In Silico Discovery and In Vitro Validation of Catechol-ContainingSulfonohydrazide Compounds as Potent Inhibitors of the DiguanylateCyclase PleD

Silvia Fernicola,a Alessandro Paiardini,b Giorgio Giardina,a Giordano Rampioni,c Livia Leoni,c Francesca Cutruzzolà,a

Serena Rinaldoa

Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italya; Department of Biology and BiotechnologyCharles Darwin, Sapienza University of Rome, Rome, Italyb; Department of Science, University Roma Tre, Rome, Italyc

ABSTRACT

Biofilm formation is responsible for increased antibiotic tolerance in pathogenic bacteria. Cyclic di-GMP (c-di-GMP) is a widelyused second-messenger signal that plays a key role in bacterial biofilm formation. c-di-GMP is synthesized by diguanylate cycla-ses (DGCs), a conserved class of enzymes absent in mammals and hence considered attractive molecular targets for the develop-ment of antibiofilm agents. Here, the results of a virtual screening approach aimed at identifying small-molecule inhibitors ofthe DGC PleD from Caulobacter crescentus are described. A three-dimensional (3D) pharmacophore model, derived from themode of binding of GTP to the active site of PleD, was exploited to screen the ZINC database of compounds. Seven virtual hitswere tested in vitro for their ability to inhibit the activity of purified PleD by using circular dichroism spectroscopy. Two drug-like molecules with a catechol moiety and a sulfonohydrazide scaffold were shown to competitively inhibit PleD at the low-mi-cromolar range (50% inhibitory concentration [IC50] of �11 �M). Their predicted binding mode highlighted key structural fea-tures presumably responsible for the efficient inhibition of PleD by both hits. These molecules represent the most potent in vitroinhibitors of PleD identified so far and could therefore result in useful leads for the development of novel classes of antimicrobi-als able to hamper biofilm formation.

IMPORTANCE

Biofilm-mediated infections are difficult to eradicate, posing a threatening health issue worldwide. The capability of bacteria toform biofilms is almost universally stimulated by the second messenger c-di-GMP. This evidence has boosted research in the lastdecade for the development of new antibiofilm strategies interfering with c-di-GMP metabolism. Here, two potent inhibitors ofc-di-GMP synthesis have been identified in silico and characterized in vitro by using the well-characterized DGC enzyme PleDfrom C. crescentus as a structural template and molecular target. Given that the protein residues implied as crucial for enzymeinhibition are found to be highly conserved among DGCs, the outcome of this study could pave the way for the future develop-ment of broad-spectrum antibiofilm compounds.

In the last decade, the nucleotide cyclic di-GMP (c-di-GMP) hasemerged as the most common bacterial second messenger able

to elicit different cellular responses, including virulence, motility,adhesion, and biofilm development (1, 2). c-di-GMP promotesbiofilm formation by stimulating the biosynthesis of adhesins andexopolysaccharide matrix substances and by inhibiting variousforms of motility (3). Intracellular levels of c-di-GMP are modu-lated by the opposite activities of diguanylate cyclase (DGC) en-zymes (containing the conserved GGDEF domain), which synthe-tize this second messenger from two GTP molecules, and ofphosphodiesterase (PDE) enzymes (containing either the EAL orthe HD-GYP domain), which hydrolyze it to pGpG and GMP,respectively. DCGs and PDEs usually contain signaling domainsthat function as sensors of environmental or cellular cues to mod-ulate their activity and, consequently, c-di-GMP intracellular lev-els (4).

The lack of conserved domains involved in c-di-GMP turnoverin mammalian genomes suggests that small molecules targetingDGCs may represent promising hits for the development of anti-biofilm drugs. Currently, different approaches to inhibit c-di-GMP signaling have been described (5), which are based mainlyon whole-cell assays or in vitro screening of small-molecule librar-ies (6–9). Besides these strategies, structure-based rational design

represents an important tool to retrieve novel molecules and gainmechanistic knowledge to target c-di-GMP signaling in bacteria.A repertoire of such approaches belongs to studies aimed at tar-geting the catalytic site (10) or the inhibitory site (I site) (wherec-di-GMP binds as a negative allosteric regulator) of DGCs(11–13).

