Thiophenesulfonamide compounds are potent and specific inhibitors of Vibrio quorum sensing regulators LuxR/HapR that block their activity in vivo, thus preventing transcription of genes for virulence, motility, and biofilms. This protocol details how these compounds are synthesized, modeled in silico, and assayed in vivo for activity against LuxR/HapR.
Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.
Bacteria sense population density and the type of cells nearby using a cell-cell communication process called quorum sensing (QS)1. Diverse clades of bacteria use QS to control various behaviors, such as motility, biofilm formation, virulence factor secretion, and more. The proteins and signals involved in QS differ widely among bacteria. In Vibrio species, the QS signaling system predominantly uses membrane-bound hybrid histidine-kinase receptors that recognize specific cognate small molecule signals called autoinducers2 (Figure 1). These receptors control the flow of phosphate through the system to a response regulator that transcribes small RNAs. The production of sRNAs alters the production of the master quorum sensing regulator, which is defined as a conserved group of proteins collectively called LuxR/HapR3. Thus, at low cell densities, the mRNA encoding LuxR/HapR is degraded via sRNA targeting, and at high cell density, LuxR/HapR protein is produced at maximal levels (reviewed in Ball et al.3).
The LuxR/HapR group of proteins belongs to the large group of TetR proteins, which is defined by the presence of a helix-turn-helix in the DNA binding domain, formation of a functional homodimer, and typically the inclusion of a ligand binding domain4. The Vibrio LuxR/HapR proteins fit all these criteria, though a ligand has not yet been identified. The LuxR/HapR protein in all studied Vibrio species controls numerous downstream behaviors, many of which are known to be important in pathogenesis: production of biofilms, proteases, cytotoxins, hemolysins, type III secretion, and type VI secretion complexes, and more3. Deletion of the LuxR/HapR protein leads to a decrease or loss of virulence in host systems5,6,7, leading to the hypothesis that inhibition of these proteins is a viable strategy for counteracting disease progression. Vibrio species cause vibriosis disease in marine organisms, including fish, shellfish, and corals, as well as in humans who contact or ingest certain species.
Previous research has identified a panel of thiophenesulfonamide compounds that specifically bind to the ligand binding domain of LuxR/HapR proteins in multiple Vibrio species to block their function5,8,9 (Figure 1). Using a reporter screen in E. coli, the compounds were identified and subsequently tested in the native Vibrio, which showed a high correlation between the effect in the heterologous E. coli and the efficacy in the Vibrio9. These compounds are simple to synthesize in a single step, making them ideal for small library synthesis in the context of a chemical biology lab course. A three-week course-based undergraduate research experience (CURE) that was designed around this molecular scaffold has been previously reported8. This three-week module has been further optimized, streamlined, and broadened in this year-long CURE designed to target the inhibition of LuxR/HapR proteins in diverse Vibrio species.
The details of the reagents and the equipment used for the study are listed in the Table of Materials.
1. Design and synthesis of thiophenesulfonamide libraries
NOTE: Thiophenesulfonamide inhibitors such as 3-phenyl-1-(thiophen-2-ylsulfonyl)-1H-pyrazole (PTSP) are synthesized via the one-step base-promoted condensation8,9, as shown in Figure 2. To design new compound libraries for study, researchers must procure appropriate amines and sulfonyl chlorides and follow the steps summarized below. If the structure of the amine differs greatly from the aromatic pyrazole derivative, then alternative reaction conditions may need to be identified by searching the literature. This procedure is robust, and bases such as sodium hydroxide, triethyl amine, and pyridine have also worked well. If this is performed in a course, students may be given the freedom to choose their own procedure with likely favorable outcomes.
