Structure-Aided Design of a LuxR-Type Quorum Sensing SuFEx-Based Potential Inhibitor: Covalent or Competitive Inhibition?

TL;DR

This study designs and tests new quorum sensing inhibitors that may competitively block bacterial communication.

Contribution

The paper introduces novel sulfonyl-based quorum sensing inhibitors and identifies their competitive inhibition mechanism.

Findings

The fluorosulfonyl derivative showed strong inhibition with an IC50 of 15 ± 2 µM.

Kinetic studies confirmed the fluorosulfonyl derivative's stability.

Experiments suggest the inhibition mechanism is competitive, not covalent.

Abstract

New N-benzoyl-l-homoserine lactone derivatives bearing a meta-fluorosulfonyl or a meta-methylsulfonyl group have been designed, synthesized and evaluated as quorum sensing (QS) inhibitors. Docking simulations involving the structure of several targeted LuxR-type receptors suggested that a sulfonyl substituent on the benzene ring can trigger interactions within the binding site, possibly consistent with either covalent SuFEx reaction targeting a tyrosine residue or competitive interaction with additional hydrogen bonding. Biological evaluation of the two meta- methyl or fluorosulfonyl-benzoyl acylhomoserine lactone (AHL) analogs as LuxR-regulated quorumsensing inhibitors showed a significant effect for the fluorosulfonyl derivative with an IC50 value of 15 ± 2 µM, while the methylsulfonyl was found to be a weak inhibitor. The stability of the fluorosulfonyl derivative was confirmed by kinetic studies based on 19F NMR experiments. Investigations dedicated to defining the mechanism of action, either covalent or competitive, were achieved through experiments including inhibition assays without or with pre-incubation in the bacterial medium, and LC/MS analysis with the ExpR protein. The results strongly suggest that the type of inhibition is a competitive one.

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Full text

1. Introduction

Bacterial quorum sensing (QS) is a specific communication system enabling bacteria to coordinate their behavior according to their population density. This system allows them to adapt their life to the environment [1]. In Gram-negative bacteria, the molecular process regulating this system is based on small molecules named autoinducers and structurally related to acylhomoserine lactones (AHLs) [2]. These compounds, biosynthesized by LuxI-type proteins, can diffuse out of the cells to the external medium, reaching a critical concentration. Diffusion into bacteria is leading to gene expression through the interactions of AHLs with LuxR-type transcriptional regulators. QS may be modulated using different strategies, aiming at activating this process with the addition of external LuxR-type autoinducers or agonists. On the contrary, the process may be inhibited through AHLs hydrolysis or sequestration, or by using small molecules with antagonistic activity. The latter approach is largely employed through the design and synthesis of AHL analogs. The structure of AHLs can be described with three structural parts, which can be structurally modified: the lactone part, the amide functional group and the alkyl chain. AHLs analogs with heterocyclic systems [3] have been studied as well as the modifications of the amide functional group with bioisosteric replacements [4], such as a triazole heterocycle obtained via click chemistry.

Click chemistry has indeed revolutionized chemistry with the synthesis of 1,4-triazole using the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition between azides and alkynes. This type of heterocycle was used not only to mimic the central amide functional group [5] but also the 3-oxo group or to study the potentiality of multibranched AHL platforms [6,7]. Other click reactions have emerged, such as the thio-ene reaction [8,9], Diels–Alder reaction [10,11] and more recently sulfur (VI) fluoride exchange (SuFEx) [12,13,14,15]. This new click reaction is of interest in bio-organic/medical chemistry for the design of covalent inhibitors targeting residues such as lysine or tyrosine [16,17,18,19,20,21,22,23,24]. This SuFEx approach, which is currently attracting growing interest, has not yet been applied to the design of QS inhibitors, in particular for the LuxR-type transcription factor.

Quorum sensing is considered a promising target for controlling bacterial behaviors, particularly bacterial virulence and adaptive processes [25,26,27,28,29]. Studies aiming at the covalent inhibition of QS already exist, but this inhibition requires the presence of at least one nucleophilic cysteine residue in the binding site. The design, synthesis and evaluation of warheads with a terminal electrophilic group such as a halogen atom or isothiocyanate functional group targeting cysteine residues of the transcriptional factors LasR (Cys79, P. aeruginosa) [30,31] and more recently SdiA (E. coli) [32] have been reported. In another study by Perez and coworkers, they were inspired by a structurally AHL-unrelated LasR agonist with a triphenyl scaffold to design irreversible LasR inhibitors with an isocyanate or maleimide functional group [33].