Regarding the I site, a series of c-di-GMP analogues have beendesigned, synthesized, and tested for their ability to lock the DGC

Received 4 September 2015 Accepted 21 September 2015

Accepted manuscript posted online 28 September 2015

Citation Fernicola S, Paiardini A, Giardina G, Rampioni G, Leoni L, Cutruzzolà F,Rinaldo S. 2016. In silico discovery and in vitro validation of catechol-containingsulfonohydrazide compounds as potent inhibitors of the diguanylate cyclasePleD. J Bacteriol 198:147–156. doi:10.1128/JB.00742-15.

Editor: G. A. O’Toole

Address correspondence to Serena Rinaldo, serena.rinaldo@uniroma1.it.

S.F. and A.P. contributed equally to this work.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.00742-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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enzymes in an inactive conformation (5). However, these com-pounds are likely ineffective against those DGCs lacking an I site(14) and are linearized in vitro by the EAL subtype of PDEs, if oneof the natural phosphodiester bonds is conserved (15).

As for the active site of DGCs, virtual screening studies werepreviously attempted. Virtual hits for Rv1354c, a potential targetof antituberculosis drugs containing both GGDEF and EAL do-mains, have been identified but not tested in vitro (10). In a pre-vious study, four molecules targeting the active site of the DGCPleD from Caulobacter crescentus were identified by virtual screen-ing (16). These molecules were able to weakly inhibit in vitro theDGC WspR from Pseudomonas aeruginosa only at high concen-trations (50% inhibitory concentration [IC50] ranging from 45�M to 100 �M) but were able to decrease biofilm levels in both P.aeruginosa and Acinetobacter baumannii (16), providing a proof ofconcept that targeting DGCs is a feasible strategy to interfere withbiofilm formation, thus encouraging further in silico screeningcampaigns.

In the present work, a virtual screening approach was under-taken to identify molecules targeting DGC enzymes, using theactive site of the DGC PleD from C. crescentus as a structuraltemplate. Overall, seven compounds were selected as potentialinhibitors of DGCs. Two of these compounds, inhibitors 2 and 7,drastically reduced DGC activity in vitro. At present, the moleculesidentified in this work represent the most efficient compoundstargeting the active site of the DGC enzyme, showing an IC50 of�11 �M.

MATERIALS AND METHODSVirtual screening of the ZINC database. Pharmacophore modeling cal-culations were performed by using LigandScout (17). Default values forthe radii of atom-based chemical features (1.0 Å) and projections (1.4 Å)were used. An excluded volume, comprising residues at the active site ofPleD located within a radius 15 Å from any atom of the ligands, wasinserted into the pharmacophore. The obtained pharmacophore was thenused to screen the purchasable subset of the ZINC database (18), using theZincPharmer server (19). Several runs were carried out, systematicallydischarging one or two features of the pharmacophore each time. Thecompounds retrieved from the pharmacophore searches were dockedinto the active site of PleD, by means of Molegro Virtual Docker (MVD)software (CLCbio). To this end, the obtained three-dimensional (3D)structure of PleD (PDB accession number 2V0N) was prepared by auto-matically assigning bond orders and hybridization and adding explicithydrogens, charges, and Tripos atom types. A search space with a 15-Åradius, centered on the active-site groove, was used for docking. The grid-based MolDock score with a grid resolution of 0.30 Å was used as a scoringfunction, and MolDock SE was used as a docking algorithm (20). For eachligand, 10 runs were defined. The retrieved compounds were ranked ac-cording to their score (obtained with the scoring function implemented inMVD). The means and standard deviations of the scores were then calcu-lated, and those compounds scoring at �2.0 standard deviations frommean were selected and redocked with AutoDock v.4.2.5.1 (21). To thisend, the Lamarckian genetic algorithm (LGA) implemented in AutoDockwas used, using the following values: number of individuals in populationof 150, maximum number of energy evaluations of 2,500,000, maximumnumber of generations of 27 � 103, and rate of gene mutation of 0.02. Allother parameters were kept at their default values. Only those compoundsshowing a similar (root mean square deviation [RMSD] of �2.0 Å)docked pose, as assessed by MVD and AutoDock, were kept. Finally, theobtained compounds were ranked on the basis of predicted affinity andcommercial availability, and the most potent compounds were chosen forin vitro testing.

DGC expression and purification. The pET11-PleD vector was trans-formed into Escherichia coli BL21(DE3)/pLysS cells, and protein expres-sion and purification were performed as described previously (12).

