Synthesis of substrates and inhibitors of botulinum neurotoxin type A metalloprotease.
Synthesis of substrates and inhibitors of botulinum neurotoxin type A metalloprotease.
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A 型肉毒杆菌神经毒素金属蛋白酶的底物和抑制剂的合成。
DOI：
10.1111/j.1399-3011.2004.00124.x
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Rich,DH
中科院分区：
文献类型：
--
作者：
Sukonpan,C;Oost,T;Goodnough,M;Tepp,W;Johnson,EA;Rich,DH
肉毒杆菌神经毒素（BoNT）金属蛋白酶及其相关蛋白酶是已知的最具选择性的蛋白酶。X射线晶体结构表明，天然酶的活性位点以催化活性不强的形式存在，必须通过底物结合来激活。为了表征假定的底物诱导的酶活化构象变化，我们合成了一系列过渡态类似物抑制剂，其中二肽切割位点被最小底物肽序列内的四面体中间类似物取代。在本文中，我们报告了我们的努力，设计抑制剂的BoNT/A金属蛋白酶。我们确认BoNT/A金属蛋白酶的有效底物序列是对应于SNAP-25的残基187-203的17聚体肽。合成了一种更稳定的底物Nle 202 SNAP-25 [187-203]，以开发BoNT/A金属蛋白酶的蛋白水解活性测定法，该测定法可用于监测时间依赖性抑制。通过固相肽合成将Gln-197的α-巯基酰胺类似物掺入两种17聚体最小肽底物序列中。描述了全毒素A底物的α-硫代酰胺类似物的合成、表征和抑制动力学。这些底物衍生的抑制剂被证明是BoNT/A催化活性的亚微摩尔抑制剂。
Botulinum neurotoxin (BoNT) metalloproteases and related proteases are the most selective proteases known. X‐ray crystal structures suggest that the active sites of the native enzymes exist in catalytically incompetent forms that must be activated by substrate binding. In order to characterize the postulated substrate‐induced conformational changes for enzyme activation, we synthesized a series of transition‐state analog inhibitors in which the dipeptide cleavage site is replaced by tetrahedral intermediate analogs within the minimal substrate peptide sequence. In this paper, we report our efforts to design inhibitors of BoNT/A metalloprotease. We confirm that an effective substrate sequence for BoNT/A metalloprotease is a 17‐mer peptide corresponding to residues 187–203 of SNAP‐25. A more stable substrate, Nle202SNAP‐25 [187–203] was synthesized in order to develop an assay for proteolytic activity of BoNT/A metalloprotease that can be used to monitor time‐dependent inhibition.α‐Thiol amide analogs of Gln‐197 were incorporated via solid‐phase peptide synthesis into both 17‐mer minimal peptide substrate sequences. The synthesis, characterization and inhibition kinetics for theα‐thiol amide analogs of holotoxin A substrate are described. These substrate‐derived inhibitors were shown to be submicromolar inhibitors of BoNT/A catalytic activity.