The work by Blackwell and co-workers on SdiA irreversible inhibitors [32], which included studies on substituted benzoyl-l-homoserine lactone, has attracted our attention. Depending on the substitution of the benzene ring, this class of compounds is indeed known as QS modulators in the LuxR family of receptors being either antagonists of LuxR (3-oxo-C6-HSL receptor of Alvibrio fischeri), LasR (3-oxo-C12-HSL receptor of Pseudomonas aeruginosa), RhlR (C4-HSL receptor of Pseudomonas aeruginosa), QscR (3-oxo-C12-HSL receptor of Pseudomonas aeruginosa), and agonists or antagonists for SdiA (3-oxo-C6-HSL receptor of E. coli) and CepR (C8-HSL receptor of Burkholderia cepacian) [34] or inactive for TraR (Agrobacterium tumefaciens). Building on this background, the work reported herein is devoted to studying new potential inhibitors structurally related to benzoyl-l-homoserine lactone substituted with a methylsulfonyl or fluorosulfonyl group, this latter possibly enabling either a SuFEx-type inhibition by targeting tyrosine residues conserved in the LuxR family [35,36] or at least offering advantageous competitive interaction through a modification of the hydrogen bond network between the ligand and the binding site. The work reported below describes the synthesis of 3-methylsulfonyl and 3-fluorosulfonyl-benzoyl-l-homoserine lactone, their biological evaluation as QS inhibitors and the study of the possible mode of action, either covalent or competitive.

2. Materials and Methods

2.1. Molecular Modeling: Structure-Aided Design

The structure-oriented design was achieved using three LuxR-type proteins, SdiA (PDB code 4Y15) [37], the LuxR model constructed as previously described [38], and the Dickeya dadantii ExpR model obtained with AlphaFold [39]. For the latter, the natural ligand was first docked within the binding site of the ExpR model as described for LuxR [40]. Vega ZZ [41] was used to obtained tridimensional structure of compounds 1 and 2 for which Gasteiger charges were calculated. Prior to being saved as mol file, the structures were minimized using the SP4 force field with default parameters. Compounds 1 and 2 were docked within the binding site of the three proteins using a docking box 15 × 15 × 15 Å centered on the natural ligand with the docking module of ArgusLab [42] and the genetic algorithm with default parameters [43]. The docking results for the best docking poses showing a distance compatible for a nucleophilic substitution (<4 Å) [44] between the oxygen atom of the phenol group of Tyr71 (SdiA and ExpR) or Tyr70 (LuxR) and the sulfur atom of the sulfonyl fluoride group of compound 2.

2.2. Synthesis

(S)-3-(Methylsulfonyl)-N-(2-oxotetrahydrofuran-3-yl) benzamide (1). To a solution of 3-(methylsulfonyl)benzoic acid (0.2 g, 1 mmol) in dichloromethane (5 mL) was added dropwise at 0 °C oxalyl chloride (0.24 g, 1.32 mmol). One drop of dimethylformamide was added, and the mixture was then stirred for 1.5 h at ambient temperature. The mixture was evaporated under reduced pressure, and the residue was dissolved in dichloromethane. The resulting solution was added dropwise at 0 °C to a solution of l-homoserine lactone hydrobromide (0.18 g, 1 mmol) and triethylamine (0.29 mL, 2 mmol) in dichloromethane (10 mL). The reaction mixture was stirred for 3 h, and the solvent was evaporated. The residue was purified using column chromatography (ethyl acetate) to give a white solid (0.163 g, 53%). ^1^H NMR (300 MHz, acetone-d6): 8.55 (d, J = 6.7 Hz, 1 H), 8.42 (td, J = 1.8 and 0.4 Hz, 1 H), 8.23–8.27 (m, 1 H), 8.12–8.15 (m, 1 H), 7.80 (td, J = 7.8 and 0.5 Hz, 1 H), 4.89–4.99 (m, 1 H), 4.34–4.42 (m, 1 H), 4.45–4.52 (m, 1 H), 3.18 (s, 3 H), 2.64–2.74 (m, 1 H), 2.42–2.56 (m, 1 H); ^13^C NMR (75 MHz, acetone-d6): 176.3, 166.8, 143.7, 136.9, 134.1, 132.0, 131.6, 127.8, 67.1, 50.8, 45.2, 30.3; HR-MS for C_12_H_14_NO_5_S calculated 284.0587, found 284.0582 [M+H]^+^.

