Name: Author Spotlight: Advancing Therapeutics to Treat Vibriosis in Humans and Aquatic Organisms
Uploaded: 2024-05-31T13:10:00
Description: Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In ...

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 Fig...

Biological Assessment of Thiophenesulfonamides in Quorum Sensing Inhibition

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	<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>

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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.

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

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors

Our research focuses on designing, synthesizing, and analyzing potential inhibitors of Vibrio bacteria that cause disease in marine animals and humans. Our previous studies identified thiophenesulfonamide compounds as potent inhibitors of pathogenicity in Vibrio species. We aim to develop compounds for the treatment of Vibrio disease.We have synthesized a panel of thiophenesulfonamide molecules active against several Vibrio species. We've solved the X-ray crystal structure of a thiophenesulfonamide molecule in the binding pocket of the target Vibrio. And we determined the thiophenesulfonamide mechanism of action in blocking the protein target and preventing pathogenicity in vivo.This protocol is designed with the novice researcher in mind. Every step was optimized for simplicity and error reduction, while still allowing for teaching concepts and the scientific method. Our protocol bridges microbiology, organic chemistry, structure modeling, and drug design in a novel, teaching-based experimental system.Novel therapeutics that target Vibrio bacteria are needed to treat the increasing cases of Vibriosis in humans and aquaculture. Because our compounds are not antibiotics and don't kill cells, they have the potential to treat disease without generating resistance. We will focus on optimizing the inhibitors of Vibrio species that do not have a therapeutically relevant inhibitor yet.We will also explore the chemical space afforded by substituted aromatic sulfonyl chloride starting materials.

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...

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 ...