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Abstract

The gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen associated with drug resistance complications and, as such, an important object for drug discovery efforts. One attractive target for development of therapeutics is the ADP-ribosyltransferase Exotoxin-S (ExoS), an early effector of the type III secretion system that is delivered into host cells to affect their transcription pattern and cytoskeletal dynamics. The purpose of this study was to formulate a real-time assay of purified recombinant ExoS activity for high-throughput application. We characterized the turnover kinetics of the fluorescent dinucleotide 1,N⁶-etheno-NAD+ as co-substrate for ExoS. Further, we found that the toxin relied on any of five tested isoforms of human 14-3-3 to modify vH-Ras and the Rho-family GTPases Rac1, -2, and -3 and RhoC. We then used 14-3-3β–stimulated ExoS modification of vH-Ras to screen a collection of low-molecular-weight compounds selected to target the poly-ADP ribose polymerase family and identified 3-(4-oxo-3,5,6,7-tetrahydro-4H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-2-yl)propanoic acid as an ExoS inhibitor with micromolar potency. Thus, we present an optimized method to screen for inhibitors of ExoS activity that is amenable to high-throughput format and an intermediate affinity inhibitor that can serve both as assay control and as a starting point for further development.

Keywords

ADP-ribosylation bacterial toxins drug discovery enzyme inhibitors

Introduction

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that is best known as a common cause of hospital infections. Pseudomonas species feature both intrinsic and acquired mechanisms of antibiotic resistance and thereby pose a serious threat to human and livestock health. At an early stage in infection, the pathogen makes contact with a cell surface and delivers a small set of effectors into the cytosol using a type III secretion system. These effectors include four exotoxins: the bimodular ExoS and ExoT, the lipase ExoU, and the adenylate cyclase ExoY.^1,2 ExoS and ExoT share 76% amino acid homology and are bifunctional enzymes containing an N-terminal GTPase activating protein (GAP) domain and a C-terminal ADP-ribosyltransferase (ART) domain.² Together, their activities elicit effects that contribute to creating a replication competent niche for the bacteria. The GAP activities of the toxins inactivate Rho GTPases Rho, Rac, and Cdc42, thereby modulating the state of actin polymerization in the host cell.³ The ART activities are directed toward diverse proteins; ExoS ART domain targets include Ras- and Rho-family GTPases^4–6 and ezrin/radixin/moesin proteins.⁷ Expression of ExoS ADP-ribosylation activity is dependent on the association with a mammalian cofactor, originally termed factor activating exoenzyme-S,⁸ and later identified as the zeta/delta isoform of 14-3-3 (YWHAZ).⁹ The interaction with 14-3-3 proteins is mediated by a C-terminal segment of ExoS.^10–12

The prominent early effects of ExoS ART activity, combined with the fact that this activity is required for inhibition of phagocytosis in pneumonia,¹³ make the ExoS ART domain a promising target for intervention strategies. A chemical probe directed at this domain would be useful both for studying cellular pathology and for development of a therapeutic entity to overcome antibiotic resistance.

Methods to detect toxin-mediated ADP-ribosylation include the use of either radioisotope-labeled or biotinylated NAD⁺ for detection of ADP-ribosyl groups on target proteins and the use of fluorescent NAD analog, 1,N⁶-etheno-NAD⁺ (εNAD⁺) for detection of εNAD⁺ hydrolysis. The latter has been used to study a variety of enzymes with NAD-glycohydrolase activity, including phosphodiesterase,¹⁴ Diphtheria toxin,¹⁵ Pertussis toxin,¹⁶ Vibrio splendidus Vis toxin,¹⁷ and ecto-ARTs.¹⁸ A real-time assay using εNAD⁺ has been developed for mono-ARTs¹⁹ and for the Diphtheria-class Pseudomonas toxin ExoA.²⁰ Here, our aim was to formulate an assay of ExoS activity that was amenable to compound screening in a high-throughput format. We verified functional cleavage of εNAD⁺ by recombinant ExoS ART domain and quantified the stimulating effects of several 14-3-3 isoforms on the modification of several small soluble GTPases. Then, we performed a pilot screen and identified a commercially available compound that mimics nicotinamide and that inhibits ExoS activity with micromolar potency in vitro.

Materials and Methods

Materials

Fine chemicals and media were purchased from SigmaAldrich. 1,N⁶-etheno-NAD⁺ was obtained from SigmaAldrich and 1,N⁶-etheno-AMP from Jena Bioscience. Commercial sources of the poly-ADP ribose polymerase (PARP) inhibitor-like compounds are given in Supplementary Table S1. Exosins were purchased from ChemDiv, and their integrity was confirmed by liquid chromatography–mass spectrometry and proton nuclear magnetic resonance analyses. Experimental procedures and analytical data for resynthesized STO1101 and compounds 1–6 are provided in the Supplementary Data.