2. Structural modeling to predict the binding of thiophenesulfonamides to Vibrio LuxR/HapR regulator
NOTE: This protocol utilizes the web-based version of AutoDock Vina called Webina11 to dock small molecule inhibitors (PTSP in this case) into the ligand binding pocket of the LuxR/HapR homolog called SmcR from Vibrio vulnificus12. The outcomes of this protocol are (1) calculated binding affinities and (2) a structure file that can be opened in PyMol or a related program to visualize the ligand-protein interactions. This protocol can be adapted for any small molecule and any protein with a ligand binding pocket. It takes minutes to dock into SmcR because the search area for this receptor is defined below. If the search area needs to be defined for a new receptor (steps 2.15-2.20), this can be completed in one 3 h lab period. Once the search area is defined, subsequent dockings will take minutes.
3. Biological assessment of thiophenesulfonamides in quorum sensing inhibition
NOTE: The procedure specifically for assaying the Vibrio campbellii protein LuxR is described here. However, this procedure can be adapted for use with any of the Vibrio LuxR/HapR proteins, all of which are available on ectopically replicating plasmids that are compatible with the reporter plasmid pJV0649. The "red-green screen" assay is performed in a heterologous bacterial background using E. coli cells containing the two plasmids. The LuxR/HapR expression plasmid (conferring kanamycin resistance) is available expressing different LuxR genes (Figure 4A). The pJV064 plasmid (conferring chloramphenicol resistance) encodes a gfp gene under the control of a LuxR-activated promoter and a mCherry gene under the control of a LuxR-repressed promoter (Figure 4A). Both promoters were chosen due to their identification as LuxR-regulated genes with large changes in expression in vivo15. The LuxR/HapR E. coli strains and plasmid pJV064 have been published previously9,15.
4. Washing the black assay plates for re-use
As representative results, data is included from three thiophenesulfonamide compounds synthesized by undergraduate students for compounds 1A, 2B, and 3B (Figure 5A–C; described in detail in Newman et al.9). Each compound was tested in the E. coli strain expressing V. campbellii LuxR and using the pJV064 reporter plasmid. The normalized fluorescence per cell is shown for each assay. The assay was performed with a 1:10 dilution series to assay concentrations 1 µM, 10 µM, and 100 µM of each compound compared to the solvent control. Compounds 1A and 3B were each inhibitory though to different extents: 1A only exhibited inhibition of LuxR at 100 µM, as evident by the loss of GFP expression and increase in mCherry expression. Conversely, 3B inhibited LuxR activity at all three tested concentrations. Compound 2B did not have any activity; thus, all concentrations tested appeared similar to those of the DMSO solvent control. A minimum of three biological replicates of each assay is recommended to perform statistics and determine the data distribution and error.
Figure 1: LuxR/HapR proteins are the master QS regulators in Vibrios. (A) Schematic of the generic QS circuit in Vibrio species. Membrane-bound histidine kinase receptors bind autoinducers, which leads to the production of the master QS transcriptional regulator LuxR/HapR. Group behaviors are activated, which typically include numerous virulence factors that contribute to vibriosis disease. Thiophenesulfonamide inhibitors such as compound PTSP (3-phenyl-1-(thiophen-2-ylsulfonyl)-1H–pyrazole) bind to LuxR/HapR and block its function, thus eliminating group behaviors. (B) Pymol rendering of a docked structure: V. vulnificus SmcR protein in which PTSP has been modeled into the binding pocket using Webina. Please click here to view a larger version of this figure.
Figure 2: Synthesis of thiophenesulfonamides. (A) General scheme for the synthesis of thiophenesulfonamide compounds. (B) Scheme for the synthesis of PTSP. Please click here to view a larger version of this figure.
Figure 3: Modeling of PTSP in SmcR. (A) Output from Webina that shows PTSP docked into the ligand binding pocket of SmcR from Vibrio vulnificus. (B) A figure from a student report showing a Pymol rendering of the same interaction. Please click here to view a larger version of this figure.
Figure 4: E. coli screen for thiophenesulfonamide inhibitors of LuxR/HapR. (A) Diagram of the E. coli red-green screen reporter plasmid and LuxR/HapR expression plasmid. (B) Diagram of the 96-well assay plate setup for screening thiophenesulfonamide compounds. Please click here to view a larger version of this figure.
