Design of potent inhibitors for human brain memapsin 2 (β-secretase)
Design of potent inhibitors for human brain memapsin 2 (β-secretase)
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DOI：
10.1021/ja000300g
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发表时间：
2000-04-12
影响因子：
15
通讯作者：
Tang, J
中科院分区：
文献类型：
--
作者：
Ghosh, AK;Shin, DW;Tang, J
人脑中通过膜锚定 β-淀粉样前体蛋白 (APP) 的蛋白水解产生 40/42 残基的 β-淀粉样蛋白 (Aβ) 肽，是阿尔茨海默病进展的关键事件。 1 参与 Aβ 肽生成的蛋白酶称为 γ 分泌酶和 β 分泌酶。 β-分泌酶催化Aβ产生的限速步骤，水解APP管腔侧容易到达的位点，被视为抑制剂药物设计的主要治疗靶点。我们的实验室最近克隆了一种名为 memapsin 2 的人脑天冬氨酸蛋白酶，我们证明它是人们长期寻找的 β-分泌酶。 2 其他几个实验室也独立发现了相同的酶。 3 关于 memapsin 22 动力学和特异性的新知识使我们能够设计和测试两种有效的人 memapsin 2 抑制剂。重组 memapsin 2 很难水解 APP (SEVKM/DAEFR) 的 β-分泌酶位点 (kcat/Km) 40 s-1 M-1)。然而，来自瑞典突变体 APP (SEVNL/DAEFR) 的同一位点是优秀的 基材（kcat/Km）2450 s-1 M-1）。 2 因此，在 memapsin 2 抑制剂的初始设计中，我们利用瑞典 APP 的 β 分泌酶位点模板，将 P1' Asp 更改为 Ala。P1' 位点的特异性不仅表明 Ala 是高度优选的残基，2 这样的变化还降低了抑制剂的极性并增加了亲脂性，这是穿透血脑屏障的重要因素。 4 抑制剂中 P1 和 P1' 位点之间的肽键被羟基乙烯过渡态电子等排体取代，这是一种用于抑制天冬氨酸蛋白酶的高效过渡态类似物。 5 最初设计的两种抑制剂 OM99-1 (1) 和 OM99-2 (2) 的结构如图 1 所示。这些抑制剂的合成策略是首先合成 Leu* Ala 二肽等排体（星号代表羟基乙烯等排体），然后将其用于抑制剂的固态肽合成6。 Leu* Ala 的合成如方案 1 所示。通过用氯甲酸异丁酯和 N-甲基哌啶处理，然后用 N,O-二甲基羟胺处理所得混合酸酐，将商业 Boc-亮氨酸转化为 Weinreb 酰胺 3。 7 在乙醚中用氢化铝锂还原 3，得到醛 4，醛 4 与由丙炔酸乙酯和二异丙基氨基锂处理得到的丙炔酸锂反应，生成
The generation of the 40/42-residue amyloid β (Aβ) peptide in human brain by proteolysis of the membrane anchored β-amyloid precursor protein (APP) is a key event in the progression of Alzheimer’s disease. 1 Proteases involved in the production of Aβ peptide are known as γ-and β-secretases. β-Secretase, which catalyzes the rate-limiting step in Aβ production, hydrolyzes an easily accessible site in the luminal side of APP, and is regarded as the major therapeutic target for the design of inhibitor drugs. Our laboratory recently cloned a human brain aspartic protease called memapsin 2, which we demonstrated to be the long sought β-secretase. 2 Several other laboratories also independently discovered the same enzyme. 3 The new knowledge on kinetics and specificity of memapsin 22 has enabled us to design and test two potent inhibitors for human memapsin 2. The β-secretase site of APP (SEVKM/DAEFR) is hydrolyzed poorly (kcat/Km) 40 s-1 M-1) by recombinant memapsin 2. However, the same site from the Swedish mutant APP (SEVNL/DAEFR) is an excellent substrate (kcat/Km) 2450 s-1 M-1). 2 Thus, for the initial design of memapsin 2 inhibitors, we utilized the template of the β-secretase site of Swedish APP with a change of P1′ Asp to Ala. Not only does the specificity at the P1′ site indicate that Ala is a highly preferred residue, 2 such a change also reduces polarity and increases lipophilicity of the inhibitor, factors important for blood-brain barrier penetration. 4 The peptide bond between P1 and P1′ sites in the inhibitors is replaced by a hydroxyethylene transition-state isostere, which is a highly effective transition-state analogue for the inhibition of aspartic proteases. 5 The structures of two of the initially designed inhibitors, OM99-1 (1) and OM99-2 (2), are shown in Figure 1. The strategy for the synthesis of these inhibitors is to first synthesize Leu* Ala dipeptide isostere (the asterisk represents hydroxyethylene isostere) which was then used in solid-state peptide synthesis6 of the inhibitors. The synthesis of Leu* Ala is outlined in Scheme 1. Commercial Boc-leucine was converted to Weinreb amide 3 by treatment with isobutyl chloroformate and N-methylpiperidine followed by treatment of the resulting mixed anhydride with N, O-dimethylhydroxylamine. 7 Reduction of 3 with lithium aluminum hydride in diethyl ether provided the aldehyde 4 which was reacted with lithium propiolate derived from the treatment of ethyl propiolate and lithium diisopropylamide to