Purification of the DGCs from P. aeruginosa, i.e., (i) YfiNHAMP-GGDEF

(cytosolic portion containing the HAMP-GGDEF domain), (ii)YfiNGGDEF (construct containing the GGDEF domain), and (iii) WspR,was performed as previously described (14, 22).

Measurement of diguanylate cyclase activity. The inhibitory activityof synthetized compounds was measured by circular dichroism (CD)spectroscopy as described previously (23). PleD (0.5 �M) in 20 mM Tris(pH 8.0) and 100 mM NaCl was incubated for 10 min at room tempera-ture with metals (10 mM MgCl2 and 2.5 mM MnCl2), activated by theaddition of 1 mM BeCl2 and 10 mM NaF, and kept at room temperaturefor 30 min. The enzyme was incubated for 15 min with different concen-trations of compounds. The same experiment was performed without aninhibitor as a control (with 5% dimethyl sulfoxide [DMSO]). The digua-nylate cyclase reaction was started by the addition of 100 �M GTP to themixture, and 100 mM CaCl2 was added after 30 min to stop the enzymaticreaction. Within this time frame, the enzyme is not completely inhibited,and it is still working. Samples were analyzed by using a 1-cm quartzcuvette (Hellma) on a Jasco J-710 spectropolarimeter at 25°C. The valuesare the means of data from at least three independent experiments, andthe errors are within 15%.

Spectra were then baseline corrected by subtracting values for thesame samples without the substrate from the raw data, and the signal wasadjusted to zero at 340 nm, as no optical activity is expected at this wave-length. The c-di-GMP content was extrapolated by using the calibrationcurve of c-di-GMP obtained in the presence of compound 2 or 7 (see Fig.S1 in the supplemental material).

The inhibitory activity of 100 �M compounds 2 and 7 against PleDwas also evaluated by reverse-phase high-performance liquid chromatog-raphy (HPLC), as previously described (24), to probe the effect of the soleMn2� or Mg2� ion on controlling catalysis.

Compounds 2 and 7 were also tested on the YfiNHAMP-GGDEF

and WspR DGCs from P. aeruginosa, two DGCs which are in the “on”state and therefore do not require preactivation. Six micromolarYfiNHAMP-GGDEF (in a buffer of 20 mM Tris [pH 7.5], 100 mM NaCl, 1%glycerol) was incubated for 10 min at room temperature with metals (10mM MgCl2 and 2.5 mM MnCl2). Two micromolar WspR (in a buffer of 25mM Tris [pH 7.5], 100 mM NaCl) was incubated for 10 min at roomtemperature with metals (2 mM MgCl2 and 2.5 mM MnCl2). If indicated,this assay was also carried out in the presence of 100 �M or 1 mM EDTAas a chelating agent.

The inhibition assays were performed under the experimental condi-tions described above for PleD.

Fluorescence experiments. Titration of YfiNGGDEF with MANT [2=/3=-O-(N-methyl-anthraniloyl)]-GTP (Life Technologies) was carried outas described in the supplemental material (see Fig. S2 in the supplementalmaterial); displacement of MANT-GTP was then carried out followingthe decrease in fluorescence of the MANT-GTP/YfiNGGDEF complexupon the addition of different amounts of inhibitor 7.

Briefly, protein tryptophans were excited at 280 nm, and the fluores-cence emission spectra were recorded between 400 and 540 nm, as a resultof Förster resonance energy transfer (FRET) to MANT-GTP. The exper-iment was performed by the sequential addition of different amounts ofinhibitor 7 (10 to 120 �M) to 3 �M the YfiNGGDEF/MANT-GTP complex(obtained in the presence of stoichiometric amounts of the nucleotide).The corresponding fluorescence emission signal spectra were recorded ona Fluoromax single-photon counting spectrofluorometer (Horiba Jobin-Yvon) after 8 min of incubation with the inhibitor to reach equilibrium.

