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.

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.
<ol>
	<li>Synthesis and isolation of PTSP 
	NOTE: This can be completed in one 3 h lab period.
	<ol>
		<li>Dissolve 822 mg of phenylpyrazole (5.7 mmol of the amine, 1.5 eq.) in 15 mL of tetrahydrofuran in a 50 mL round bottom flask with a stir bar.</li>
		<li>Slowly add 306 mg of sodium hydride (60% in oil, 7.65 mmol, 2 eq.) to create a suspension. Let this suspension stir on a stir plate for 10 min.</li>
		<li>In a vial, prepare a solution of thiophenesulfonyl chloride (3.82 mmol of the sulfonyl chloride, 1 eq.) in 5 mL of THF.</li>
		<li>Add the solution of sulfonyl chloride prepared in step 1.1.3 to the suspension prepared in step 1.1.2 and allow the resulting suspension to stir for an additional 30 min.</li>
		<li>Add 20 mL of DI water to the reaction mixture in the 50 mL round bottom flask.</li>
		<li>Transfer all contents of the reaction flask except the stir bar into a 250 mL separatory funnel10.</li>
		<li>Add 20 mL of ethyl acetate (EtOAc) to the separatory funnel and shake.</li>
		<li>Remove the bottom (aqueous) layer and set aside.</li>
		<li>Remove the top (organic) layer and set aside in a 200 mL Erlenmeyer flask.</li>
		<li>Return the aqueous layer to the separatory funnel and extract with 20 mL of ethyl acetate.</li>
		<li>Repeat steps 1.1.8 and 1.1.9.</li>
		<li>Return the combined organic layers to the separatory funnel and wash with 20 mL of saturated NaCl (brine).</li>
		<li>Remove the bottom (aqueous) layer and set aside.</li>
		<li>Drain the top (organic) layer back into the Erlenmeyer flask.</li>
		<li>Dry the combined organic layers over MgSO4 in the Erlenmeyer flask and filter into a 250 mL round bottom flask using qualitative filter paper10.</li>
		<li>Remove the organic solvent on a rotary evaporator.</li>
	</ol>
	</li>
	<li>Analyze the crude reaction mixture by 1H NMR to ensure the product has formed10. 
	NOTE: The time necessary for this step will depend on the student&#39;s comfort level with NMR spectroscopy.</li>
	<li>Purify the product by silica gel column chromatography (10:1 hexanes: ethyl acetate)10. 
	NOTE: This can be completed in one 3 h lab period.</li>
	<li>Characterize the product spectroscopically. 
	NOTE: The time necessary for this step will depend on the student&#39;s comfort level with NMR spectroscopy. 
	NOTE: Characterization data for 3-phenyl-1-(thiophen-2-ylsulfonyl)-1H-pyrazole (PTSP)10: 
	1H NMR (400 MHz, CDCl 3 ):&#160;&#948; 8.11 (d, J = 2.8 Hz, 1H), 7.88 - 7.79 (m, 3H), 7.68 (dd, J = 5.0, 1.4 Hz, 1H), 7.44 - 7.31 (m, 3H), 7.07 (dd, J = 5.0, 3.9 Hz, 1H), 6.71 (d, J = 2.8 Hz, 1H). 
	13C NMR (101 MHz, CDCl 3 ):&#160;&#948; 157.17, 136.84, 135.40, 135.36, 132.55, 131.28, 129.36, 128.72, 127.79, 126.47, 106.90. 
	HRMS (ESI): Calculated for C13H10O2N2NaS2 [M+Na+]: 313.0076. Found: 313.0078.</li>
</ol>
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.
<ol>
	<li>Download the AutoDock Tools. Select the appropriate file for the operating system and install it on the computer. 
	NOTE: This protocol utilizes a web-based version of AutoDock Vina. The docking can also be performed locally if this program is installed (see the manufacturer&#39;s instructions).</li>
	<li>Download the structure for apo SmcR.</li>
	<li>Navigate to the Protein Data Bank.</li>
	<li>In the search bar, enter the PDB ID 3KZ9.</li>
	<li>Click on the Download Files from the dropdown menu toward the top right corner of the page and select pdb format.</li>
	<li>After downloading the file, save it to a new folder dedicated to the docking work.</li>
	<li>Create a .mol structure file of the small molecule inhibitor (PTSP).</li>
	<li>Navigate to molview and click on the trash can icon to remove the structure that is there.</li>
	<li>Draw PTSP in the structure window.</li>
	<li>Click on 2D to 3D.</li>
	<li>Click on Tools &#8594; export&#160;&#8594;&#160;MOL file.</li>
	<li>A file titled &#34;Molview.mol&#34; will be downloaded to the computer. Give the file a name that makes sense and save it to the folder that contains the protein pdb file.</li>
	<li>Define the search area.</li>
	<li>Define the area in Webina as the binding pocket of the protein (SmcR) to facilitate docking in the correct region. 
	NOTE: Coordinates for SmcR are provided and generated using the following protocol. If SmcR is being used, then steps 2.15-2.20&#160;can be skipped. The protocol can be adapted for any protein with a known ligand binding pocket. Coordinates: center_x = 17, center_y = 40, center_z = 56, size_x = 12, size_y = 12, size_z = 12.</li>