Molecular Cloning

The cDNA fragment encoding ExoS residues 233–453, subcloned in the NdeI and EcoRI sites of pET28a, was contributed by Yngve Östberg (Umeå University). The cDNA fragments encoding ExoS residues 199–453, 215–453, 233–416, 242–416, 242–435, 242–453, 266–416, 266–435, and 266–453 were PCR-amplified from extracts of P. aeruginosa PAK cells and inserted into pNIC28-Bsa4 by ligase independent cloning. An expression plasmid (pGEX-2TR) for human vH-Ras was provided by Anne Ridley (King’s College, London). The cDNA fragments encoding all other small GTPases were contributed by Pontus Aspenström (Karolinska Institutet, Stockholm), and these were subcloned into pNIC28-Bsa4. Expression plasmids containing the 14-3-3 encoding genes YWHAB (pTvHR21-SGC), YWHAE (pLIC-SGC1), YWHAG (pTvHR21-SGC), YWHAH (pNIC28-Bsa4), and YWHAQ (pNIC28-Bsa4) were contributed by the Structural Genomics Consortium (Oxford University) and obtained from Addgene.

Protein Expression and Purification

All proteins except vH-Ras were expressed as hexahistidine fusion proteins in Escherichia coli strains BL21(DE3) or C41 in rich medium, by induction during logarithmic growth phase using 0.5 mM isopropyl β-D-1-thiogalactopyranoside and overnight bacterial growth at 18 °C. Purification of ExoS constructs, 14-3-3 isoforms, and GTPases was done using immobilized metal affinity chromatography followed by size exclusion chromatography (SEC). vH-Ras was expressed as a glutathione S-transferase fusion protein in E. coli strain BL21(DE3) and purified using glutathione sepharose 4B (GE Healthcare) followed by SEC. Protein containing fractions was pooled after analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and target protein integrity was verified by time-of-flight mass spectrometry.

Enzymatic Assay

Assays were carried out in black flat-bottom 384-well plates (Corning 3573 or Greiner 781900) with a final assay volume of 50 µL. The optimized conditions for compound screening assays contained 100 nM ExoS, 500 nM 14-3-3β, 2 µM vH-Ras, and 25 µM εNAD⁺ in 20 mM HEPES pH 7.5, 50 mM NaCl, 4 mM MgCl₂, and 0.5 mM TCEP. For positive inhibition assay controls, ExoS^233-453 was substituted for ExoS^242-416 or 14-3-3 protein was omitted. The DMSO concentration was kept at 1% when testing compounds. Enzymatic reactions, at ambient temperature (22 °C), were started by addition of εNAD⁺, and fluorescence was followed over time in a CLARIOstar multiplate reader (BMG Labtech) using λ_ex = 302/10 nm (filter) and λ_em = 410/10 nm (monochromator). Fluorescence increase was linear over 30 min. Where applicable, fluorescence was related to concentrations of fluorescent product using dilution series of 1,N⁶-etheno-AMP. Fluorescence data were analyzed and kinetic parameters calculated using Graph Pad (Prism Software). K_M values for ExoS determined based on either fluorescence increase in the linear time range or using endpoint assays at 30 min were identical within experimental error.

Screening Library

A screening set of compounds with molecular weights up to 300 Da was chosen from a previously assembled library of PARP inhibitors and chemically related compounds²¹ (see Suppl. Table S1 for details).

Results and Discussion

To formulate a real-time fluorescence-based assay for ExoS activity, we used the most widely studied toxin fragment (ExoS ART domain residues A233–A453) and its ability to ADP-ribosylate human vH-Ras protein using εNAD⁺ as co-substrate. First, we assessed the ability of five of the seven human 14-3-3 isoforms to activate the ExoS ART domain. The beta, gamma, epsilon, eta, and theta isoforms of human 14-3-3 displayed similar efficiency in activation of ExoS activity, when present in five-molar excess (Suppl. Fig. S1A). Next, we constructed eight different versions of the ExoS ART domain by stepwise extension or truncation of the N-terminal and stepwise truncation of the C-terminal end of the classical ART domain construct (Suppl. Fig. S1B). None of these proteins was purified more efficiently, or displayed higher activity, than ExoS^233–453. A construct spanning residues V242-S416 (lacking the 14-3-3 binding site)¹² showed no ADP-ribosyltransferase activity in the presence of 14-3-3β in five-molar excess. This construct could be produced at satisfactory yields from recombinant E. coli and was found useful as a negative control in the assay. We then tested the ability of ExoS to modify several mammalian Rho-family GTPases that contained an arginine residue in the position homologous to the ExoS target residue R41 of Ras.²² Assays of εADP-ribosyltransfer showed that Rac1, Rac2, Rac3, and RhoC, present in 20-molar excess, were modified to a similar extent as vH-Ras (Suppl. Fig. S1C).