(S)-3-(Sulfonylfluoride)-N-(2-oxotetrahydrofuran-3-yl) benzamide (2). To a solution of 3-(sulfonylfluoride)benzoic acid (0.1 g, 0.49 mmol) in dichloromethane (2.5 mL) was added dropwise at 0 °C oxalyl chloride (0.06 g, 0.49 mmol). One drop of dimethylformamide was added, and the mixture was then stirred for 1.5 h at ambient temperature. The mixture was evaporated under reduced pressure, and the residue was dissolved in dichloromethane. The resulting solution was added dropwise at 0 °C to a solution of l-homoserine lactone hydrobromide (0.089 g, 0.49 mmol) and diisopropylethylamine (0.17 mL, 0.98 mmol) in dichloromethane (4 mL). The reaction mixture was stirred for 3 h, and the solvent was evaporated. The residue was purified using column chromatography (ethyl acetate) to give a white solid (0.075 g, 53%). ^1^H NMR (300 MHz, acetone-d6): 8.66 (d, J = 7.0 Hz, 1 H), 8.56 (s, 1 H), 8.46 (d, J = 8.8 Hz, 1 H), 8.31 (d, J = 7.9 Hz, 1 H), 7.97 (t, J = 7.9 Hz, 1 H), 4.91–5.00 (m, 1 H), 4.46–4.53 (m, 1 H), 4.34–4.40 (m, 1 H), 2.56–2.76 (m, 1 H), 2.42–2.56 (m, 1 H); ^13^C NMR (75 MHz, acetone-d6): 29.3, 49.9, 66.2, 127.8, 131.6, 131.9, 133.8, (d, J = 25 Hz) 135.7, 136.6, 165.0, 175.2; ^19^F NMR (282 MHz, acetone-d6): 65 (s, 1F); HR-MS for C_11_H_11_FNO_5_S calculated 288.0336, found 288.0339 [M+H]^+^.

2.3. Biological Evaluation

2.3.1. QS Modulation

Solutions of compounds were made in dimethyl sulfoxide, and experiments were conducted with a maximum of 2% dimethyl sulfoxide (v/v). The recombinant Escherichia coli strain DH5 alpha containing the sensor plasmid pSB401 was used to measure the induction of luminescence by various acyl-HSL analogs. This plasmid with a fusion of luxRluxl′::luxCDABE indeed responds to exogenous AHLs by producing bioluminescence [45]. Bacterial cultures were grown in Luria broth, in the presence of tetracycline (10 µg mL^−1^) at 30 °C.

Agonistic activity: detection of biological activity using the biosensor [46]

The inducing activity of compounds 1 and 2 was monitored using the E. coli biosensor strain. Acyl-HSL activity was measured in a 96-well microtiter plate format, with bioluminescence quantified using a TECAN Spark luminometer. Concentrations of compounds, ranging from 1 µM to 200 µM (final concentration in 0.2 mL), were made up to 0.1 mL volumes with growth medium. The same volume (0.1 mL) of a 1:10 dilution of an overnight culture of the biosensor strain was then added, and the plate was incubated at 30 °C. The amount of light produced by the bacteria was detected after 5 h, when the ratio of induced to background light was at its maximum. The amount of light measured was expressed in relative light units (RLU).

Antagonistic activity: competition assays using the biosensor strain

The influence of compounds 1 and 2 on bioluminescence induced by 3-oxo-C6-HSL was determined as described above, except that 3-oxo-C6-HSL was included at a final concentration of 200 nM together with the compounds.

2.3.2. Pre-Incubation Assays

Compound 2 was incubated at room temperature for 2 h at concentrations ranging from 1 µM to 200 µM (final concentration in 0.2 mL) in 0.1 mL volume of a 1:10 dilution of an overnight culture of the biosensor strain with growth medium. The same volume of LB (0.1 mL) was then added, with 3-oxo-C6-HSL included to obtain a final concentration of 200 nM (final volume 200 µL).

