Analogs of GTP as novel inhibitors of bacterial c-di-GMP-synthesizing enzymes

描述（申请人提供）：细菌第二信使c-di-GMP（环-（GMP）2）负责诱导某些病原性细菌形成生物膜，一种或多种细菌菌株的复杂结构，其抵抗常规抗生素并与许多感染性疾病如细菌性肺炎、支架阻塞、导管定植、第二信使c-di-GMP在霍乱弧菌、鼠疫耶尔森氏菌、铜绿假单胞菌、大肠杆菌等重要病原菌生物膜形成的调控中起重要作用。coli、S.肠型和S.金黄色。由两个GTP分子生物合成c-di-GMP是由被称为二鸟苷酸环化酶（DGC）的高度保守的酶催化的。已经发表了一些研究，描述了c-di-GMP类似物对其功能的影响，但没有报道描述GTP类似物对其合成的抑制作用或作为替代底物。一种GTP类似物（Rp）1-硫代-GMP已与新月柄杆菌DGC的催化形式共结晶。 我们推测，竞争性抑制剂的GTP转换为c-di-GMP可以开发为新的药物，以抑制生物膜的形成或稳定性。我们将专注于GTP类似物作为c-di-GMP生物合成的竞争性抑制剂的结构-活性关系（SAR）的第一阶段可行性研究。我们将利用申请人在作为核酸代谢酶的抑制剂/替代底物的鸟嘌呤核苷酸的合成和研究方面的背景，以及我们的分包商在DGC的酶和基于细胞的研究方面的背景，以发现将抑制相关细菌利用GTP的新型化合物。我们认识到，这只是发现新的抗生物膜抗菌剂的第一步，因为如此鉴定的核苷酸不太可能是“药物样的”。因此，该项目的第二阶段将致力于对选定的GTP类似物进行化学修饰，以发现抗生物膜抗菌剂的线索。 本项目的具体目标是：1、合成或获得29种GTP类似物，其中对碱基（N2-和8-取代、氮杂和脱氮电子等排体）、糖（O-甲基、阿拉伯糖基、无环）和磷酸部分（亚甲基、二氟亚甲基、亚氨基、硫代）进行修饰。所有新化合物将通过反相或离子交换色谱法纯化，并通过NMR和LCMS方法表征; 2，测试GTP类似物抑制绿脓杆菌DGC“PA 3702”将GTP环化为c-di- GMP的能力;测量焦磷酸盐释放的偶联测定和测量产物形成的HPLC测定将用于该目的。在表明类似物实际上是酶的底物的情况下，即在已经产生修饰的c-di-GMP的情况下，LCMS将用于定量和确定产物的分子结构;和3，选择性地测试有效抑制剂在铜绿假单胞菌测定中对c-di-GMP产生和生物膜形成的影响，以及在铜绿假单胞菌和哺乳动物细胞（HeLa）增殖测定中的选择性。 上述实验的结果将首次详细表征底物类似物与重要DGC结合的SAR，并将用于设计更多的药物样化合物进行合成和测试，可能在该项目的第二阶段进行。该项目的影响至少是双重的。首先，将首次描述CDG的底物结合位点和SAR。第二，将开发用于干扰重要病原菌的生物膜和毒力的药物的先导代。拟议的研究将导致改性底物类似物作为该领域的新研究工具，并导致开发用于治疗生物膜相关疾病的新型治疗剂。 公共卫生相关性：某些致病菌形成生物膜，这是一种抵抗常规抗生素的复杂结构，与肺炎、支架堵塞、导管定植等众多感染性疾病有关，其中一种分子“c-di-GMP”在生物膜形成的调控中起着至关重要的作用，抑制其生物合成是本项目的基础。我们将合成或采购底物GTP的29个类似物，并确定它们作为竞争性抑制剂的结构-活性关系（SAR）和可能的替代底物的酶从铜绿假单胞菌负责c-di-GMP生产。这些研究的结果将在两个领域产生影响。首先，将首次描述生物膜相关酶的底物结合位点和SAR。第二，将开发用于干扰重要病原菌的生物膜和毒力的药物的先导代。

DESCRIPTION (provided by applicant): The bacterial second messenger c-di-GMP (cyclo-(GMP)2) is responsible for inducing certain pathogenic bacteria to form biofilms, complex structures of one or more bacterial strains that resist conventional antibiotics and are associated with numerous infectious diseases such as bacterial pneumonia, stent blockage, catheter colonization, etc. The second messenger c-di-GMP plays an essential role in the regulation of biofilm formation of important pathogens as Vibrio cholerae, Yersinia pestis, P. aeruginosa, E. coli, S. enteric and S. aureus. The biosynthesis of c-di-GMP from two molecules of GTP is catalyzed by highly conserved enzymes known as diguanylate cyclases (DGCs). A few studies have been published describing the effects of analogs of c-di-GMP on its functions, but no reports are available describing the effects of GTP analogs on inhibition of its synthesis or as alternate substrates. One GTP analog, (Rp) 1-thio-GMP, has been cocrystallized with the catalytic form of Caulobacter crescentus DGC. We hypothesize that competitive inhibitors of conversion of GTP to c-di-GMP could be developed as novel drugs to inhibit biofilm formation or stability. We will focus on the structure-activity relationships (SAR) of GTP analogs as competitive inhibitors of c-di-GMP biosynthesis for this phase I feasibility study. We will capitalize on the applicant's background in synthesis and study of guanine nucleotides as inhibitors/alternate substrates of nucleic acid metabolizing enzymes and that of our subcontractor in enzymatic and cell-based study of DGCs to discover novel compounds that will inhibit