has also been broken, and Glu270 is rotated away from its original position to accommodate, depending on the resonance structure ( 24 , Scheme 6), either a hydrogen bonding interaction between the formyl group and Arg445 or a weak electrostatic interaction between the enolate of the formyl group in 24 and Arg445 (Figure 5 shows the structure with 1 bound, and Supplementary Information Figure S11 shows the structure with 2 bound). The rotation of Glu270, however, is less than that in the case of CPP-115, in which Glu270 completely rotates away to accommodate a full second guanidinium−carboxylate electrostatic interaction with Arg445 (Figure 7 shows an overlay of 1 -inactivated GABA-AT and CPP-115-inactivated GABAAT; Supplementary Information Figure S14 shows an overlay of 2 -inactivated GABA-AT and CPP-115-inactivated GABAAT).…”

“…17 The inactivation was initiated by Schiff base formation of CPP-115 with the active site PLP, followed by γ-proton removal and catalytic hydrolysis of the difluoromethylenyl group to give the PLP-bound dicarboxylate metabolite. We also discovered that the Glu270-Arg445 salt bridge in the active site was disrupted, leading to the formation of a new binding pocket for the inactivator.…”