We went on to evaluate whether the N⁶-etheno group on the adenine moiety of NAD affected the kinetics of the ExoS^233–453-catalyzed ADP-ribosyltransferase reaction. First, we determined the catalytic properties of 14-3-3β–stimulated modification of vH-Ras with εNAD⁺ as co-substrate. Based on five independent experiments, we determined an apparent K_M of 22.5 ± 6 µM ( Fig. 1A ; Table 1 ). This is higher than a previous determination using the same method and substrate (9 µM)²³ and lower than the value determined by the use of radiolabeled NAD⁺ and soy bean trypsin inhibitor as target (185.3 ± 32.5 µM).²⁴ Together, these data suggested that the etheno ring in εNAD⁺ might affect the rates of ADP-ribosyl transfer. Therefore, we tested the dependence of the apparent K_M on the percentage of εNAD⁺ in the reaction mixture. As shown in Figure 1B , apparent K_M values increased slightly with increasing [εNAD⁺]/[NAD⁺]. Thus, our data indicate that εNAD⁺ is turned over somewhat less efficiently that NAD⁺. We conclude that differences between kinetic parameters measured here and published previously likely are attributable to assay variations. The simplicity and robustness of the assay warranted consideration of the method for high-throughput screening assays to discover novel inhibitors of ExoS ADP-ribosyltransferase activity.

Figure 1.

Enzymatic properties of ExoS^233–453. (A) Dependence of ExoS ADP-ribosyltransferase activity on εNAD⁺ concentration. K_M values determined from this and similar experiments are reported in Table 1 . (B) Dependence of K_M values, determined as in panel A, on the percentage of εNAD⁺ in the assay mixture.

Table 1.

ExoS assay parameters and statistics.

ExoS^233–453	100 nM
14-3-3β	500 nM
vH-Ras	2 µM
εNAD+	25 µM
Format	384-well plates
Assay volume	50 µL
Enzymatic properties
K_M^εNAD (mean ± SD, N = 5; n = 11)	22.53 ± 5.96 µM
Screening assay statistics
Standard error positive control	8.4%
Standard error negative control	1.58%
Z′-factor	0.70

To test whether this assay of ExoS activity was indeed useful for the identification of ExoS inhibitors, we screened part of a previously described library of PARP inhibitors and chemically related compounds.²¹ A screening set of compounds with molecular weights up to 300 Da was chosen from this library (Suppl. Table S1). The rationale for choosing this screening set was the likelihood of identifying low-potency inhibitors. ExoS shares the general features of the ADP-ribosyl transfer catalysis with PARP enzymes; therefore, we reasoned that some of the smaller nicotinamide-mimicking PARP inhibitors might bind to the ExoS active site.

First, we tested the DMSO tolerance of the assay. Enzymatic activity was unaffected, within experimental error, by DMSO in the range of 0% to 2% (Suppl. Table S1). We then commenced and tested the compounds (at 25 µM; 1% DMSO) for inhibition of ExoS ADP-ribosyltransferase activity under the conditions summarized in Table 1 . Assay statistics were favorable, with a Z′-factor²⁵ of 0.7 ( Table 1 ). The majority of the compounds tested, and likewise three previously reported ExoS inhibitors termed exosins,²³ showed weak or no significant inhibition of ExoS activity (Suppl. Table S1). However, we identified three compounds that inhibited ExoS activity by more than three standard deviations of the positive control reaction relative to the negative control containing no 14-3-3 protein ( Fig. 2 ).

Figure 2.

A screen for enzymatic inhibitors of ExoS^233–453 using a small collection of PARP inhibitors and chemically related compounds. Correlation plot of data from three independent screens, carried out with independent protein batches. Data are shown as mean percentages ± SD (N = 2, n = 4) of control reactions (containing 1% DMSO), corrected for background signal in the absence of 14-3-3β. The chemical structure of STO1101, the most active compound, is shown. Full data along with compound information are given in Supplementary Table S1.