the utilization of GTP by relevant bacteria. We recognize that this is but the first step in the discovery of new anti-biofilm antibacterials, because the nucleotides so identified will not likely be "drug-like". Consequently, phase II of this project will be devoted to chemical modification of selected GTP analogs to discover leads for anti-biofilm antibacterials. The specific aims of this project are to: 1, synthesize or procure 29 analogs of GTP in which modifications in the base (N2- and 8-substitution, aza and deaza isosteres), sugar (O-methyl, arabino, acyclo) and phosphate moieties (methylene, difluoromethylene, imido, thio) are made. All new compounds will be purified by reverse phase or ion exchange chromatography, and characterized by NMR and LCMS methods; 2, test GTP analogs for their ability to inhibit cyclization of GTP to c-di- GMP by the Pseudomonas aeruginosa DGC "PA3702"; a coupled assay measuring pyrophosphate release and a HPLC assay measuring product formation will be used for this purpose. Where it is suggested that the analog is actually a substrate for the enzyme, i.e. where a modified c-di-GMP has been produced, LCMS will be used to quantitate and determine the molecular structure of the product(s); and 3, selectively test potent inhibitors for effects on c-di-GMP production and biofilm formation in P. aeruginosa assays, and selectivity in P. aeruginosa and mammalian cell (HeLa) proliferation assays. Results of the above experiments will be the first detailed characterization of the SAR for binding of substrate analogs to an important DGC and will be used to design more drug-like compounds for synthesis and testing, likely in Phase II of the project. The impacts of this project will be at least twofold. First, the substrate binding site and SAR for a CDG will be described for the first time. Second, lead generation for drugs that can be developed to interfere with biofilms and virulence of important pathogenic bacteria will be developed. The proposed study will result in modified substrate analogs as new research tools in the field and lead to development of novel therapeutic agents for treatment of biofilm-related diseases. PUBLIC HEALTH RELEVANCE: Certain pathogenic bacteria form biofilms, complex structures that resist conventional antibiotics and are associated with numerous infectious diseases such as pneumonia, stent blockage, catheter colonization, etc. One molecule "c-di-GMP" plays an essential role in the regulation of biofilm formation, and the inhibition of its biosynthesis is the basis for this project. We will synthesize or procure 29 analogs of the substrate GTP, and determine their structure-activity relationships (SAR) as competitive inhibitors and possible alternate substrates of the enzyme from Pseudomonas aeruginosa responsible for c-di-GMP production. Results of these studies will have impact in two areas. First, the substrate binding site and SAR for a biofilm-related enzyme will be described for the first time. Second, lead generation for drugs that can be developed to interfere with biofilms and virulence of important pathogenic bacteria will be developed.