RESULTSVirtual screening for identification of novel PleD inhibitors. Onthe basis of the binding mode of the substrate analogue GTP-�-S(guanosine-alpha-thio-triphosphate) bound to PleD from Caulo-

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bacter crescentus CB15 (PDB accession number 2V0N) (12), thechemical features responsible for the key binding interactionswere derived by using a pharmacophore-based approach. A 3Dpharmacophore query resulted in a 14-point pharmacophore hy-pothesis (Fig. 1), including four hydrogen bond acceptor featuresengaged by the triphosphate moiety of GTP-�-S (F1 to F4) andpointing to the side chain of K442 (F1) or to the main chain of thebinding cleft formed by residues F330 and F331 (F2 to F4); an-other hydrogen bond acceptor feature engaged by the carbonylatom of the guanine moiety with the side chain of R366 (F5); twohydrogen bond donor features engaged by the N1- and 2-aminonitrogen atoms of the guanine moiety and pointing at the sidechain of D344 (F6 and F7); two aromatic centroids located at thegeometric center of the bicyclical aromatic moiety of the purine ringand their normal projections (F8 and F9); two metal binding featuresprojecting from the ,-bisphosphate moiety and pointing to theMg601 ion atom (F10 and F11); and three negative charges located atthe geometric center of the three phosphate moieties, respectively(F12 to F14). Finally, in order to take into account the shape of theactive site of PleD, excluded volumes were derived and included inthe final pharmacophore, which was then used to filter the purchas-able subset of the ZINC database (�2.3 � 107 compounds) (18). Thesame 14-point pharmacophore hypothesis was obtained by using an-other GGDEF domain (PDB accession number 4H54) in complexwith GTP-�-S (25).

A total of 9,418 compounds matching at least 6 chemical fea-tures were retrieved in this first step. These virtual hits underwenta second docking-based filtering step by means of the MVD tool. Atotal of 328 molecules with significant scores (�2.0 standard de-viations from the mean) were selected and resubmitted to a fur-ther docking step by means of a second docking tool, AutoDock4.0 (21). Thirty-five compounds showed comparable poses whendocked with both AutoDock 4.0 and MVD (RMSD value of �2Å). The most promising virtual screening hits were selected on thebasis of their predicted affinity for PleD and commercial availabil-ity. This protocol gave us seven molecules (compounds 1 to 7)(Table 1) that were further subjected to in vitro testing.

Effect of compounds selected by virtual screening on in vitroactivity of DGC. The seven compounds identified by in silicoscreening (compounds 1 to 7) (Table 1) were tested in vitro fortheir ability to inhibit the DGC activity of PleD from C. crescentus.Inhibitory activity was measured by using circular dichroismspectroscopy (23).

Two out of the seven compounds tested, namely, compounds 2and 7 (Table 1), were found to significantly reduce the activity ofthe PleD DGC; this result was independently confirmed by assay-ing the residual PleD activity by HPLC (Fig. 2).

To quantitatively evaluate the inhibitory effects of these twocompounds, the IC50 was measured by CD spectroscopy, whosereal-time measurements are not affected by the heat enzyme inac-tivation step, contrary to HPLC analysis. Compounds 2 and 7inhibited PleD activity, showing IC50 values of 11.1 � 1.2 �M and11.1 � 1.1 �M, respectively (Fig. 2).

The high level of similarity between IC50 values displayed bycompounds 2 and 7 is not surprising considering the structuralsimilarity of these molecules (Tanimoto coefficient of �0.9),based on a sulfonohydrazide scaffold carrying a catechol moiety.Binding of compounds 2 and 7 to PleD (Fig. 3) is stabilized bypolar interactions with residues surrounding the GTP bindingpocket, namely, N335, D344, and R366.

The metal binding catechol moiety of both compounds is pre-dicted to chelate the Mg ion, similarly to the -phosphate group ofGTP. To probe this, we tested a series of compounds (compounds8 to 13) with slight substitutions at the benzene ring of the catecholmoiety, obtaining in every case a complete loss of inhibitory po-tency of the compounds.

Given the proposed role of the catechol moiety in binding themetal in the active site, we tested the effects of compounds 2 and 7during catalysis in the presence of the sole Mn2� or Mg2� ion.Interestingly, compound 2 is strongly sensitive to the nature of themetal, being unable to inhibit both PleD or WspR in the presenceof the sole Mg2� ion. On the other hand, compound 7 exerts thesame inhibitory role independently of the divalent ion (data notshown). Therefore, we focused our biochemical characterizationon compound 7.

To exclude unspecific effects of inhibitor 7 on the cyclase ac-tivity, we performed different control experiments aimed at dem-onstrating the competitive binding of such a compound to theactive site of DGCs. Due to the complexity of PleD (nucleotide-dependent oligomerization, extra metal binding site, and preacti-vation by beryllium-induced dimerization [11, 12]), some of theexperiments reported below were carried out with other DGCs.