2.3.3. Kinetic Experiments

The hydrolysis reaction of compound 2 was monitored using ^19^F NMR spectroscopy using a 400 MHz JEOL spectrometer (scan numbers: 4, relaxation time: D1 = 30 s, impulsion angle 90°). To a solution of compound 2 (1.3 mg, 4.5 µmol) in DMSO (50 µM) was added 450 µL of a D_2_O PBS buffer pH = 7.4. ^19^F NMR spectra were then recorded over time on the 400 MHz spectrometer to follow the decrease in the signal at 65 ppm corresponding to the sulfonyl fluoride of compound 2.

2.3.4. Mass Spectrometry

The D. dadantii ExpR protein was purified as previously described [47] and was obtained at a concentration of 2.44 mg/mL in a 10 mM Tris-HCl buffer, pH 7.9, 1 mM EDTA, 100 mM KCl, 1 mM dithiothreitol (DTT), 1 mM phenylmethyl-sulphonyl fluoride (PMSF), 40% glycerol v/v [47]. LC/MS experiments were achieved using a UHPLC Ultimate 3000 (Thermo Fisher Scientific, Waltham, MA, USA) associated with a mass spectrometer, Quadrupole—Time-of-Flight (Q-TOF) (Impact II, Bruker, Billerica, MA, USA). The LC method was set up with the following conditions: column Waters—Xbridge Protein BEH C4—150 × 4.6 mm; 3.5 µm. mobile phases: A: water + 0.1% formic acid B: acetonitrile + 0.1% formic acid. Prior to the injection, the protein solution was diluted with water (1/1) without inhibitor or in the presence of 100 µM of compound 2 for 2 h (2% DMSO final v/v), and a volume of 10 µL was injected (0.5 mL/min, 80 °C, gradient A/B: 98/2 to 0/100 over 20 min). The mass spectrometer was equipped with an ESI source operated in positive mode. The capillary voltage was set to 4500 V, the nebulizer gas was set to 3.4 bar, the dry heater was set to 200 °C, and the dry gas was set to 9.0 L/min. The calibration was performed with a sodium formate solution, and the data were processed by the DataAnalysis 6.1 software from Bruker.

3. Results and Discussion

3.1. Structure-Aided Design

Considering our approach, we studied the compounds 3-(methylsulfonyl)benzoyl-l-homoserine lactone (1) as a reference compound and 3-(fluorosulfonyl)benzoyl-l-homoserine lactone (2) as a readily accessible compound because substituted benzoyl-l-homoserine lactones are common LuxR-type inhibitors, as mentioned in the introduction [32]. The covalent inhibition based on Sulfur (VI) Fluoride exchange (SuFEx) is obtained as a result of the reaction of a sulfonyl fluoride derivative with a nucleophilic residue, such as tyrosine, lysine or cysteine [48] (Figure 1). As for a common covalent inhibition process, SuFEx-based inhibitors should first bind to the protein and then react with the nucleophilic of the target residue close to the electrophilic sulfonyl fluoride moiety. The structure-aided design of the inhibitors can be achieved through molecular docking studies and by monitoring the nucleophilic–electrophilic distance [44]. Examination of known structure of LuxR-type proteins especially their AHL binding site reveals the presence of two tyrosine residues strictly conserved in this family of transcription factor [38]. This observation could lead to the discovery of SuFEx-based LuxR Quorum Sensing inhibitors with a broad spectrum of inhibition, given the conservation of these tyrosine residues.

The 3D structure of LuxR-type proteins is relatively similar for all Gram-negative species, with a receiver domain for AHLs and a domain allowing the recognition of the DNA sequence. The binding site of AHLs can be described with two hydrophobic pockets interacting with the lactone part and the acyl chain. The two pockets are separated by a hydrophilic part interacting with the amide functional group, with an aspartic residue involved in an H-bond with the NH and two tyrosine residues, one being involved in a H-bond with the C=O of the amide (Figure 2). The lactone interacting in a hydrophobic pocket is also involved in an H-bond with the C=O of the ester with a tryptophan residue. This binding mode is similar in the LuxR protein family with these three conserved residues (Trp, Asp and Tyr).