	<li>In AutoDock Tools, click on File &#8594; Read Molecule and open the pdb protein file downloaded in step 2.5.</li>
	<li>Navigate to the dashboard and click on the blue arrow next to the name of the protein. This will bring up a list of chains. Click on the blue arrow next to one of the protein chains.</li>
	<li>To highlight the amino acids of the binding pocket, find the amino acid of interest in the list that appeared. Next to the amino acid, go to the triangle in the Cl (color) column at the far right. Click on the triangle and select by rainbow. 
	NOTE: Use this method to highlight the following amino acids:12 Phe75, Phe78, Leu79, Ile96, Met100, Trp114, Phe129, Asn133, Gln137, Val140, Ala163, Phe166, His167, Cys170 (If Met100 is missing, this can be skipped).</li>
	<li>Next, click on Grid &#8594; GridBox. A box should appear over the protein structure with a red, green, and blue face. Before making any changes, change Spacing (angstrom) to 1. This will ensure the box is correctly scaled for the next steps.
	<ol>
		<li>Then, change the Center Grid Box &#60;offset&#62; dials to translate the box in the x, y, and z directions to place it over the highlighted amino acids. Click and drag on the protein to rotate the structure in three dimensions. 
		NOTE: This will help ensure that the box is in the correct position. If necessary, use the top three dials for the number of points to change the size of the box.</li>
	</ol>
	</li>
	<li>Once the box position and dimensions are set, click on File&#160;&#8594; Close Saving Current.</li>
	<li>In the menu, click on Grid&#160;&#8594; Output&#160;&#8594; Save GPF. Name the file and save it in the same folder as the two structure files. This GPF file has the box coordinates.</li>
	<li>Perform &#34;Docking&#34; in Webina to generate a calculated binding energy.</li>
	<li>Open the folder that contains the .mol ligand file and the .pdb protein file. Webina requires .pdbqt files, and will do the file conversion.</li>
	<li>Navigate to Webina.</li>
	<li>Receptor: Drag over the file downloaded from the pdb called 3kz9.pdb. Click on Add hydrogen atoms, and click on convert. Click on Ok on the next pop-up to work with a monomer.</li>
	<li>Ligand: Drag over the .mol file for PTSP (or other small molecule inhibitor). Ensure no boxes are selected and click on Convert. Leave &#34;Correct Pose&#34; empty and scroll down to the Docking Box. Enter the appropriate box parameters (either those listed above for SmcR or a set that was generated by the protocol in steps 2.15-2.20&#160;for a different protein).</li>
	<li>Click on Start Webina and wait.</li>
	<li>A series of calculated binding energies will be reported [Affinity (kcal/mol)] directly below a visualization of the docked molecule. Ensure that the first value is the most negative.</li>
	<li>To visualize the docked ligand in pymol or another similar program, download the Output PDBQT File by clicking on the appropriate download button. This file is just the ligand (PTSP) in the docked coordinates.</li>
	<li>Visualize the docked ligand-SmcR complex in PyMol13.
	<ol>
		<li>Download and open PyMol. 
		NOTE: Full-time students and educators may receive a PyMol License free of charge. When asked for a license after starting PyMol for the first time, click on Buy License. Select Student/Teacher.</li>
		<li>Open the pdb file called 3kz9.pdb.</li>
		<li>Open the pdbqt&#160;output file created by Webina.</li>
		<li>Visualize the best conformation (the one with the lowest binding affinity) by clicking on the small pointing looking arrows at the bottom right of the PyMol screen. The first conformation will be the most favorable and will be showing when the file is opened.</li>
		<li>Manipulate the protein with the docked ligand in PyMol using standard commands14. 
		NOTE: The previous study by Newman et al. compared the predicted binding affinities of several compounds determined by Webina to in vitro and in vivo assays9. These binding affinities serve as references for compounds that are likely to be good inhibitors with high affinities (which correlate to lower kcal/mol numbers) for the binding pocket. An example of the Webina output data is included in Figure 3A,B for PTSP binding in the pocket of SmcR.</li>
	</ol>
	</li>
</ol>
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 &#34;red-green screen&#34; 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.
<ol>
	<li>Liquid media preparation: Prepare 1 L of LB medium.
	<ol>
		<li>Weigh out 10 g of NaCl, 10 g of bactotryptone, and 5 g of yeast extract.</li>
		<li>Mix the components in a beaker or graduated cylinder using a stir plate.</li>