The capability of compound 7 to chelate metals may affectmetal availability in the active-site pocket of DGCs, thus leading tounspecific inhibition of the cyclase activity, even though experi-ments have been carried out in the presence of excess metals; if so,a similar effect in the presence of excess metal-chelating agents,such as EDTA, can be expected. Nevertheless, we found that theactivity of the DGC WspR (which requires magnesium for catal-ysis but, contrary to PleD, does not contain an extra metal bindingsite or beryllium metal for activation) is not affected by the pres-ence of excess EDTA (both 100 �M and 1 mM) (data not shown),thus suggesting that, in this context, metal binding to the activesite of WspR is not affected by any chelation from compound 7.

Moreover, we exclude any folding effect of compound 7 ontarget enzymes, by means of CD thermal melting experiments. Weperformed the experiment with YfiNGGDEF, which is a more suit-

FIG 1 Pharmacophore hypothesis of a competitive inhibitor of GTP bound toPleD. Protein is shown as gray lines, with the binding site shown as a Van der Waalssurface and colored according to polarity. GTP-�-S is also shown as sticks. Resi-dues interacting with the pharmacophore map are represented as sticks and col-ored by atom type. F1 to F5, hydrogen bond acceptors (red arrows); F6 and F7,hydrogen bond donors (green arrows); F8 and F9, aromatic centroids (blue tori);F10 and F11, metal binding groups (cyan cones); F12 to F14, negative charges (redcones). Exclusion volume spheres are not shown for clarity.

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TABLE 1 Virtual screening hits tested in vitro on PleDa

Compound Catalogue no. 2D representation PleD residual activity (%) at 100 �M

1 Amb343033 108.0

2 Amb2250085 16.6

3 Amb13892792 89.0

4 Amb9689011 96.0

5 Amb442340 106.0

(Continued on following page)

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TABLE 1 (Continued)

Compound Catalogue no. 2D representation PleD residual activity (%) at 100 �M

6 Amb9795870 118.0

7 Amb379455 15.0

8 Amb1861868 103

9 Amb2267567 115

(Continued on following page)

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TABLE 1 (Continued)

Compound Catalogue no. 2D representation PleD residual activity (%) at 100 �M

10 Amb2253897 83

11 Amb2247754 104

12 Amb2247600 87

13 Amb2331527 94

a 2D, two dimensional.

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able system for denaturation experiments than PleD or WspR,given that its oligomeric state is not nucleotide dependent (14).We found that the presence of excess compound 7 (100 �M) doesnot affect the secondary-structure content and the overall stabilityof the protein, thus indicating that the observed inhibitory prop-erty of compound 7 with DGCs is not due to unspecific foldingeffects (see Fig. S3 in the supplemental material); rather, the slightincrease of the melting temperature (see Fig. S3, gray trace) isindicative of binding of compound 7 to the protein, with subse-quent protein stabilization.

These data and the evidence that compound 7 strongly inhibitsother DGCs such as WspR from P. aeruginosa (Fig. 4B) indicatethat the effect of this compound on this class of protein is specific.Moreover, according to our structural model, compound 7should target the active site of DGCs. As mentioned above, manyDGCs, including PleD and WspR, are allosterically inhibited byc-di-GMP (via the I site); to exclude that compound 7 targets the

I site, we tested its effect on a third DGC, i.e., the cytosolic portionof YfiN (here YfiNGGDEF-HAMP) from P. aeruginosa, which isknown to be insensitive to feedback inhibition of c-di-GMP (dueto the presence of a degenerated I site) (14). The residual activityof YfiNHAMP-GGDEF in the presence of 100 �M compound 7 (Fig.4C) is close to zero, similarly to what was observed for the residualactivity of PleD and WspR (Fig. 4A and B), thus confirming thatthe I site is not involved in such DGC inhibition.

In light of these results, it is likely that compound 7 targets theactive sites of both PleD and YfiN, whose degrees of structural simi-larity and conservation are very high (Fig. 5), in particular residuesD344 and N335 (D304 and N295 in YfiN, respectively); therefore, weexpect that this compound is a competitive inhibitor of DGCs.

