Identification of c-di-GMP Derivatives Resistant to an EAL Domain Phosphodiesterase

Shanahan, Carly A.; Gaffney, Barbara L.; Jones, R. A. C.; Strobel, Scott A.

doi:10.1021/bi301510v

Cited by 28 publications

(23 citation statements)

References 56 publications

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“…Chemical analogs of cdiGMP have been developed which are able to inhibit receptor binding to cdiGMP and alter the activity of these proteins. 13–16 However, many such cdiGMP analogues are not membrane permeable and therefore have limited bioactivity. High-throughput screens for allosteric inhibitors often utilize luminescent, fluorescent, or colorimetric readouts for protein binding of small molecules.…”

Section: Introductionmentioning

confidence: 99%

High-Throughput Screening Using the Differential Radial Capillary Action of Ligand Assay Identifies Ebselen As an Inhibitor of Diguanylate Cyclases

et al. 2013

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The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains.

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Section: Introductionmentioning

confidence: 99%

High-Throughput Screening Using the Differential Radial Capillary Action of Ligand Assay Identifies Ebselen As an Inhibitor of Diguanylate Cyclases

et al. 2013

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“…The presence of these phosphodiesterases, and the absence of cdiA-or cAG-specific phosphodiesterases, could have led to the lower observed levels of cdiG in the cell lysate. The enzyme TBD1265, for instance, is a canonical EAL domain-containing phosphodiesterase that is 33-fold more selective for cdiG over cAG, with no activity toward cdiA hydrolysis (33). Furthermore, of the 28 HD-GYP and EAL proteins in V. cholera El Tor, only three HD-GYP enzymes showed cleavage activity for cAG (34).…”

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confidence: 99%

Hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3′, 3′-cGAMP)

Hallberg

Wang

Wright

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

104

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Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3′, 3′-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling.bacterial signaling | surface sensing | second messengers | cyclic dinucleotides | fluorescent biosensor

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“…We have previously demonstrated that dithiophosphate analogs of c-di-GMP are resistant to enzymatic degradation by EAL domain proteins. 66 . Together, these data suggest that replacing both of the phosphodiester bonds with phosphorothioate linkages results in a PDE-resistant analog.…”

Section: Resultsmentioning

confidence: 99%

Nuclease-Resistant c-di-AMP Derivatives That Differentially Recognize RNA and Protein Receptors

Meehan¹,

Torgerson²,

Gaffney

et al. 2016

Biochemistry

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The ability of bacteria to sense environmental cues and adapt is essential for their survival. The use of second-messenger signaling molecules to translate these cues into a physiological response is a common mechanism employed by bacteria. The second messenger 3’-5’-cyclic diadenosine monophosphate (c-di-AMP) has been linked to a diverse set of biological processes involved in maintaining cell viability and homeostasis, as well as pathogenicity. A complex network of both protein and RNA receptors inside the cell activate specific pathways and mediate phenotypic outputs in response to c-di-AMP. Structural analysis of these RNA and protein receptors has revealed the different recognition elements employed by these effectors to bind the same small molecule. Herein, using a series of c-di-AMP analogs, we probed the interactions made with a riboswitch and a phosphodiesterase protein to identify the features important for c-di-AMP binding and recognition. We found that the ydaO riboswitch binds c-di-AMP in two discrete sites with near identical affinity and a Hill coefficient of 1.6. The ydaO riboswitch distinguishes between c-di-AMP and structurally related second messengers by discriminating against an amine at the C2 position, more than a carbonyl at the C6 position. We also identified phosphate-modified analogs that bind both the ydaO RNA and GdpP protein with high affinity, while symmetrically-modified ribose analogs exhibited a substantial decrease in ydaO affinity, but retained high affinity for GdpP. These ligand modifications resulted in increased resistance to enzyme-catalyzed hydrolysis by the GdpP enzyme. Together, these data suggest that these c-di-AMP analogs could be useful as chemical tools to specifically target subsections of the second-messenger signaling pathways.

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Identification of c-di-GMP Derivatives Resistant to an EAL Domain Phosphodiesterase

Cited by 28 publications

References 56 publications

High-Throughput Screening Using the Differential Radial Capillary Action of Ligand Assay Identifies Ebselen As an Inhibitor of Diguanylate Cyclases

High-Throughput Screening Using the Differential Radial Capillary Action of Ligand Assay Identifies Ebselen As an Inhibitor of Diguanylate Cyclases

Hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3′, 3′-cGAMP)

Nuclease-Resistant c-di-AMP Derivatives That Differentially Recognize RNA and Protein Receptors

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