		<li>Bring the total volume to 1 L using graduated cylinders.</li>
		<li>If desired, aliquot into 100 mL per bottle before autoclaving.</li>
		<li>Autoclave for 15 min for each 1 L of media. Alternatively, if an autoclave is not available, an Instant Pot may be used16.</li>
	</ol>
	</li>
	<li>Strain inoculation (Day 1)
	<ol>
		<li>Add antibiotics to the LB medium at final concentrations of kanamycin (40 &#956;g/mL) and chloramphenicol (10 &#956;g/mL).</li>
		<li>Aliquot 5 mL of LB/Kan40/CM10 medium into sterile test tubes with caps.</li>
		<li>Inoculate E. coli strains from freezer stocks: E. coli culture expressing LuxR/HapR and pJV064 plasmid (LuxR+)9,15; E. coli empty vector control&#160;culture and pJV064 plasmid (LuxR-)9,15.</li>
		<li>Shake the culture overnight at 275 rpm at 30 &#176;C for 16-18 h.</li>
	</ol>
	</li>
	<li>Assay of thiophenesulfonamides (Day 2)
	<ol>
		<li>Weigh out ~30 mg of the compound. Resuspend to 100 mM in DMSO and vortex to mix. Next, prepare a 10 mM stock.</li>
		<li>Mix 20 &#956;L of the 100 mM stock with 180 &#956;L of DMSO to dilute it (1:10) to yield a 10 mM working stock. Vortex to mix.</li>
		<li>Back-dilute LuxR+ culture and LuxR- cultures (1:100) in 10 mL of LB/Kan/CM media into 15 mL conical tubes and vortex briefly to mix.</li>
		<li>Use a black-well, clear-bottomed, sterile 96-well plate for the assay.</li>
		<li>Prepare a dilution series for all columns. A diagram of the 4-fold dilution series is included as a reference (Figure 4B). 
		NOTE: A digital multi-channel pipet is recommended for this process. This method is for a 4-fold dilution series (1:4). However, different dilution series may be used (e.g., 1:10, 1:5).
		<ol>
			<li>Pipet 200 &#181;L of the LuxR+ culture mix into a 96-well plate in row A, 1-6 (Figure 4B; green).</li>
			<li>Pipet 200 &#181;L of the LuxR- culture mix into a 96-well plate in row A, 7-12 (Figure 4B; orange).</li>
			<li>Pipet 150 &#181;L of the LuxR+ culture mix into a 96-well plate in rows B-E, 1-6 (Figure 4B; green).</li>
			<li>Pipet 150 &#181;L of the LuxR- culture mix into a 96-well plate in rows B-E, 7-12 (Figure 4B; orange).</li>
			<li>Add 2 &#181;L of the compound or DMSO to each well of row A according to Figure 4B.</li>
			<li>For every column, pipet up and down 3 times, then transfer 50 &#181;L from row A into row B, in the same column.</li>
			<li>Repeat the mixing and pipetting for rows B, C, D, E. After mixing row E, remove 50 &#181;L and put in waste. In the end, one should have 150 &#956;L in each well.</li>
		</ol>
		</li>
		<li>Cover the plate with microporous tape.</li>
		<li>Incubate the plate by shaking at 275 rpm at 30 &#176;C overnight for 16-18 h.</li>
		<li>Measure the fluorescence and optical density of the assay plates (Day 3).
		<ol>
			<li>Remove the tape carefully, and do not spill liquid out of the wells.</li>
			<li>On a plate reader, read the optical density at 600 nm (OD600), GFP, and mCherry. 
			NOTE: A set gain for both fluorescence channels is recommended to enable comparisons between assays.</li>
		</ol>
		</li>
		<li>Record data as normalized fluorescence per cell: GFP/OD600 and mCherry/OD600.</li>
		<li>Plot the data, as shown in Figure 5, to illustrate the results for the DMSO control in comparison to the test samples.</li>
	</ol>
	</li>
</ol>
4. Washing the black assay plates for re-use
<ol>
	<li>Soak the plates in bleach.
	<ol>
		<li>Label a 1 L plastic beaker as &#34;biological waste&#34;. Dump liquid from plates into this waste container (carefully clean up any drips with 70% EtOH). Rinse the plates with DI water from the squirt bottle over the waste and dump it into it.</li>
		<li>Label another 1 L plastic beaker as &#34;30% bleach&#34;. Put plates/lids in this container and cover with 30% bleach. Wrap the top with a plastic sheet and weigh the plates with a tip box or something similar. Let sit submerged in the bleach solution overnight.</li>
	</ol>
	</li>
	<li>Rinse and soak in ethanol.
	<ol>
		<li>Thoroughly rinse the plates with DI water into the sink so that no bleach remains. Dump out any remaining water from wells into the sink.</li>
		<li>Add 70% EtOH to all wells and lids using a spray bottle. Let the plates sit upright with the lid on top but askew so that the EtOH can evaporate, but nothing falls into the wells. Let the plate sit overnight.</li>
	</ol>
	</li>
	<li>Allow the plates to dry.
	<ol>
		<li>Dump out any remaining EtOH.</li>
		<li>Flip over the plate onto a paper towel and let the rest of the EtOH evaporate. Leave the plates this way in a fume hood. The plates will be ready for the next use.</li>
	</ol>
	</li>
</ol>

