Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (1S,3S)-3-Amino-4-difluoromethylene-1-cyclopentanoic Acid (CPP-115)
Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (1S,3S)-3-Amino-4-difluoromethylene-1-cyclopentanoic Acid (CPP-115)
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DOI：
10.1021/ja512299n
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发表时间：
2015-02-25
影响因子：
15
通讯作者：
Silverman, Richard B.
中科院分区：
文献类型：
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作者：
Lee, Hyunbeom;Doud, Emma H.;Silverman, Richard B.
γ-氨基丁酸转氨酶 (GABA-AT) 是一种吡哆醛 5'-磷酸 (PLP) 依赖性酶，可降解哺乳动物细胞中主要的抑制性神经递质 GABA。当 GABA 浓度低于阈值水平时，就会发生抽搐。抑制 GABA-AT 会提高大脑中的 GABA 水平，从而终止癫痫发作，并在治疗其他神经系统疾病（包括药物成瘾）方面具有潜在的治疗应用。在我们之前开发的类似物中，(1S,3S)-3-氨基-4-二氟亚甲基-1-环戊酸 (CPP-115) 的效力比氨己烯酸强 187 倍，氨己烯酸是已知的 GABA-AT 灭活剂，也是批准用于治疗婴儿痉挛症和难治性成人癫痫的药物 (Sabril)。最近，CPP-115在一期临床试验中显示没有不良反应。在这里，我们报告了 CPP-115 的一种新的失活机制，这是一种基于机制的失活剂，它经历 GABA-AT 催化的二氟亚甲基水解为羧酸，同时损失两个氟离子和辅酶转化为吡哆胺 5'-磷酸 (PMP)。 CPP-115与GABA-AT的分配比约为2000，释放出环戊酮-2,4-二羧酸酯(22)和该化合物的另外两种前体(20和21)。时间依赖性失活是由于 4-氨基环戊烷-1,3-二甲酸和 PMP 的醛亚胺形成引起的构象变化而发生的 (20)，这会破坏 Glu270 和 Arg445 之间的静电相互作用，从而在 Arg445 和 CPP-115 中二氟亚甲基水解产生的新形成的羧酸盐之间形成静电相互作用，从而形成非共价的紧密结合的复合物。这代表了一种新的 GABA-AT 失活机制，也是一种设计基于机制的失活剂的新方法。
gamma-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.