Development of small molecule inhibitors and stabilizers of dimerization of tRNA-guanine transglycosylase to treat Shigellosis

开发治疗志贺菌病的tRNA-鸟嘌呤转糖基酶二聚化小分子抑制剂和稳定剂

• 批准号: 324043133
• 负责人: Professor Dr. Gerhard Klebe
• 金额: --
• 依托单位: Institut für Pharmazeutische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/324043133?language=en
• 关键词: Development; small; molecule; inhibitors; stabilizers

Interference with protein-protein interfaces (PPI) is a new option for therapeutic intervention. Inhibitors either perturbing or stabilizing PPI formation can modulate protein function and will arrest the protein in a non-functional state. From an experimental point of view, the study of homodimers is particularly challenging, as manipulations requiring separate monomer units are impossible. We want to study a tRNA-modifying enzyme, target to fight Shigellosis, which is only functional as homodimer. In solution, the protein is a stable dimer, which exchanges monomer units slowly over many hours. Via mutagenesis, we could produce interface variants with partial to complete dissociation in solution, remarkably in the crystalline state all variants still assemble in C2-symmetrical homodimer packing. This prevents access to structural information about the interface in monomeric state and makes development of modulators breaking-up the homodimer difficult. A Tyr/Cys interface variant results in monomerization in solution, in the crystal the customary homodimer packing is formed. However, upon oxidation the introduced cysteine residues undergo disulfide linkage and merge the monomer units together in a completely different packing arrangement, now exposing a crucial loop-helix motif of the old interface in a new conformation. Importantly, this loop is known to trigger dimer formation and it now occurs in a geometry incompatible with the original dimer packing. Below this loop, a small binding pocket is opened; ready to accommodate a small-molecule modulator to stabilize its geometry in the state incompatible with homodimerization. In the proposed project, we want to develop such allosteric surface binders capable to prevent protein-protein formation. By chemical expansion of our active-site inhibitors, we succeeded to induce a transition of the customary homodimer into an alternative, structurally completely different homodimer packing characterized by an equally large contact interface. The novel interface is incompetent to recognize a tRNA molecule, which is enzymatically only processed by the original homodimer. Thus, the novel dimer arrangement arrests the enzyme in a catalytically inactive state. Via modifications of our active-site inhibitors, we want to develop stabilizers freezing the newly discovered dimer in the functionally inactive state. The development of such protein-protein interface stabilizers is an alternative concept to block enzyme function. The project is accomplished by site-directed mutagenesis, protein crystallography, fragment-based lead discovery, chemical synthesis and biophysical characterization by mass spectrometry, ESR-spin resonance and isothermal titration calorimetry. The experiments will be supported by computer simulations. Collaborations with the groups of F. Diederich (synthesis, ETH Zurich), S. Cianferani (mass spectrometry, Univ. Strasbourg) and J. Klare (ESR, Univ. Osnabrück) are planned.

干扰蛋白质-蛋白质界面（PPI）是治疗干预的新选择。干扰或稳定PPI形成的抑制剂可以调节蛋白质功能，并将蛋白质阻滞在非功能状态。从实验的角度来看，同二聚体的研究特别具有挑战性，因为需要单独单体单元的操作是不可能的。我们想研究一种tRNA修饰酶，靶向对抗志贺氏菌病，它只作为同源二聚体起作用。在溶液中，蛋白质是一个稳定的二聚体，它在许多小时内缓慢地交换单体单元。通过诱变，我们可以产生在溶液中部分至完全解离的界面变体，值得注意的是，在结晶状态下，所有变体仍然组装在C2-对称同源二聚体包装中。这阻止了获得关于单体状态下的界面的结构信息，并且使得难以开发分解同二聚体的调节剂。Tyr/Cys界面变体导致溶液中的单体化，在晶体中形成常规的同二聚体堆积。然而，在氧化时，引入的半胱氨酸残基经历二硫键连接并将单体单元以完全不同的包装排列合并在一起，现在以新的构象暴露旧界面的关键环螺旋基序。重要的是，已知该环会触发二聚体形成，并且它现在以与原始二聚体包装不相容的几何形状发生。在该环下方，打开了一个小的结合口袋;准备容纳小分子调节剂以将其几何形状稳定在与同二聚化不相容的状态。在拟议的项目中，我们希望开发能够防止蛋白质-蛋白质形成的变构表面结合剂。通过我们的活性位点抑制剂的化学膨胀，我们成功地诱导了传统的同二聚体转变成一种替代的、结构上完全不同的同二聚体包装，其特征在于具有同样大的接触界面。这种新的界面不能识别tRNA分子，而tRNA分子只能由原始的同源二聚体进行酶促加工。因此，新的二聚体排列将酶抑制在催化失活状态。通过我们的活性位点抑制剂的修改，我们希望开发稳定剂冻结新发现的二聚体在功能上无活性的状态。此类蛋白质-蛋白质界面稳定剂的开发是阻断酶功能的另一种概念。该项目是通过定点诱变，蛋白质晶体学，基于片段的铅发现，化学合成和生物物理表征质谱，ESR-自旋共振和等温滴定量热法完成的。这些实验将得到计算机模拟的支持。与F. Diederich（synthesis，ETH苏黎世），S. Cianferani（质谱，斯特拉斯堡大学）和J. Kemperor（ESR，奥斯纳布吕克大学）计划。

项目成果

Sugar Acetonides are a Superior Motif for Addressing the Large, Solvent-Exposed Ribose-33 Pocket of tRNA-Guanine Transglycosylase.

糖丙酮化物是解决 tRNA-鸟嘌呤转糖基酶大、溶剂暴露的核糖 33 袋的优越基序

• DOI: 10.1002/chem.201801756
• 发表时间: 2018
• 期刊: Chemistry
• 作者: L. D. Movsisyan;E. Schäfer;A. Nguyen;F. R. Ehrmann;A. Schwab;T. Rossolini;D. Zimmerli;B. Wagner;H. Daff;A. Heine;G. Klebe;F. Diederich
• 通讯作者: F. Diederich