Molecular docking studies show that the orientation of the meta-sulfonyl fluoride moiety in compound 2 within the SdiA binding site, taken as a reference for LuxR-type transcription factors, is compatible in terms of distance with a nucleophilic attack of the phenol group of the residue Tyr71 (Figure 3). Docking simulations using compound 2 and the LuxR model described previously [38] or the alphafold model of ExpR gave the same results with binding modes compatible with a SuFEx inhibition of both proteins with compound 2 by targeting the corresponding tyrosine residues, namely Tyr70 (LuxR) and Tyr71 (ExpR), respectively (Figure 3). Docking simulations led to similar best docking poses for the three proteins. The predicted binding modes revealed an H-bond between the tyrosine residues mentioned above and the oxygen atom of the sulfonyl group. Interestingly, in the case of SdiA (E. coli), the sulfur of the residue Cys45 is located at a distance of 3.43 Å, which could induce a nucleophilic attack.

This molecular modeling study suggests that compound 2 is thus suitable for our study by interacting with LuxR-type proteins with H-bond network modifications compared to the natural ligands, with a Tryptophan and a tyrosine residue conserved in the family. Moreover, the fluorosulfonyl group is located in the vicinity of the tyrosine residue to potentially react with this residue. In the present study, we have focused our work on tyrosine residues, which can be targeted to design a broad range of inhibitors due to their conservation in the LuxR family.

3.2. Synthesis

The synthesis of compound 2 was achieved by standard acylation of l-homoserine lactone hydrobromide, synthesized as previously described [6], with the acyl chloride obtained from the 3-(fluorosulfonyl)benzoic acid with oxalyl chloride. Compound 1 with a methyl group replacing the fluorine atom was also synthesized using the same procedure with the 3-(methylsulfonyl)benzoic acid (Scheme 1).

3.3. Quorum Sensing Modulation

Agonistic and antagonistic activities towards the LuxR protein were evaluated using E. coli equipped with a pSB401 plasmid responding to exogenous AHLs. Compounds 1 and 2 were not found to be LuxR agonists, i.e., they do not induce bioluminescence, confirming the observation by Helen Blackwell and coworkers for substituted benzoyl-l-homoserine lactone [32]. For antagonistic activity (Figure 4), both compounds 1 and 2 were found to decrease the bioluminescence induced by the 3-oxohexanoyl-l-homoserine lactone, with moderate activity for compound 1 (IC_50_ > 150 µM) and significant activity for compound 2 with an IC_50_ value of 15 ± 2 µM (standard deviation). These latter experiments showed the inhibitory activity of the compounds. For both compounds, no effect on bacterial growth was observed based on OD_600_ measurements, showing no toxicity of the sulfonyl fluoride group. The difference in activity for these two compounds may be attributed to either a favorable interaction of the fluorosulfonyl group compared to the more sterically hindered methylsulfonyl substituent, or to a SuFEx-type inhibition for compound 2.

3.4. Studies of the Potential Mechanism for the QS Inhibition

3.4.1. Pre-Incubation Assays

As compound 2 was identified as a LuxR inhibitor (Section 3.3), an experiment was envisioned to investigate the LuxR inhibition by pre-incubation experiments with various concentrations of inhibitor 2 and then the addition of OHHL. This type of experiment was chosen, allowing the enzyme to react with the inhibitor, prior to the addition of the natural ligand [49]. With this experiment, an increase inhibition should be expected for a covalent inhibition by an irreversible reaction with compound 2 rendering impossible the activation of OHHL. Hence, this experiment was performed by first pre-incubation of compound 2 with the sensor bacteria strain for 2 h, then activation with OHHL. However, a significant decrease in the inhibition with an IC_50_ value > 120 µM suggests that compound 2 was lower in availability to inactivate LuxR, perhaps because of some non-specific hydrolysis or other non-specific reactions with other biomolecules within the bacteria.

3.4.2. In Vitro Kinetic Experiments

To potentially differentiate between hydrolysis and unspecific reactions, ^19^F NMR spectroscopy was used to monitor in vitro hydrolysis of compound 2 in a D_2_O PBS buffer, pH 7.4, with 10% DMSO. In these conditions, the half-life was estimated as 54.5 h, showing the relative stability of this compound in these conditions (Supplementary data Figure S1). This suggests that compound 2 is less available when preincubated with bacteria, probably due to nonspecific reactions with biomolecules.

