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

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, (1<i>S</i>,3<i>S</i>)-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 (<b>22</b>) and two other precursors of this compound (<b>20</b> and <b>21</b>). Time-dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP (<b>20</b>), 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

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

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 (1<i>S</i>,3<i>S</i>)-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 (<i>E</i>)- and (<i>Z</i>)-(1<i>S</i>,3<i>S</i>)-3-amino-4-fluoromethylenyl-1-cyclopentanoic acid (<b>1</b> and <b>2</b>, 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 <b>1</b> and <b>2</b>. 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

Suppression of Hepatocellular Carcinoma by Inhibition of Overexpressed Ornithine Aminotransferase

Hepatocellular carcinoma is the second leading cause of cancer death worldwide. DNA microarray analysis identified the ornithine aminotransferase (OAT) gene as a prominent gene overexpressed in hepatocellular carcinoma (HCC) from <i>Psammomys obesus</i>. <i>In vitro</i> studies demonstrated inactivation of OAT by gabaculine (<b>1</b>), a neurotoxic natural product, which suppressed in vitro proliferation of two HCC cell lines. Alpha-fetoprotein (AFP) secretion, a biomarker for HCC, was suppressed by gabaculine in both cell lines, but not significantly. Because of the active site similarity between GABA aminotransferase (GABA-AT) and OAT, a library of 24 GABA-AT inhibitors was screened to identify a more selective inhibitor of OAT. (1<i>S</i>,3<i>S</i>)-3-Amino-4-(hexafluoropropan-2-ylidene)­cyclopentane-1-carboxylic acid (<b>2</b>) was found to be an inactivator of OAT that only weakly inhibits GABA-AT, l-aspartate aminotransferase, and l-alanine aminotransferase. <i>In vitro</i> administration of <b>2</b> significantly suppressed AFP secretion in both Hep3B and HepG2 HCC cells; <i>in vivo</i>, <b>2</b> significantly suppressed AFP serum levels and tumor growth in HCC-harboring mice, even at 0.1 mg/kg. Overexpression of the OAT gene in HCC and the ability to block the growth of HCC by OAT inhibitors support the role of OAT as a potential therapeutic target to inhibit HCC growth. This is the first demonstration of suppression of HCC by an OAT inactivator

(1<i>S</i>, 3<i>S</i>)-3-Amino-4-difluoromethylenyl-1-cyclopentanoic Acid (<b>CPP-115</b>), a Potent γ-Aminobutyric Acid Aminotransferase Inactivator for the Treatment of Cocaine Addiction

Vigabatrin, a GABA aminotransferase (GABA-AT) inactivator, is used to treat infantile spasms and refractory complex partial seizures and is in clinical trials to treat addiction. We evaluated a novel GABA-AT inactivator (1<i>S</i>, 3<i>S</i>)-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid (CPP-115, compound <b>1</b>) and observed that it does not exhibit other GABAergic or off-target activities and is rapidly and completely orally absorbed and eliminated. By use of in vivo microdialysis techniques in freely moving rats and microPET imaging techniques, <b>1</b> produced similar inhibition of cocaine-induced increases in extracellular dopamine and in synaptic dopamine in the nucleus accumbens at ¹/₃₀₀ to ¹/₆₀₀ the dose of vigabatrin. It also blocks expression of cocaine-induced conditioned place preference at a dose ¹/₃₀₀ that of vigabatrin. Electroretinographic (ERG) responses in rats treated with <b>1</b>, at doses 20–40 times higher than those needed to treat addiction in rats, exhibited reductions in ERG responses, which were less than the reductions observed in rats treated with vigabatrin at the same dose needed to treat addiction in rats. In conclusion, <b>1</b> can be administered at significantly lower doses than vigabatrin, which suggests a potential new treatment for addiction with a significantly reduced risk of visual field defects