<meta charset="utf8"></head><body>噻吩磺酰胺在群体感应抑制中的生物学评价

<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>

<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 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.

<meta charset="utf8"></head><body>作者聚焦：推进治疗人类和水生生物弧菌病的疗法

弧菌群体感应抑制剂的合成和测定

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

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors

325 次观看.  Indiana University. 我们的研究重点是设计、合成和分析导致海洋动物和人类疾病的弧菌的潜在抑制剂。我们以前的研究确定噻吩磺酰胺化合物是弧菌物种致病性的有效抑制剂。我们的目标是开发用于治疗弧菌病的化合物。我们合成了一组对几种弧菌物种具有活性的噻吩磺酰胺分子。我们已经解决了目标弧菌结合口袋中噻吩磺酰胺分子的 X 射线晶体结构。我们确定...

视频: 作者聚焦：推进治疗人类和水生生物弧菌病的疗法

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.

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.