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。抑制剂扰动或稳定PPI形成可以调节蛋白质功能，并将蛋白质以非功能状态阻止。从实验的角度来看，对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。我们想研究一种拟型酶的酶，靶向智毛病，这仅是同二聚体的功能。在溶液中，蛋白质是稳定的二聚体，在许多小时内缓慢交换单体单位。通过诱变，我们可以产生具有部分分离溶液的界面变体，在晶体状态下，所有仍在C2符合均应型二聚体填料中的变体都非常分离。这样可以防止访问单体状态下界面的结构信息，并使调制器的开发变得困难。 Tyr/Cys界面变体导致溶液中的单体化，在晶体中形成了习惯均应二聚体堆积。但是，氧化后，引入的半胱氨酸保留了二硫键连接，并以完全不同的填充布置将单体单元合并在一起，现在揭露了新构型中旧界面的重要环路螺旋图案。重要的是，已知该循环会触发二聚体的形成，现在它发生在与原始二聚体堆积不符的几何形状中。在此循环下方，打开一个小装订口袋。准备容纳小分子调节器，以稳定其与同二聚化不相容的状态中的几何形状。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粘合剂。通过化学膨胀我们的活性位点抑制剂，我们成功地诱导了习惯同型二聚体的过渡为替代性，结构上完全不同的同型二聚体填料，其特征在于同样大的接触界面。新颖的界面无法识别tRNA分子，该分子仅由原始同型二聚体进行酶促。这是新颖的二聚体布置使酶以催化性的无活性状态捕获。通过修改我们的活性位点抑制剂，我们希望开发稳定器，使新发现的二聚体在功能不活跃状态下。这种蛋白质 - 蛋白质界面稳定剂的发展是阻断酶功能的替代概念。该项目是通过定位的诱变，蛋白质晶体学，基于碎片的铅发现，化学合成和生物物理表征，通过质谱，ESR-SPIN共振和等温滴定热量法来完成。实验将由计算机模拟支持。与F. diDerich（合成，Eth Zurich），S。Cianferani（质谱法，史坦斯堡大学）和J. Klare（ESR，Univ。Osnabrück）的合作。

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