As previously reported (11, 26), the standard Michaelis-Mentenkinetic scheme is inadequate for the kinetic profile of GGDEF en-zymes, and therefore, the corresponding inhibition profile could notbe informative. In order to bypass this limitation, we designed aFRET-based displacement experiment aimed at demonstrating qual-itatively that inhibitor 7 competes for the GTP binding site. The ex-periment was done with the DGC YfiNGGDEF, for which the bindingaffinity for GTP is quantitatively demonstrated (14), in complex witha stoichiometric amount of the GTP analogue MANT-GTP, whichsaturated the protein (see Fig. S2 in the supplemental material fordetails), and addition of different concentrations of inhibitor 7.

Tryptophan excitation at 280 nm of the MANT-GTP/proteincomplex results in a peak at 440 nm (Fig. 6A, black continuousline), which is minimal in the absence of the YfiNGGDEF protein orabsent in the nucleotide-free protein (see also Fig. S2 in the sup-plemental material). Upon the addition of increasing amounts ofcompound 7 (10 �M to 120 �M), we observed a fluorescencedecrease (Fig. 6A, dashed lines), which reached a baseline valueupon the addition of 120 �M compound 7. The hyperbolic shapeof such a transition indicates that replacement equilibrium is oc-curring (i.e., the fluorophore MANT-GTP dissociates from andcompound 7 binds to the protein) with an apparent dissociationconstant value of �80 �M.

DISCUSSION

It is becoming evident that c-di-GMP is a pivotal second messen-ger involved in bacterial virulence-related phenotypes, includingvirulence factor production, motility, adhesion, and biofilm de-

FIG 2 PleD inhibition assay with various concentrations of inhibitors 2 (A) and 7 (B). Each point of the concentration-response curve represents the mean valueand standard deviation of data from three independent experiments by CD spectroscopy. The line is the best-fit curve generated by Prism software, using thelog-dose-versus-response equation. Points falling on the y axis represent the 100% residual activities obtained in the absence of an inhibitor. Data fitting allowedextrapolation of the IC50s as 11.05 � 1.2 �M and 11.07 � 1.1 �M for inhibitors 2 and 7, respectively. An inhibition assay with 100 �M compounds 2 and 7 wasalso performed by HPLC (gray points).

FIG 3 Predicted binding pose of the sulfonohydrazide and catechol moietiesin the active site of PleD. PleD is shown as a gray ribbon. Compound 2 is shownas sticks as a reference. Conserved residues in the active site interacting withcompounds 2 and 7 are indicated; the interactions are displayed in the box.GTP is shown superposed on the compounds as gray lines, for reference. TheMg601 ion is shown as a green sphere.

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velopment. Therefore, targeting the enzymes involved in c-di-GMP synthesis represents an appealing strategy for the develop-ment of antibiofilm drugs (5). In this study, two novel moleculeswith a 3-nitro-benzenesulfonamide scaffold that inhibit PleD inthe low-micromolar range (IC50 of �11 �M) are described. Toour knowledge, these are the most potent competitive inhibitorsof PleD identified to date.

The active site of the DGC domain of PleD from C. crescentus(PDB accession number 2V0N) was used for in silico screening.Since the mechanism of catalysis of DGC enzymes is not yet fullyunderstood at the atomic level, for the development of a 3D phar-macophore, we focused on the interactions between the nonhy-drolyzable GTP analogue (GTP-�-S) and PleD, as found in the

crystal structure. Three residues have been pinpointed for thestrict specificity of the guanine base for the active site of PleD (8,16): N335, making a hydrogen bond between the N3 of the gua-nine base with its amino group and a hydrogen bond between theN2 and its side-chain carbonyl group; D344, making a hydrogenbond between the N1 of the base and the oxygen of the side chainof its carboxyl group; and R366, making a hydrogen bond with theoxygen atom of the guanine ring. Notably, as assessed by the pre-dicted binding poses, N335 and R366 also interact with the twoidentified inhibitors, even if the chemical entities involved arehighly dissimilar compared to GTP-�-S: a hydrogen bond be-tween the sulfohydrazine moiety of compounds 2 and 7 and N335and another hydrogen bond between the nitro group of the inhib-itors and the side chain of R366. D344 displays no interactionswith the inhibitors, although it is placed at a suitable distance(�3.6 Å) to interact with the nitro group of the compounds.

