Perturbance of enzyme function by blocking dimer interface formation: Novel route to specific antibiotics

通过阻断二聚体界面形成扰动酶功能：特异性抗生素的新途径

• 批准号: 164232547
• 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 Units
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/164232547?language=en
• 关键词: Perturbance; enzyme; function; blocking; dimer

The tRNA modifying enzyme, tRNA-guanine transglycosylase (Tgt), constitutes a putative target for new selective antibiotics against Shigella bacteria. Based on several crystal structures of Tgt in complex with tRNA the formation of a Tgt homodimer was suggested. Non-covalent nanoESI mass spectrometry that can study protein complexes under non-degrading conditions confirms the dimeric oligomerisation state in solution and 2:1 binding stoichiometry with its Substrate tRNA. Point mutations were introduced to destabilize the protein-protein dimer interface. Enzyme kinetics reveal a reduced catalytic activity of these mutated variants supposedly related to the destabilization of the dimer as further evidenced by both, non-covalent mass spectrometry and X-ray crystallography. Extended active site inhibitors penetrating into the dimer interface region perturb the interaction geometry of the protein-protein contact. These inhibitors will be further developed into sole interface binders preventing dimer formation and thus blocking protein function. Alternatively, virtual and experimental screening will be applied to discover novel interface binders. A tethering approach connecting appropriate disulfides with Cys residues engineered into surface-exposed positions will be performed to identify weak binders in the interface region. These will then be optimized to result in potent interface binders.

TRNA修饰酶，tRNA-鸟氨酸转糖基酶(TGT)，构成了新的选择性抗志贺氏菌抗生素的靶点。根据TGT与tRNA形成的络合物的几种晶体结构，提出了TGT均二聚体的形成。非共价纳米ESI质谱仪可以在非降解条件下研究蛋白质复合体，证实了溶液中的二聚齐聚状态，并与其底物tRNA进行了2：1的结合化学计量比。引入点突变来破坏蛋白质-蛋白质二聚体界面的稳定性。酶动力学显示这些突变变体的催化活性降低，推测与二聚体的不稳定有关，非共价质谱学和X射线结晶学都进一步证明了这一点。延伸的活性部位抑制剂渗透到二聚体界面区域，扰乱了蛋白质-蛋白质接触的相互作用几何形状。这些抑制剂将进一步发展成为唯一的界面结合物，防止二聚体的形成，从而阻断蛋白质的功能。或者，将应用虚拟和实验筛选来发现新的界面粘结剂。将执行一种将适当的二硫化物与设计到表面暴露位置的半胱氨酸残基连接起来的系链方法，以识别界面区域中的弱结合。然后，这些将被优化，以产生有效的界面粘结剂。