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首页> 外文期刊>Journal of Molecular Biology >Recruiting Zn2+ to mediate potent, specific inhibition of serine proteases.
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Recruiting Zn2+ to mediate potent, specific inhibition of serine proteases.

机译：招募Zn2 +来调节丝氨酸蛋白酶的有效，特异性抑制。

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As regulators of ubiquitous biological processes, serine proteases can cause disease states when inappropriately expressed or regulated, and are thus rational targets for inhibition by drugs. Recently we described a new inhibition mechanism applicable for the development of potent, selective small molecule serine protease inhibitors that recruit physiological Zn2+ to mediate high affinity (sub-nanomolar) binding. To demonstrate some of the structural principles by which the selectivity of Zn2+-mediated serine protease inhibitors can be developed toward or against a particular target, here we determine and describe the structures of thrombin-BABIM-Zn2+, -keto-BABIM-Zn2+, and -hemi-BABIM-Zn2+ (where BABIM is bis(5-amidino-2-benzimidazolyl)methane, keto-BABIM is bis(5-amidino-2-benzimidazolyl)methane ketone, and hemi-BABIM is (5-amidino-2-benzimidazolyl)(2-benzimidazolyl)methane), and compare them with the corresponding trypsin-inhibitor-Zn2+ complexes. Inhibitor binding is mediated by a Zn ion tetrahedrally coordinated by two benzimidazole nitrogen atoms of the inhibitor, by N(epsilon2)His57, and by O(gamma)Ser195. The structures of Zn2+-free trypsin-BABIM and -hemi-BABIM were also determined at selected pH values for comparison with the corresponding Zn2+-mediated complexes. To assess some of the physiological parameters important for harnessing Zn2+ as a co-inhibitor, crystal structures at multiple pH and [Zn2+] values were determined for trypsin-keto-BABIM. The Kdvalue of Zn2+ for the binary trypsin-keto-BABIM complex was estimated to be <12 nM at pH 7.06 by crystallographic determination of the occupancy of bound Zn2+ in trypsin-keto-BABIM crystals soaked at this pH in synthetic mother liquor containing inhibitor and 100 nM Zn2+. In synthetic mother liquor saturated in Zn2+, trypsin-bound keto-BABIM is unhydrated at pH 9.00 and 9.93, and has an sp2 hybridized ketone carbon bridging the 5-amidinobenzimidazoles, whereas at pH 7.00 and 8.00 it undergoes hydration and a change in geometry upon addition of water to the bridging carbonyl group. To show how Zn2+ could be recruited as a co-inhibitor of other enzymes, a method was developed for locating in protein crystals Zn2+ binding sites where design of Zn2+-mediated ligands can be attempted. Thus, by soaking trypsin crystals in high concentrations of Zn2+ in the absence of a molecular inhibitor, the site where Zn2+ mediates binding of BABIM and analogs was identified, as well as another Zn2+ binding site. Copyright 1999 Academic Press.
机译：作为普遍存在的生物过程的调节剂，丝氨酸蛋白酶在表达或调节不当时会引起疾病状态，因此是药物抑制的合理靶点。最近，我们描述了一种新的抑制机制，可用于开发有效的，选择性的小分子丝氨酸蛋白酶抑制剂，这些抑制剂募集生理上的Zn2 +以介导高亲和力（亚纳摩尔级）结合。为了证明可以针对特定靶标开发Zn2 +介导的丝氨酸蛋白酶抑制剂选择性的一些结构原理，在此我们确定并描述了凝血酶-BABIM-Zn2 +，-酮-BABIM-Zn2 +和-hemi-BABIM-Zn2 +（其中BABIM为双（5-ami基-2-苯并咪唑基）甲烷，keto-BABIM为双（5-ami基-2-苯并咪唑基）甲烷酮，半-BABIM为（5-（基-2 -苯并咪唑基）（2-苯并咪唑基）甲烷），并将其与相应的胰蛋白酶-抑制剂-Zn2 +复合物进行比较。抑制剂的结合是由抑制剂的两个苯并咪唑氮原子，N（ε2）His57和OγSer195四面体配位的Zn离子介导的。还确定了在选定的pH值下不含Zn2 +的胰蛋白酶-BABIM和-hemi-BABIM的结构，以便与相应的Zn2 +介导的复合物进行比较。为了评估对于利用Zn2 +作为共抑制剂重要的一些生理参数，对于胰蛋白酶-酮-BABIM，在多个pH和[Zn2 +]值下确定了晶体结构。通过结晶学测定在此pH值下浸泡在含有抑制剂和的合成母液中的胰蛋白酶-酮-BABIM晶体中结合的Zn2 +的占有率，估计二元胰蛋白酶-酮-BABIM络合物中Zn2 +的Kd值<12 nM。 100 nM Zn2 +。在饱和Zn2 +中的合成母液中，胰蛋白酶结合的酮-BABIM在pH值为9.00和9.93时未水合，并具有与5-2基苯并咪唑桥连的sp2杂化酮碳，而在pH 7.00和8.00时会发生水合作用并改变几何形状将水加到桥接羰基上。为了显示如何将Zn2 +募集为其他酶的共抑制剂，开发了一种用于定位蛋白质晶体Zn2 +结合位点的方法，可以尝试设计Zn2 +介导的配体。因此，通过在没有分子抑制剂的情况下将胰蛋白酶晶体浸泡在高浓度的Zn2 +中，可以确定Zn2 +介导BABIM和类似物结合的位点，以及另一个Zn2 +结合位点。版权所有1999，学术出版社。

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