The immunosuppressive drug azathioprine inhibits biosynthesis of the bacterial signal molecule cyclic-di-GMP by interfering with intracellular nucleotide pool availability

Abstract

In Gram-negative bacteria, production of the signal molecule c-di-GMP by diguanylate cyclases (DGCs) is a key trigger for biofilm formation, which, in turn, is often required for the development of chronic bacterial infections. Thus, DGCs represent interesting targets for new chemotherapeutic drugs with anti-biofilm activity. We searched for inhibitors of the WspR protein, a Pseudomonas aeruginosa DGC involved in biofilm formation and production of virulence factors, using a set of microbiological assays developed in an Escherichia coli strain expressing the wspR gene. We found that azathioprine, an immunosuppressive drug used in the treatment of Crohn's disease, was able to inhibit WspR-dependent c-di-GMP biosynthesis in bacterial cells. However, in vitro enzymatic assays ruled out direct inhibition of WspR DGC activity either by azathioprine or by its metabolic derivative 2-amino-6-mercapto-purine riboside. Azathioprine is an inhibitor of 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase, an enzyme involved in purine biosynthesis, which suggests that inhibition of c-di-GMP biosynthesis by azathioprine may be due to perturbation of intracellular nucleotide pools. Consistent with this hypothesis, WspR activity is abolished in an E. coli purH mutant strain, unable to produce AICAR transformylase. Despite its effect on WspR, azathioprine failed to prevent biofilm formation by P. aeruginosa; however, it affected production of extracellular structures in E. coli clinical isolates, suggesting efficient inhibition of c-di-GMP biosynthesis in this bacterium. Our results indicate that azathioprine can prevent biofilm formation in E. coli through inhibition of c-di-GMP biosynthesis and suggest that such inhibition might contribute to its anti-inflammatory activity in Crohn's disease.

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

“…These results correlate very well with the inhibitory activity and affinity of the compounds against WspR and YdeH measured in the biochemical assays, suggesting that a reduction in intracellular c-di-GMP levels promoted either by compounds that direct inhibits DGCs or by interference with the bacterial nucleotide pool, might be responsible for the reducing of cell aggregates. Finally, in agreement with previous results reported by Antoniani et al,26,27 the potent anti-biofilm agent sulfathiazole almost completely abolished DGC-induced cell aggregation, validating our assays. Growing evidence has established that high c-di-GMP intracellular concentrations inhibit bacterial motility while stimulates the production of extracellular polysaccharides and other adhesion factors, leading to a transition between planktonic and biofilm lifestyles.…”

“…26,27 In this report, we used an in silico strategy to identify DGC inhibitors. Since the selected compounds are FDA-approved drugs, their scaffolds lie within a privileged chemical-biology space, where their pharmacokinetic properties and toxicological profiles are well established.…”

“…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. 28,29 In this report, we identified novel inhibitors of bacterial DGCs by virtual screening methods (VS) from the DrugBank database.…”

Purine Biosynthesis, Biofilm Formation, and Persistence of an Insect-Microbe Gut Symbiosis

“…Based on this observation, we hypothesized that Burkholderia symbionts generate biofilms in the host gut to successfully establish an insect-microbe symbiosis. Because this biofilm formation is regulated by the intracellular level of cyclic-di-GMP (c-di-GMP) (15), which is affected by the purine nucleotide pool (16)(17)(18), we aimed to generate biofilm-defective strains by manipulating the intracellular purine nucleotide pool.…”

In Silico Discovery and In Vitro Validation of Catechol-Containing Sulfonohydrazide Compounds as Potent Inhibitors of the Diguanylate Cyclase PleD

“…Currently, different approaches to inhibit c-di-GMP signaling have been described (5), which are based mainly on whole-cell assays or in vitro screening of small-molecule libraries (6)(7)(8)(9). Besides these strategies, structure-based rational design represents an important tool to retrieve novel molecules and gain mechanistic knowledge to target c-di-GMP signaling in bacteria.…”