Biological Assessment of Thiophenesulfonamides in Quorum Sensing Inhibition

<ol>
	<li>Ng, W. L., Bassler, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+quorum-sensing+network+architectures.">Bacterial quorum-sensing network architectures.</a> Annu Rev Genet. 43, 197-222 (2009).</li><li>Barrasso, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-function+quorum-sensing+systems+in+bacterial+pathogens+and+symbionts.">Dual-function quorum-sensing systems in bacterial pathogens and symbionts.</a> PLoS Pathog. 16, e1008934 (2020).</li><li>Ball, A. S., Chaparian, R. R., van Kessel, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quorum+sensing+gene+regulation+by+LuxR/HapR+master+regulators+in+Vibrios.">Quorum sensing gene regulation by LuxR/HapR master regulators in Vibrios.</a> J Bacteriol. 199 (19), 00105-00117 (2017).</li><li>Ramos, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TetR+family+of+transcriptional+repressors.">The TetR family of transcriptional repressors.</a> Microbiol Mol Biol Rev. 69, 326-356 (2005).</li><li>Kim, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QStatin,+a+selective+inhibitor+of+quorum+sensing+in+Vibrio+species.">QStatin, a selective inhibitor of quorum sensing in Vibrio species.</a> mBio. 9 (1), 02262 (2018).</li><li>Kim, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LuxR+homologue+SmcR+is+essential+for+Vibrio+vulnificus+pathogenesis+and+biofilm+detachment,+and+its+expression+is+induced+by+host+cells.">LuxR homologue SmcR is essential for Vibrio vulnificus pathogenesis and biofilm detachment, and its expression is induced by host cells.</a> Infect Immun. 81, 3721-3730 (2013).</li><li>Hasegawa, H., Hase, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TetR-type+transcriptional+regulator+VtpR+functions+as+a+global+regulator+in+Vibrio+tubiashii.">TetR-type transcriptional regulator VtpR functions as a global regulator in Vibrio tubiashii.</a> Appl Environ Microbiol. 75, 7602-7609 (2009).</li><li>Dorn, S. K., Newman, J. D., Van Kessel, J. C., Brown, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+biological+assay+of+small-molecule+quorum+sensing+inhibitors:+A+three-week+course-based+undergraduate+research+experience.">Synthesis and biological assay of small-molecule quorum sensing inhibitors: A three-week course-based undergraduate research experience.</a> J Chem Educ. 98, 3533-3541 (2021).</li><li>Newman, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acid+divergence+in+the+ligand-binding+pocket+of+Vibrio+LuxR/HapR+proteins+determines+the+efficacy+of+thiophenesulfonamide+inhibitors.">Amino acid divergence in the ligand-binding pocket of Vibrio LuxR/HapR proteins determines the efficacy of thiophenesulfonamide inhibitors.</a> Mol Microbiol. 116 (4), 1173-1188 (2021).</li><li>Mohrig, J. R. H., Schatz, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Techniques in Organic Chemistry. 3. End. , (2001).</li><li>Kochnev, Y., Hellemann, E., Cassidy, K. C., Durrant, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Webina:+An+open-source+library+and+web+app+that+runs+AutoDock+Vina+entirely+in+the+web+browser.">Webina: An open-source library and web app that runs AutoDock Vina entirely in the web browser.</a> Bioinformatics. 36, 4513-4515 (2020).</li><li>Kim, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+SmcR,+a+quorum-sensing+master+regulator+of+Vibrio+vulnificus,+provides+insight+into+its+regulation+of+transcription.">Crystal structure of SmcR, a quorum-sensing master regulator of Vibrio vulnificus, provides insight into its regulation of transcription.</a> J Biol Chem. 285, 14020-14030 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The PyMOL molecular graphics system v. 2.0. , (2023).</li><li>. PyMol wiki commands Available from: <a target="_blank" href="https://pymolwiki.org/index.php/Category:Commands">https://pymolwiki.org/index.php/Category:Commands</a> (2023)</li><li>van Kessel, J. C., Ulrich, L. E., Zhulin, I. B., Bassler, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+activator+and+repressor+functions+reveals+the+requirements+for+transcriptional+control+by+LuxR,+the+master+regulator+of+quorum+sensing+in+Vibrio+harveyi.">Analysis of activator and repressor functions reveals the requirements for transcriptional control by LuxR, the master regulator of quorum sensing in Vibrio harveyi.</a> mBio. 4 (4), 00378 (2013).</li><li>Swenson, V. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+and+verification+of+commercially+available+pressure+cookers+for+laboratory+sterilization.">Assessment and verification of commercially available pressure cookers for laboratory sterilization.</a> PLoS One. 13, e0208769 (2018).</li></ol>

