Generation of functional molecules which regulate the glutathione level in living organisms-Design and synthesis of specific inhibitors of γ-glutamylcysteine synthetase-

调节生物体内谷胱甘肽水平的功能分子的生成-γ-谷氨酰半胱氨酸合成酶特异性抑制剂的设计与合成-

• 批准号: 10680566
• 负责人: HIRATAKE Jun
• 金额: $ 2.18万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680566/
• 关键词: glutathione biosynthesis; γ-glutamylcysteine synthetase; transition-state analogue inhibitor; γ-glutamylphosphate intermediate analogue; mechanism-based enzyme inactivator; enzyme suicide substrate; γ-glutamyltranspeptidase; 遷移状態アナログ; γ-グルタミルリン酸中間

The purpose of this research is to design and synthesis of functional molecules which affects the glutathione level in living organisms. The biosynthesis and degradation of glutathione are mediated by γ-glutamylcysteine synthetase (γ-GCS) and γ-glutamyltranspeptidase (GGT), respectively. Based on the reaction mechanisms of these enzymes, specific inhibitors of these two enzymes were designed, synthesized and evaluated in terms of inhibition potency and profile. γ-GCS catalyzes the reaction by activating the γ-carboxyl group of L-Glu by phosphorylation, followed by nucleophilic substitution with L-Cys. As a transition-state analogue of the substitution step, tetrahedral phosphinic acid and sulfoximine analogues were synthesized. Each compound was found to be a potent time-and ATP-dependent inactivator of γ-GCS. In particular, the sulfoximine served as an extremely powerful slow-binding inhibitor, in which the sulfoximine S=NH nitrogen was phosphorylated by ATP in a mechanism-based manne … More r to form a phosphorylated sulfoximine which is highly analogous to the putative transition state. Two diastereomeric sulfoximines with respect to the chiral sulfur atom were synthesized and evaluated as an inhibitor of γ-GCS. The diastereomer with (R)-sulfur atom served as a highly potent ATP-dependent inactivator (KィイD2iィエD2=39 nM), but the (S)-diastereomer was a rather weak reversible inhibitor (KィイD2iィエD2=12μM). Thus, the enzyme recognized the chirality of the sulfur atom and phosphorylated the (R)-sulfoximine solely. The (R)-sulfoximine inhibited γGCS 1000 times more strongly than buthionine sulfoximine, a famous inhibitor of γ-GCS. In an attempt to obtain an enzyme-suicidal substrate of γ-GCS, the effect of L-glutamic acid γ-hydroxamate was examined. This compound was found to inhibit γ-GCS very slowly, but irreversibly in the presence of ATP, and the inhibition profile suggested that the hydroxamate was phosphorylated enzymatically by ATP to form an isocyanate via a Rossen-type rearrangement. A transition-state analogue inhibitor of GGT was also synthesized. A γ-phosphonofluoridate analogue of Glu was synthesized and was found to serve as a mechanism-based labeling agent of GGT to phosphonylate the active site catalytic nucleophile. Ion-spray MS of the labeled GGT revealed that the N-terminal Thr-391 in the small subunit was the catalytic nucleophile of GGT. Less

本研究的目的是设计和合成影响生物体内谷胱甘肽水平的功能分子。谷胱甘肽的生物合成和降解分别由γ-谷氨酰半胱氨酸合成酶（γ-GCS）和γ-谷氨酰转肽酶（GGT）介导。根据这两种酶的反应机理，设计、合成了这两种酶的特异性抑制剂，并对其抑制效力和特性进行了评价。γ-GCS通过磷酸化活化L-Glu的γ-羧基，然后用L-Cys进行亲核取代来催化反应。作为取代步骤的过渡态类似物，合成了四面体次膦酸和亚砜亚胺类似物。发现每种化合物都是γ-GCS的有效时间和ATP依赖性灭活剂。特别是，亚砜亚胺作为一种非常强大的慢结合抑制剂，其中亚砜亚胺S=NH氮以基于机制的方式被ATP磷酸化， ...更多信息 R形成与推定的过渡态高度类似的磷酸化亚砜亚胺。合成了两个与手性硫原子对映的亚砜亚胺，并评价了它们作为γ-GCS抑制剂的活性。具有（R）-硫原子的非对映体作为高度有效的ATP依赖性失活剂（K_（12）D2=39 nM），但（S）-非对映体是相当弱的可逆抑制剂（K_（12）D2=12μM）。因此，该酶识别硫原子的手性并仅磷酸化（R）-亚砜亚胺。（R）-亚砜亚胺对γ-GCS的抑制作用比著名的γ-GCS抑制剂丁噻呋亚砜亚胺强1000倍。为了获得γ-GCS的酶自杀底物，检查了L-谷氨酸γ-异羟肟酸盐的作用。发现该化合物在ATP存在下非常缓慢地抑制γ-GCS，但不可逆，抑制曲线表明异羟肟酸酯被ATP酶促磷酸化，通过Rossen型重排形成异氰酸酯。本文还合成了一种GGT过渡态类似物抑制剂。合成了一种Glu的γ-膦酰氟类似物，并发现其可作为GGT的基于机理的标记剂，将活性位点催化亲核试剂膦酰化。对标记的GGT进行离子喷雾质谱分析，结果表明小亚基N端的Thr-391是GGT的催化亲核试剂。少

项目成果

M.Inoue, J.Hiratake et al.: "Iedntification of Catalytic Nucleaophile of E.coli V-Glutamyltranspeptidase by γ-Monofluorophosphono Derivative of Glutamic Acid : N-Terminal Thr-391 in Small Subunit is the Nucleophile"Biochemistry. (印刷中). (2000)

M.Inoue、J.Hiratake 等人：“通过谷氨酸的 γ-单氟膦酰基衍生物对大肠杆菌 V-谷氨酰转肽酶的催化亲核试剂进行鉴定：小亚基中的 N 末端 Thr-391 是亲核试剂”（生物化学）。 2000)

N.Tokutake: "Design,Synthesis and Evaluation of Transition-State Analogne Inhibitor of Escherichta coli γ-Glutamylcysteine Synthetase" Bioorg & Med.Chem.6. 1935-1953 (1998)

N.Tokutake：“大肠杆菌γ-谷氨酰半胱氨酸合成酶的过渡态类似物抑制剂的设计、合成和评估”Bioorg & Med.Chem.6 (1998)。

N. Tokutake, J. Hiratake et al: "Design, Synthesis and Evaluation of Transition-State Analogue Inhibitors of Escherichia coli γ-Glutamyl-cysteine Synthetase"Bioorg. & Med. Chem.. 6. 1935-1953 (1998)

N. Tokutake、J. Hiratake 等人：“大肠杆菌 γ-谷氨酰半胱氨酸合成酶的过渡态类似物抑制剂的设计、合成和评估”Chem.. 6. 1935-1953 (1998)。

M.Koizumi,J.Hiratake et al.: "A potent trunsition-state annlogue inhibitors of Escherichia coli asparagine synthetase A"J.Am.Chem.Soc.. 121(24). 5799-5800 (1999)

M.Koizumi、J.Hiratake 等人：“大肠杆菌天冬酰胺合成酶 A 的有效扭转态环系物抑制剂”J.Am.Chem.Soc. 121(24)。

J. Hiratake: "Probing the Function of Biocatalysts and Their Applications by Organic Synthetic Methods"Nippon Nogeikagaku Kaishi. 73. 1261-1272 (1999)

J. Hiratake：“通过有机合成方法探讨生物催化剂的功能及其应用”N​​ippon Nogeikagaku Kaishi。