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

蛋白-蛋白界面干扰（PPI）是一种新的治疗干预方法。干扰或稳定PPI形成的抑制剂可以调节蛋白质的功能，并使蛋白质处于非功能状态。从实验的角度来看，同型二聚体的研究尤其具有挑战性，因为需要单独的单体单元的操作是不可能的。我们想要研究一种trna修饰酶，以对抗志贺氏菌病为目标，它只具有同型二聚体的功能。在溶液中，蛋白质是稳定的二聚体，在许多小时内缓慢地交换单体单位。通过诱变，我们可以在溶液中产生部分或完全解离的界面变体，在结晶状态下，所有变体仍然以c2对称的同型二聚体排列。这阻碍了在单体状态下获取界面的结构信息，并使调制剂的开发难以分解同型二聚体。Tyr/Cys界面的变化导致溶液中单体化，在晶体中形成习惯的同二聚体堆积。然而，在氧化后，引入的半胱氨酸残基发生二硫键并以完全不同的包装排列将单体单元合并在一起，现在以新的构象暴露出旧界面的关键环-螺旋基序。重要的是，已知该环可以触发二聚体的形成，并且现在以与原始二聚体堆积不相容的几何形状发生。在这个环的下面，打开一个小的装订袋；准备容纳一个小分子调节剂，以稳定其几何形状在不相容的同二聚化状态。在提议的项目中，我们希望开发这种能够阻止蛋白质-蛋白质形成的变构表面粘合剂。通过活性位点抑制剂的化学膨胀，我们成功地诱导了习惯的同型二聚体向另一种结构完全不同的同型二聚体的转变，其特征是具有同样大的接触界面。新的界面不能识别tRNA分子，tRNA分子只能由原同源二聚体进行酶处理。因此，新的二聚体排列使酶处于催化非活性状态。通过对活性位点抑制剂的修饰，我们希望开发稳定剂，将新发现的二聚体冻结在功能失活状态。这种蛋白质-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。这些实验将得到计算机模拟的支持。计划与F. Diederich（合成，苏黎世联邦理工学院），S. Cianferani（质谱，斯特拉斯堡大学）和J. Klare （ESR，奥斯纳布尔<e:1>大学）小组合作。

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.