JVK and LCB disclose financial interests in Quornix, LLC, that may benefit from the outcomes of research on thiophenesulfonamide compounds.

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&#39;s Arts and Sciences Undergraduate Research Ex...

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.

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	<tbody>
		<tr>
			<td>2-thiophensulfonyl chloride</td>
			<td>Ambeed</td>
			<td>A258464</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Phenyl-1H-pyrazole</td>
			<td>Ambeed</td>
			<td>A104401</td>
			<td>98%</td>
		</tr>
		<tr>
			<td>96-well clear bottom black plates</td>
			<td>USA Scientific</td>
			<td>5665-5090Q</td>
			<td>96-well polystyrene uClear black TC plate with lid, clear flat bottom, sterile, 8/sleeve, 32/case&#160;</td>
		</tr>
		<tr>
			<td>Autodock Tools</td>
			<td></td>
			<td></td>
			<td>http://mgltools.scripps.edu/downloads</td>
		</tr>
		<tr>
			<td>Autodock Vina</td>
			<td></td>
			<td></td>
			<td>https://vina.scripps.edu&#160;</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ethyl acetate</td>
			<td>Fisher Scientific</td>
			<td>AA31344M4</td>
			<td>Reagent grade</td>
		</tr>
		<tr>
			<td>hexanes</td>
			<td>Fisher Scientific</td>
			<td>H291</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>magnesium sulfate</td>
			<td>Fisher Scientific</td>
			<td>M65-500</td>
			<td>Anhydrous</td>
		</tr>
		<tr>
			<td>Microporous Film</td>
			<td>USA Scientific</td>
			<td>2920-1010</td>
			<td>Microporous Film, -20degC to +80degC, 50/box, Sterilized</td>
		</tr>
		<tr>
			<td>molview</td>
			<td></td>
			<td></td>
			<td>molview.org</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Databank</td>
			<td></td>
			<td></td>
			<td>https://www.rcsb.org/</td>
		</tr>
		<tr>
			<td>Pymol</td>
			<td></td>
			<td></td>
			<td>https://pymol.org/2/</td>
		</tr>
		<tr>
			<td>Qualitative filter paper</td>
			<td>Fisher Scientific</td>
			<td>09-805-342</td>
			<td>Cytiva Whatman&#8482; Qualitative Filter Paper: Grade 1 Circles, 47 mm</td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>Sorbtech</td>
			<td>30930M-25</td>
			<td>Silica Gel, Standard Grade, 60A, 40-63um (230 x 400 mesh)</td>
		</tr>
		<tr>
			<td>Sodium hydride</td>
			<td>Millipore Sigma</td>
			<td>452912</td>
			<td>60 % dispersion in mineral oil</td>
		</tr>
		<tr>
			<td>Tetrahydrofuran</td>
			<td>Fisher Scientific</td>
			<td>MTX02847</td>
			<td>Tetrahydrofuran, anhydrous, 99.9%, ACS Grade, DriSolv</td>
		</tr>
		<tr>
			<td>TLC Plates</td>
			<td>Sorbtech</td>
			<td>1634067</td>
			<td>Silica gel TLC plates, aluminum backed</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>webina</td>
			<td></td>
			<td></td>
			<td>https://durrantlab.pitt.edu/webina/</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis and assay of vibrio quorum sensing inhibitors

