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

描述（由申请人提供）：本研究的长期目标是探索c-di-GMP在调节细菌信号传导中的作用，并开发干扰c-di-GMP信号传导途径的小分子。C-di-GMP是细菌第二信使，其通常调节许多人类病原体中的生物膜形成，所述人类病原体包括弧菌属，鼠伤寒沙门氏菌、鼠疫耶尔森氏菌和绿脓杆菌。c-di-GMP的细胞水平由合成c-di-GMP的二鸟苷酸环化酶和水解它的磷酸二酯酶的相反活性控制。C-di-GMP、二鸟苷酸环化酶和c-di-GMP磷酸二酯酶在人类中未发现，使得c-di-GMP信号传导系统成为抗细菌干预的有吸引力的靶点。在这里，我们建议确定X-射线晶体结构的二鸟苷酸环化酶的复合物与不同的二鸟苷酸环化酶抑制剂。使用高通量体内遗传筛选鉴定抑制剂，证明其在体外特异性抑制来自不同细菌物种的多种二胍酸环化酶的酶活性，并显示其在静态或流动条件下抑制生物膜形成。X射线晶体结构将揭示抑制剂功能的机理基础，例如，所述抑制剂是否与二鸟苷酸环化酶活性位点结合并充当竞争性抑制剂，并且还鉴定介导化合物-靶相互作用的抑制剂官能团。这些信息将推动结构-活性关系研究，旨在通过反复的化合物重新设计、合成以及体内遗传和体外生化分析来产生更紧密结合的拮抗剂。

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.