Identification of Small Molecules That Antagonize Diguanylate Cyclase Enzymes To Inhibit Biofilm Formation

Abstract

c Bacterial biofilm formation is responsible for numerous chronic infections, causing a severe health burden. Many of these infections cannot be resolved, as bacteria in biofilms are resistant to the host's immune defenses and antibiotic therapy. New strategies to treat biofilm-based infections are critically needed. Cyclic di-GMP (c-di-GMP) is a widely conserved second-messenger signal essential for biofilm formation. As this signaling system is found only in bacteria, it is an attractive target for the development of new antibiofilm interventions. Here, we describe the results of a high-throughput screen to identify small-molecule inhibitors of diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP. We report seven small molecules that antagonize these enzymes and inhibit biofilm formation by Vibrio cholerae. Moreover, two of these compounds significantly reduce the total concentration of c-di-GMP in V. cholerae, one of which also inhibits biofilm formation by Pseudomonas aeruginosa in a continuous-flow system. These molecules represent the first compounds described that are able to inhibit DGC activity to prevent biofilm formation.

Identification of Anti-Inflammatory and Anti-Hypertensive Drugs as Inhibitors of Bacterial Diguanylate Cyclases

“…Sambanthamoorthy et al 24 used V. cholera carrying a luciferase c-di-GMP-responsive reporter plasmid to screen potential anti-biofilm agents and identified seven DGCs inhibitor out of 66,000 tested. Here, out of the ten compounds selected from the DrugBank database through a hybrid target-and ligand-based virtual screen consensual strategy, eprosartan and sulfasalazine presented similar DGCs inhibition to the compounds selected by HTS.…”

“…Notably, sulfasalazine shares a remarkable structural similarity with other DGCs inhibitors with potent anti-biofilm properties (Figure 8). 24 We also selected an anti-hypertensive drug (eprosartan) from our virtual screening, which revealed itself as an inhibitor of bacterial DGCs. Eprosartan and other angiotensin receptor blockers (ARBs) exert their pharmacological properties acting as a specific antagonist of type 1 angiotensin II receptor (AT1R), one of the core components of the renin-angiotensin system (RAS).…”

“…Recent studies using cell-based high throughput screening identified molecules that impair c-di-GMP intracellular levels through direct inhibition of DGCs 24 or by interference with nucleotide substrate pools. 26,27 In this report, we used an in silico strategy to identify DGC inhibitors.…”

“…23 There are an increasing number of researches for bacterial DGC inhibitors, as an example, the high throughput screening of chemical libraries led to the identification of competitive DGC inhibitors able to interfere with P. aeruginosa and Acinetobacter baumannii biofilms. 24,25 In vivo screenings showed that sulfathiazole and azathioprine have an impact on biofilm formation by indirectly decreasing c-di-GMP concentrations, probably targeting the biosynthesis of GTP. 26,27 Alternatively, strategies based on c-di-GMP modification and GTP analogs also identified specific DGCs inhibitors targeting both active and the allosteric binding sites.…”

“…24,25 In vivo screenings showed that sulfathiazole and azathioprine have an impact on biofilm formation by indirectly decreasing c-di-GMP concentrations, probably targeting the biosynthesis of GTP. 26,27 Alternatively, strategies based on c-di-GMP modification and GTP analogs also identified specific DGCs inhibitors targeting both active and the allosteric binding sites.…”