X-ray Crystallographic Analysis of Diguanylate Cyclase Enzyme-Inhibitor Complexes

描述(申请人提供)：这项研究的长期目标是探索c-di-GMP在调节细菌信号通路中的作用，并开发干扰c-di-GMP信号通路的小分子。C-di-GMP是一种细菌第二信使，通常调节许多人类病原体的生物膜形成，其中包括弧菌、鼠伤寒沙门氏菌、鼠疫耶尔森氏菌和铜绿假单胞菌。C-di-GMP的细胞水平由合成c-di-GMP的双鸟苷环化酶和水解c-di-GMP的磷酸二酯酶的相反活性控制。C-di-GMP、二鸟苷环化酶和c-di-GMP磷酸二酯酶在人类中没有发现，这使得c-di-GMP信号系统成为抗菌干预的一个有吸引力的靶点。在这里，我们建议测定与不同的双瓜尔酸盐环化酶抑制剂的络合物中的双鸟苷环化酶的X射线晶体结构。通过高通量体内遗传筛选鉴定了这些抑制剂，证明它们在体外能特异性地抑制来自不同细菌物种的多个二愈创木酸环酶的酶活性，并在静态或流动条件下抑制生物膜的形成。X-射线晶体结构将揭示抑制剂作用的机制基础，例如，抑制剂是否与二鸟苷环化酶活性部位结合并作为竞争性抑制剂，也将识别介导化合物-靶标相互作用的抑制剂官能团。这些信息将推动构效关系研究，旨在通过反复几轮化合物重新设计、合成、体内遗传和体外生化分析来产生更紧密的结合拮抗剂。

DESCRIPTION (provided by applicant): The long-term goals of this research are to explore the role of c-di-GMP in regulating bacterial signaling and to develop small molecules that interfere with c-di-GMP signaling pathways. C-di-GMP is a bacterial second messenger that commonly regulates biofilm formation in numerous human pathogens, including, among others, Vibrio sp., Salmonella typhimurium, Yersinia pestis, and Pseudomonas aeruginosa. Cellular levels of c-di-GMP are controlled by the opposing activities of diguanylate cyclases that synthesize c-di-GMP and phosphodiesterases that hydrolyze it. C-di-GMP, diguanylate cyclases, and c-di-GMP phosphodiesterases are not found in humans, making c-di-GMP signaling systems an attractive target for anti-bacterial intervention. Here we propose to determine X-ray crystal structures of diguanylate cyclases in complex with different digualylate cyclase inhibitors. The inhibitors were identified using a high-throughput in vivo genetic screen, demonstrated to specifically inhibit the enzymatic activity of multiple digualylate cyclases from different bacterial species in vitro, and shown to repress biofilm formation under static or flow conditions. The X-ray crystal structures will reveal the mechanistic basis of inhibitor function, e.g., whether the inhibitors are binding to the diguanylate cyclase active site and acting as competitive inhibitors, and also identify the inhibitor functional groups mediating the compound-target interactions. This information will drive structure-activity relationship studies designed t generate tighter binding antagonists through iterative rounds of compound redesign, synthesis, and in vivo genetic and in vitro biochemical analysis.