Inhibitors of c-di-GMP synthesis and degradation

促进细菌在多细胞群落中生长的蛋白质和碳水化合物的表达 生物被膜是许多人类病原体的重要毒力属性。假单胞菌 在铜绿假单胞菌和许多其他革兰氏阴性细菌中，这些表面结构的形成受到控制 通过细胞水平的第二信使环状双GMP(c-di-GMP)。C-di-GMP的浓度为 由两类酶的拮抗活性决定。二鸟苷环化酶(DGC)催化 C-di-GMP的形成，而磷酸二酯酶(PDE)负责这种调节的降解 二核苷酸。在这个项目中，将使用化学方法来鉴定和表征DGC的抑制剂。 和PDE。铜绿假单胞菌WSPR调节几种生物膜成分的产生，包括 富含葡聚糖的PEL多糖，而ROCR，一种PDE，已被证明调节CuPc的产生 菌毛粘附素。对这两种高效蛋白的重组形式进行了大量纯化和鉴定 建立了c-di-GMP合成和降解的特异性酶促分析方法。在这个目标1中 建议，我们将利用有机合成或酶促合成的c-di-GMP类似物。 利用WSPR的超活性突变体合成。它们不仅将作为WSPR和ROCR的抑制剂进行测试，而且 也用于它们对铜绿假单胞菌产生的其他DGC和PDE的活动，目的是 鉴定广谱抑制剂。我们还将测试候选抑制剂与c-di-GMP结合的能力。 并干扰天然c-di-GMP配体的结合。活性化合物的结构衍生物 将对化合物进行排序和测试，以建立各种化学部分的结构-活性关系。 在目标2中，我们将对体内抑制铜绿假单胞菌靶标酶的活性化合物进行表征。 并评估其对生物膜发育的生物活性。这些化合物也将被测试为潜在的 由这种生物产生的特定外排泵排出的底物。除了提供有价值的 探索c-di-GMP在毒力、DGC和PDE抑制剂鉴定中的作用的研究工具 应为临床前计划提供基础，旨在将这些试剂开发为 针对生物被膜形成的广谱抗菌剂。

Expression of proteins and carbohydrates capable of promoting bacterial growth in multicellular communities called biofilms is an important virulence property of a number of human pathogens. In Pseudomonas aeruginosa and in many other gram-negative bacteria, the formation of these surface structures is controlled by the cellular levels of the second messenger cyclic di GMP (c-di-GMP). The concentrations of c-di-GMP are determined by the antagonistic activities of two classes of enzymes. Diguanylate cyclases (DGCs) catalyze the formation of c-di-GMP while phosphodiesterases (PDEs) are responsible for the degradation of this regulatory dinucleotide. In this project a chemical approach will be used to identify and characterize inhibitors of DGCs and PDEs. The P. aeruginosa WspR regulates the production of several biofilm components including the glucan-rich PEL polysaccharide, while RocR, a PDE, has been shown to regulate the production of the CupC fimbrial adhesin. Recombinant forms of these two highly active proteins were purified in large quantities and were used to develop specific enzymatic assay for c-di-GMP synthesis and degradation. In Aim 1 of this proposal, we will utilize synthetic analogues of c-di-GMP prepared by organic synthesis or by enzymatic synthesis using hyperactive mutants of WspR. They will be tested not only as inhibitors of WspR and RocR but also for their activities against other DGCs and PDEs produced by P. aeruginosa, with the objective of identifying broad-spectrum inhibitors. We will also test the candidate inhibitors for their ability to bind c-di-GMP receptors and interfere with the binding of the natural c-di-GMP ligands. Structural derivatives of active compounds will be ordered and tested to establish structure-activity relationships of various chemical moieties. In Aim 2 we will characterize the active compounds for in vivo inhibition of the target enzymes in P. aeruginosa and assess their biological activities on biofilm development. The compounds will be also tested as potential substrates for elimination by specific efflux pumps produced by this organism. In addition to providing valuable research tools to probe the role of the c-di-GMP in virulence, the identification of DGC and PDE inhibitors should provide the basis for a preclinical program directed towards the development of these reagents into broad-spectrum antimicrobial agents targeting biofilm formation.