The most potent primary screening hit, compound STO1101, was further characterized in two different inhibition experiments ( Fig. 3 ): (1) K_M experiments were conducted in the presence of different concentrations of STO1101. We calculated a K_i of 8.6 ± 3 µM (n = 4) based on the results ( Fig. 3A ). Lineweaver-Burk plots of these data also established STO1101 as a competitive inhibitor of ExoS ( Fig. 3B ). (2) Concentration-response experiments were carried out to determine an IC₅₀ value of 19 µM ( Fig. 3C ). Considering our assay conditions and K_M = 22.5 µM ( Table 1 ; Fig. 1 ) and using the Cheng-Prusoff equation,²⁶ this translates into a K_i value of 9.0 µM, in good agreement with the previous value. For the two less potent inhibitors, we determined IC₅₀ values of 148 µM (STO1222) and 437 µM (STO1401; data not shown).

Figure 3.

Validation of the primary screening hit. (A) Michaelis-Menten kinetics experiments in presence of different concentrations of STO1101 illustrate its concentration-dependent inhibition of ExoS activity. (B) A Lineweaver-Burk plot of the data presented in panel A established STO1101 as a competitive inhibitor of ExoS with an apparent K_i of 8.6 µM. (C) Concentration-response experiments for STO1101 indicated an IC₅₀ value of 19 µM. All data are based on both commercial and resynthesized compound.

Following the finding that STO1101 inhibited ExoS ADP-ribosylation activity with reasonable potency, we resynthesized the compound and confirmed its identity and potency. Furthermore, to establish primary structure-activity relationships (SARs), we synthesized a small set of analogues to investigate the importance of the main functional groups of STO1101 ( Table 2 ; Suppl. Fig. S2). Compound 1, in which the five-membered ring at the distal end of STO1101 is expanded to a six-membered ring, showed reduced activity with an IC₅₀ value of 47.9 µM. Reducing the carboxylic acid moiety in STO1101 to a hydroxyl group attenuated ExoS inhibition (compound 2, IC₅₀ 52.4 µM), and this effect was also observed for compound 3 with a six-membered ring (IC₅₀ 169 µM). Replacement of the five-membered ring and the thiophene moieties by a benzene ring reduced potency (compound 4, IC₅₀ 135 µM). This effect could, however, be compensated for by elongating the spacer between the carboxylic acid and the central scaffold by one methylene group (compound 5, IC₅₀ 24.9 µM). Essentially, all potent PARP inhibitors harbor an amide functionality that mimics the nicotinamide in NAD⁺.²⁷ The fact that STO1101 is a competitive inhibitor ( Fig. 3B ) suggests that this holds true also for this ExoS inhibitor. To probe this, we prepared the methylated derivative 6 and concluded that this modification abrogated activity (IC₅₀ 294 µM), supporting the hypothesis that the amide functionality in STO1101 mimics nicotinamide. This primary SAR analysis indicates that structural modifications of STO1101 are tolerated and forms the basis for continued medicinal chemistry efforts to achieve potent and selective inhibitors of the ExoS ADP-ribosylation activity. Although STO1101 itself is likely not a significant inhibitor of any PARP isoform,²¹ a cell-active ExoS inhibitor based on the same chemical scaffold should be examined for PARP inhibition.

Table 2.

Structure-activity relationship study of the STO1101 scaffold.

Compound	pIC₅₀ ± SD (M)	IC₅₀ (µM)
STO1101	4.72 ± 0.08	19.0
1	4.32 ± 0.11	47.9
2	4.28 ± 0.15	52.4
3	3.77 ± 0.28	169
4	3.87 ± 0.17	135
5	4.60 ± 0.10	24.9
6	3.53 ± 0.21	294

In summary, a real-time fluorescence assay of P. aeruginosa ExoS, using εNAD⁺ as co-substrate, was optimized for high-throughput screening purposes and used to identify and validate STO1101 as a novel ExoS inhibitor with an affinity (K_i) of roughly 9 µM.

Footnotes

Acknowledgements

We thank the Protein Science Facility at Karolinska Institutet and SciLifeLab for molecular cloning.

Supplementary material for this article is available on the Journal of Biomolecular Screening Web site at .

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Swedish Foundation for Strategic Research (grant SB12-0022; Å.F., M.E., and H.S.), the IngaBritt and Arne Lundbergs Research Foundation and Karolinska Institutet (H.S.), and the Swedish Research Council (grant 521-2012-2802; M.E.).

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Supplementary Material

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Identification of Inhibitors of Pseudomonas aeruginosa Exotoxin-S ADP-Ribosyltransferase Activity

Abstract

Keywords

Introduction

Materials and Methods

Materials

Molecular Cloning

Protein Expression and Purification

Enzymatic Assay

Screening Library

Results and Discussion

Footnotes

Acknowledgements

Declaration of Conflicting Interests

Funding

References

Supplementary Material