Synthesis and Assay of Vibrio Quorum Sensing Inhibitors

Summary

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.

Abstract

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.

Introduction

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 thr....

Protocol

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 condensation^8,9, as shown in Figure 2. To design new compound libraries for study, researchers must procure appropriate amines and sulfonyl chlorides and follow the steps su....

Results

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.......

Discussion

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 Ex.......

Materials

Name	Company	Catalog Number	Comments
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