In addition, the GGDEF motif that is representative of thisfamily of enzymes forms part of the active site, and it is involved inMg ion binding, together with one of the oxygen atoms of thephosphate group of GTP. The catechol group of the identifiedinhibitors is predicted to occupy the same position of the phos-phate group involved in metal chelation, and it is suitably placedto interact with the Mg ion. Indeed, catechols are well-knownmetal binding groups (27), and slight modifications of this moietycompletely abolished the inhibitory potency of the tested com-pounds (compounds 8 to 13) (Table 1).

The lead compounds identified in this study show certainstructural similarities with some previously identified DGC inhib-itors (16). Their lengths range from 13 to 15 longest countableatomic linkages end to end. They are generally linear in shape,sharing similar steric demands.

In particular, compounds 2 and 7 share a hydrazine moietywith the previously identified inhibitors LP3134 and LP4010,which has been predicted to interact with the amino group ofN335. Finally, LP3134 also contain a catechol moiety, althoughthe latter was predicted not to be involved in any interactionwith Mg.

FIG 4 Residual DGC activity of PleD, WspR, and YfiNHAMP-GGDEF. Kinetics of PleD (A), WspR (B), and YfiNHAMP-GGDEF (C) in the presence of 100 �Mcompound 7 (gray traces) were determined. Control experiments without inhibitor are also reported (black traces); the kinetics shapes reported in panels A andB (black traces) are indicative of a product inhibition phenomenon occurring in PleD and WspR, due to the presence of the conserved I site, which is degenerated(and therefore c-di-GMP insensitive) in YfiNHAMP-GGDEF (C).

FIG 5 Structural comparison between the PleD and YfiN GGDEF domains.The PleD and YfiN GGDEF domains are shown as gray and pink ribbons,respectively. Conserved residues in the active site putatively interacting withcompounds 2 and 7 are indicated; compound 2 is shown as green sticks. TheMg ion is shown as a green sphere. The location of the PleD I site is alsoindicated.

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To discard the hypothesis that these compounds target the in-hibitory site of PleD, we also tested them on YfiN from P. aerugi-nosa, confirming that compounds 2 and 7 are also active on YfiNand that the I site is not required for their ability to inhibit DGCs.

Since bacteria usually encode multiple DGC enzymes, we alsoconsidered the possibility that the identified compounds are ableto inhibit GGDEF targets other than PleD and YfiN, involved inc-di-GMP synthesis/signaling processes. Several of these targetsare of great clinical interest (such as Rv1354c as a target of antitu-berculosis drugs [10]). Indeed, a multiple-sequence alignment ofGGDEF domains from representative bacteria shows that residuesat an interacting distance from compounds 2 and 7 are evolution-arily invariant (see Fig. S1 in the supplemental material). The highlevel of evolutionary conservation of the residues of GGDEF do-mains involved in protein-inhibitor contacts suggests that com-pounds 2 and 7 could also inhibit other proteins related to c-di-GMP synthesis/signaling and therefore represent leads for thedesign of broad-spectrum antibiofilm inhibitors.

Conclusions. In summary, we have described the identifica-tion of novel catechol-containing sulfonohydrazide compoundsas inhibitors of DGCs such as PleD, WspR, and YfiN and as po-tential lead compounds for the development of new antivirulencedrugs acting on c-di-GMP signaling. In addition, we report a newand efficient pharmacophore hypothesis for targeting of the PleDactive site. The identified compounds represent the most potent invitro inhibitors of PleD known to date. The compounds describedhere are currently ineffective in reducing c-di-GMP intracellularlevels in bacterial pathogens in which biofilm formation reliesupon c-di-GMP signaling systems (see Fig. S5 and S6 in the sup-plemental material). Further optimization of compounds 2 and 7is in progress and will be reported in due course.

ACKNOWLEDGMENT

We thank Alessio Paone for fruitful discussion on experimental data.

FUNDING INFORMATIONMinistero dell’Istruzione, dell’Università e della Ricerca (MIUR) pro-vided funding to Serena Rinaldo and Giordano Rampioni under grantnumber RBFR10LHD1. Sapienza Università di Roma (Sapienza Univer-

sity of Rome) provided funding to Alessandro Paiardini, Serena Rinaldo,and Francesca Cutruzzolà under grant number C26A149EC4. The ItalianCystic Fibrosis Research Foundation provided funding to Livia Leoni un-der grant number FFC 13/2011.

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