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是细菌的第二信使，通常在许多人类病原体中调节生物膜的形成，其中包括弧菌、鼠伤寒沙门氏菌、鼠疫耶尔森菌和铜绿假单胞菌。c-二gmp的细胞水平由合成c-二gmp的二胍酸环化酶和水解它的磷酸二酯酶的相反活性控制。C-di-GMP、二胍酸环化酶和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，我们提出确定与不同的二胍酸环化酶抑制剂配合物的二胍酸环化酶的x射线晶体结构。这些抑制剂是通过高通量体内基因筛选鉴定出来的，在体外实验中被证明能够特异性抑制来自不同细菌种类的多种双胍酸环化酶的酶活性，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功能的机制基础，例如抑制剂是否与二胍酸环化酶活性位点结合并作为竞争性抑制剂，以及确定介导化合物-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.