Analogs of GTP as Novel Inhibitors of Bacterial C-Di-GMP-Synthesizing Enzymes

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). <P>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.<P> 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. <P>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.<P> 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. <P>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. <P>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. <P>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. <P>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.