Figure 5: Student data assessing three thiophenesulfonamides in the E. coli assay. Three compounds (A–C) were synthesized and assayed as described in this protocol. The normalized GFP and mCherry fluorescence are shown for all three compounds compared to the DMSO solvent control at equal volume. These data are from a single biological assay for each compound. Please click here to view a larger version of this figure.
Figure 6: Moderate LuxR inhibitors with unexplored structural motifs. Please click here to view a larger version of this figure.
Supplementary Figure 1: ASURE lab two-semester program. Please click here to download this File.
This CURE was originally developed as an abbreviated two-stage, three-week protocol (design/synthesis and assay) and was implemented in five semesters as part of an upper-level organic laboratory course8. Since the original report, the computer modeling module was added, and the E. coli assay was optimized for novice researchers. The resulting three-stage, two-semester protocol has been implemented three times as part of Indiana University's Arts and Sciences Undergraduate Research Experience (ASURE) program, which is taken by students in their first and second years. The overall structure of the course changes year to year as research goals evolve; an example schedule is outlined in Supplementary Figure 1.
Procedures for one specific compound (PTSP) and two target proteins (LuxR and SmcR) are described herein; however, the value and longevity of this CURE lie within its inherent flexibility. More than 100 compounds have been synthesized in the context of these courses with various levels of activity or inactivity in the E. coli assay. This synthetic library does not scratch the surface of the potential chemical space yet to be explored. Most of the synthetic library consists of PTSP derivatives with variously substituted pyrazole and thiophene groups, leaving much room for investigation. For example, the benzenesulfonamide (1E), amide (3F), and pyrrolidine (P0074 H4) derivatives shown in Figure 6 have moderate activity against LuxR and SmcR, and none of these scaffolds have been explored in depth10. In addition to the unexplored chemical space, there are several LuxR homologs for which there is no effective inhibitor10. A variation of this CURE could be designed around the exploration of chemical space or, around one of the less sensitive LuxR homologs, or with a combination of these research goals.
This set of protocols is a unique combination of chemical synthesis, computer modeling, and microbiology in a single course that enables students to emulate the medicinal chemistry process for drug design. This CURE model has inherent flexibility and can be implemented in many different ways, or it can serve as a model for a laboratory course designed around small-scale drug design in any target.
The authors have nothing to disclose.
Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R35GM124698 to JVK. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
2-thiophensulfonyl chloride | Ambeed | A258464 | |
3-Phenyl-1H-pyrazole | Ambeed | A104401 | 98% |
96-well clear bottom black plates | USA Scientific | 5665-5090Q | 96-well polystyrene uClear black TC plate with lid, clear flat bottom, sterile, 8/sleeve, 32/case |
Autodock Tools | http://mgltools.scripps.edu/downloads | ||
Autodock Vina | https://vina.scripps.edu | ||
Chloramphenicol | |||
DMSO | |||
ethyl acetate | Fisher Scientific | AA31344M4 | Reagent grade |
hexanes | Fisher Scientific | H291 | |
Kanamycin | |||
magnesium sulfate | Fisher Scientific | M65-500 | Anhydrous |
Microporous Film | USA Scientific | 2920-1010 | Microporous Film, -20degC to +80degC, 50/box, Sterilized |
molview | molview.org | ||
NaCl | |||
Protein Databank | https://www.rcsb.org/ | ||
Pymol | https://pymol.org/2/ | ||
Qualitative filter paper | Fisher Scientific | 09-805-342 | Cytiva Whatman™ Qualitative Filter Paper: Grade 1 Circles, 47 mm |
Silica gel | Sorbtech | 30930M-25 | Silica Gel, Standard Grade, 60A, 40-63um (230 x 400 mesh) |
Sodium hydride | Millipore Sigma | 452912 | 60 % dispersion in mineral oil |
Tetrahydrofuran | Fisher Scientific | MTX02847 | Tetrahydrofuran, anhydrous, 99.9%, ACS Grade, DriSolv |
TLC Plates | Sorbtech | 1634067 | Silica gel TLC plates, aluminum backed |
Tryptone | |||
webina | https://durrantlab.pitt.edu/webina/ | ||
Yeast Extract |