3.4.3. Mass Spectroscopy Investigation

Further investigation was conducted to characterize a potential covalent inhibition of compound 2. For this, we used LC-MS with electrospray as an ionization mode and the ExpR protein of Dickeya dadantii as an available LuxR-type protein, displaying a comparable docking simulation with compound 2 as for the LuxR protein (Figure 3). First, analysis of the overexpressed protein was performed by acrylamide gel electrophoresis in denaturing conditions (SDS PAGE), which showed a more intense band at a molecular weight > 25 kDa corresponding to the calculated molecular weight of the protein (Figure 5). HPLC traces of the protein solution also showed a major peak at 11.2 min with a molecular weight of 25,585 Da. We then performed the experiment with incubation of the protein with compound 2 (100 µM) for 2 h. In these conditions, no protein adduct with the inhibitor could be detected, but a similar analysis was obtained compared with the protein in the absence of the inhibitor.

3.5. Dissociation Constant (Kd)

Experiments of QS inhibition suggest that compound 2 exerts a competitive inhibitor for some LuxR proteins, in particular towards LuxR, based on preincubation assays. Therefore, we were interested in the affinity of compound 2 for LuxR. We thus estimated the dissociation constant of compound 2 for the LuxR protein using the PRODIGY server [50,51] and the PDB file resulting from the docking simulation of compound 2 within the binding site of the protein. A dissociation constant value of 100 µM could be estimated. For comparison, the Kd value of the LuxR natural ligand has been determined as 100 nM [52]. There is a significant difference between the two values. Nevertheless, this compound was found to inhibit bioluminescence induced by 200 nM of the natural ligand with an IC_50_ of 15 µM.

4. Conclusions

A structure-aided design of a SuFEx inhibitor was investigated using docking simulations, and compound 2 has been identified as a potential candidate. The distance between the nucleophilic and the electrophilic center indeed measured on the resulting binding modes suggests a possible reaction enabling the covalent inhibition of the LuxR-type proteins, namely SdiA, LuxR and ExpR. Compound 2 was easily synthesized by standard acylation of the l-homoserine lactone hydrobromide with the corresponding acyl chloride in basic conditions. Compound 2 was evaluated as an inhibitor of the LuxR-regulated quorum sensing using a reporter strain, and an IC_50_ value of 15 ± 2 µM could be determined without any effect on bacterial growth based on optical density measurements (OD_600_). Further investigations of the inhibition were carried out to characterize the nature of the inhibition (covalent/competitive inhibition), in particular, pre-incubation experiments of the compound 2 with the reporter strain, leading to a significant decrease in the activity with an IC50 value > 120 µM. LC/MS experiments were achieved with the ExpR protein displaying a comparable docking simulation with compound 2 and the LuxR protein. Covalent adduct could not be detected with this experiment. Overall, considering the relative stability of compound 2 with a half-life of about 55 h, all experiments strongly suggest that the inhibition is due to a competitive binding to the proteins and not to a covalent inhibition. It can be proposed that the nucleophilic/electrophilic balance of the tyrosine residue and compound 2 may not be adapted to the reaction of both entities.

To summarize, based on a structure-aided strategy, methyl and fluorosulfonyl benzoyl AHL analogs were investigated as possible ligands of LuxR-type transcription regulators. Docking simulations indicate potential interactions, either as a SuFEx-type inhibition of LuxR-type proteins by covalent coupling or at least favorable hydrogen bond network modification of the ligand within the binding site. The fluorosulfonyl compound 2 and its methyl counterpart were thus synthesized and evaluated as LuxR-regulated QS modulators, and the results showed antagonistic activity with an IC_50_ value of 15 ± 2 µM for compound 2, while the methyl derivative was only weakly active. Experiments were then conducted to study the inhibition mode without or with preincubation assays, LC-MS experiments with the ExpR protein. The stability of compound 2 was also studied in vitro. The mode of action was checked and defined as competitive inhibition, suggesting satisfactory affinity between the ligand and the protein, but without reactivity for ensuring a covalent reaction between the fluorosulfonyl group and a tyrosine residue within the binding site. Compound 2 was found to be more efficient compared to compound 1, bearing a more sterically hindered methylsulfonyl substituent in the meta position, which may explain the decrease in the antagonistic activity. To our knowledge, this study is the first approach using sulfonyl-substituted benzoyl derivatives reported towards the inhibition of LuxR-type proteins. While the strategy towards SuFEx-based covalent inhibition remains a challenge, this first approach will be useful when designing other compounds targeting LuxR, or other proteins such as LasR and SdiA, which also possess a cysteine residue, a potential nucleophilic residue.