D-丙氨酰基转移酶与抑制剂分子的相互作用研究

In recent years, more and more antibiotic resistant bacteria are emerging worldwide, some of which even evolved into multidrug resistant bacteria those are known as ‘superbugs’. Gram-positive bacteria take up a great portion of the antibiotic resistant bacteria, and become a big threat to human. Teichoic acids are one of the major cell wall components and specifically exist in gram-positive bacteria. DltB, a multi-pass transmembrane protein, is a D-alanyl transferase that catalyzes the D-alanylation of teichoic acids in gram-positive bacteria. It has been demonstrated that the compound amsacrine, which targets DltB, can inhibit the D-alanylation of teichoic acids and dramatically sensitize Staphylococcus aureus to cationic peptides and aminoglycosides. During my postdoctoral training, I solved the crystal structure of DltB, and found the potential binding site of amsacrine is located in a highly conserved funnel shaped structure that is facing towards the extracellular side. The aim of this proposal is to obtain the crystal structure of DltB and amsacrine complex, and perform structure-based virtual screening and optimization of DltB inhibitors. The resulting potential inhibitors will be further verified through Rosetta binding mimic, biochemical and functional analysis to gain the most potent DltB inhibitors. The results of this proposal can not only reveal the binding and inhibition mechanism of inhibitors on DltB, but also help the further development of new broad-spectrum antibiotics that are specifically towards gram-positive bacteria.

近年来世界范围内出现了越来越多的抗生素耐受细菌，对人类的生命安全造成了严重威胁。 D-丙氨酰基转移酶DltB是多次跨膜蛋白，催化了革兰氏阳性菌磷壁酸的D-丙氨酰化修饰。研究发现以DltB为靶标的化合物amsacrine可以抑制磷壁酸的D-丙氨酰化，极大增强金黄色葡萄球菌对带正电的抗菌肽以及氨基糖苷类抗生素的敏感性。申请人在博士后研究期间解析了DltB的晶体结构，发现amsacrine的潜在结合位点位于高度保守的漏斗形区域。本申请旨在获得amsacrine与DltB的复合物晶体结构，基于三维结构进行计算机辅助的DltB抑制剂虚拟筛选与优化，并通过生化及功能实验验证以获得DltB的高效抑制分子。本申请的研究结果不仅能够揭示抑制剂分子与D-丙氨酰基转移酶DltB相结合并抑制其催化活性的分子机制，获得的抑制剂分子还可以进一步应用于针对革兰氏阳性菌的新型广谱抗菌药物的开发。

革兰氏阳性菌D-丙氨酰基转移酶DltB是开发抗生素的重要靶标。本项目整合结构生物学、生物化学、生物物理学、计算生物学、细胞生物学等手段，研究革兰氏阳性菌D-丙氨酰基转移酶DltB与其抑制剂分子的相互作用，并对可能的抑制剂分子进行筛选、优化和验证。部分实验还在进行中，该部分结果可以为针对革兰氏阳性菌开发新型抗生素提供基础和线索。此外DltB所属的跨膜O-酰基转移酶蛋白家族成员在序列和功能上高度保守，因此在作用机制上也相对保守，因此为了帮助了解该类跨膜蛋白和底物的作用关系，本项目还拓展研究了DltB同族蛋白Porcupine的底物Wnt及其转运蛋白WLS的结构与功能。该项研究成果，揭示了WLS介导Wnt分泌的分子基础，能帮助更好地理解Wnt信号分子的传递以及信号通路激活的机制。同时，该研究中获得的高分辨率精确结构模型也将为针对Wnt分泌途径开发抗肿瘤药物提供重要线索。