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.

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 wi...

Author Spotlight: Advancing Therapeutics to Treat Vibriosis in Humans and Aquatic Organisms

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.

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In ...

synthesis-and-assay-of-vibrio-quorum-sensing-inhibitors

Research

JoVE Journal

Biology

355 Views.  Indiana University. 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.

Video: Author Spotlight: Advancing Therapeutics to Treat Vibriosis in Humans and Aquatic Organisms

Vibrio cholerae: Model Organism to Study Bacterial Pathogenesis - Interview

Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described. Triplicate runs of 48 parallel experiments are reported, varying concentration, solvent and the excess of some of the reagents. The isolation of the product was achieved by a simple filtration and wash procedure. The highest yield obtained was 66% from 0.4 M methanol with 1.2 eq. of imine. This is significantly above the 49% yield obtained from the initial reaction under equimolar concentration at 0.4 M in methanol. Methanol solutions with reagent concentrations of 0.4M or 0.2M gave superior yields while all solvent systems at 0.07M performed poorly. At 0.2M, methanol and ethanol/methanol (60/40) mixtures were statistically equally good while THF/methanol (60/40) was poor and acetonitrile/methanol (60/40) was intermediate. Good reproducibility of the precipitate yields was obtained in these replicate experiments, allowing for subtle interaction effects to be positively identified.

The Ugi reaction has proved to be a convenient way to quickly create diverse libraries of compounds. It involves the reaction of an amine, an aldehyde, a carboxylic acid and an isonitrile typically in methanol at room temperature. In this video, we utilize a 48-slot Mettler-Toledo MiniBlock equipped with filtration tubes and a Mettler-Toledo MiniMapper automated liquid handler was used to deliver the reagents and solvent. The parameters of interest were the concentration, the solvent composition and the excess of some of the reagents.

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described.  Triplicate runs ...

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides1.
Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides2, which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield1, but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation.
Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an &alpha;-amine)3,4. Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.

The efficient solid-phase peptide synthesis of a functionalized bis-peptide trimer utilizing a "safety catch" cleavage procedure from HMBA resin is described.

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters&#160;and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA (dsDNA) which has the requisite characteristics to stimulate the cytoplasmic DNA sensing pathways can be generated cheaply and with ease. By the concatemerization of short, synthetic oligonucleotides (which lack CpG motifs), dsDNA can be generated to be of sufficient length to activate the cytosolic DNA sensing pathway. This protocol involves blunt end ligation of the oligonucleotides in the presence of polyethylene glycol (PEG), which provides an environment for efficient ligation to occur. The dsDNA concatemers can be used, following purification by phenol/chloroform extraction, to simulate the innate immune response in vitro by standard transfection protocols. This DNA can also be used to stimulate innate immunity in vivo by intradermal injection into the ear pinna of a mouse, for example. By standardizing the concatemerization process and the subsequent stimulation protocols, a reliable and reproducible activation of the innate immune system can be produced.

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

The aim of the protocol is to use optimal methods for stimulating the cytoplasmic DNA sensing pathways in cells and in vivo. This is achieved by improving the generation of long, double-stranded DNA during blunt-end ligation. Cells or mice are then transfected using a lipid transfection reagent.

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...