Inhibitors of c-di-GMP synthesis and degradation

c-di-GMP 合成和降解抑制剂

• 批准号: 7847648
• 负责人: STEPHEN LORY
• 金额: $ 29.66万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-22 至 2012-01-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7847648
• 关键词: Antibiotic Resistance; Binding; Binding Sites; Biological; Biological Assay; Biology; Carbohydrates; Cells; Chemicals; Chronic; Communities; Development; Dinucleoside Phosphates; Enzymes; Fimbrial Adhesins; Genes; Genome; Glucans; Gram-Negative Bacteria; Growth; Guanidines; Host Defense; Human; Lead; Life Style; Ligands; Link; Maintenance; Maps; Microbial Biofilms; Organic Synthesis; Organism; Pathway interactions; Phosphodiesterase Inhibitors; Polysaccharides; Production; Property; Proteins; Pseudomonas aeruginosa; Reagent; Recombinants; Relative (related person); Research; Respiratory Tract Infections; Role; Second Messenger Systems; Specificity; Structure; Structure-Activity Relationship; Surface; Testing; Therapeutic; Tissues; Virulence; Work; analog; antimicrobial drug; base; bis(3' ,5' )-cyclic diguanylic acid; chemical genetics; cofactor; crosslink; cystic fibrosis patients; diguanylate cyclase; efflux pump; extracellular; high throughput screening; in vivo; inhibitor/antagonist; mutant; novel; novel therapeutics; pathogen; phosphoric diester hydrolase; pre-clinical; programs; protein expression; receptor; second messenger; small molecule; small molecule libraries; tool

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.

蛋白质和能够促进多细胞群落细菌生长的蛋白质和碳水化合物的表达 称为生物膜是许多人类病原体的重要毒力特性。在假单胞菌中 在铜绿和许多其他革兰氏阴性细菌中，控制这些表面结构的形成 通过第二信使循环DI GMP（C-DI-GMP）的细胞水平。 C-DI-GMP的浓度为 由两类酶的拮抗活性决定。二甘氨酸酸环酶（DGC）催化 C-DI-GMP的形成，而磷酸二酯酶（PDES）负责该调节的降解 二核苷酸。在这个项目中，将使用化学方法来识别和表征DGC的抑制剂 和PDE。铜绿假单胞菌WSPR调节了几种生物膜成分的生产 富含葡聚糖的PEL多糖，而PDE的ROCR已被证明可以调节CUPC的产生 纤维化粘附素。这两种高度活性蛋白的重组形式大量纯化， 用于开发用于C-DI-GMP合成和降解的特定酶试验。在目标1中 提案，我们将利用有机合成制备或酶促制备的C-DI-GMP的合成类似物 使用WSPR的多动突变体合成。它们将不仅被视为WSPR和ROCR抑制剂的测试 同样是针对铜绿假单胞菌生产的其他DGC和PDE的活动，目的 识别广谱抑制剂。我们还将测试候选抑制剂的结合能力 受体并干扰天然C-DI-GMP配体的结合。活性的结构衍生物 将订购和测试化合物，以建立各种化学部分的结构活性关系。 在AIM 2中，我们将表征用于体内抑制铜绿假单胞菌中靶酶的活性化合物 并评估他们在生物膜开发方面的生物学活动。这些化合物还将被测试为潜力 通过该生物产生的特定外排泵消除的底物。除了提供有价值的 探测C-DI-GMP在毒力中的作用，DGC和PDE抑制剂鉴定的研究工具 应该为针对这些试剂开发到的临床前计划提供基础 靶向生物膜形成的广谱抗菌剂。