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BioAssay: AID 651905

CqsS In vitro Kinase Assay Measured in Biochemical System Using Imaging - 2132-10_Agonist_Dose_DryPowder_Activity

In order to investigate how CAI-1 binding regulates quorum sensing signaling events, we used a biochemical approach to reconstitute the CqsS->LuxU->LuxO circuit in vitro. We cloned and over-expressed the CqsS receptor in recombinant E. coli, and examined in vitro auto- phosphorylation of CqsS using inverted membrane vesicles and [gamma-32P] ATP. In this assay, wild type CqsS exhibits more ..
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 Tested Compounds
 Tested Compounds
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Active(3)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Active(3)
 
 
AID: 651905
Data Source: Broad Institute (2132-10_Agonist_Dose_DryPowder_Activity)
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-12-11
Hold-until Date: 2013-07-17
Modify Date: 2013-07-17

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 3
Related Experiments
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AIDNameTypeProbeComment
588521Broad Institute Quorum Sensing in Vibrio cholera: CqsS/LuxQ Agonist Probe ProjectSummary1 depositor-specified cross reference: Summary assay
588436Cholera Quorum: HTS for inducers of light production in the absence ofautoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Agonist_SinglePoint_HTS_ActivityScreening same project related to Summary assay
602243Cholera Quorum: HTS for inducers of light production in the absence of autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Agonist_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
624140Cytotoxicity at 24 hours in dose with HeLa cells Measured in Cell-Based System Using Plate Reader - 2132-02_Agonist_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
624254Quorum LuxO: Assay for inducers of light production in the absence of#autoinducers using BH1651 (luxOD47E) Measured in Microorganism System Using Plate Reader - 2132-03_Agonist_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
624269Quorum LuxO: Assay for inducers of light production in the absence of#autoinducers using BH1651 (luxOD47E) Measured in Microorganism System Using Plate Reader - 2132-03_Agonist_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
624270Cholera Quorum: HTS for inducers of light production in the absence of#autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Agonist_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
624275Quorum CqsS/ LuxQ: Assay for inducers of light production in the absence of#autoinducers using WN1103 (delta luxQ) Measured in Microorganism System Using Plate Reader - 2132-05_Agonist_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
624281Quorum CqsS/ LuxQ: Assay for inducers of light production in the absence#of autoinducers using DH231 (delta cqsS) Measured in Microorganism System Using Plate Reader - 2132-04_Agonist_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651774Cytotoxicity at 24 hours in dose with HeLa cells Measured in Cell-Based System Using Plate Reader - 2132-02_Agonist_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651808Quorum CqsS/ LuxQ: Assay for inducers of light production in the absence of autoinducers using DH231 (delta cqsS) Measured in Microorganism System Using Plate Reader - 2132-04_Agonist_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651809Cholera Quorum: HTS for inducers of light production in the absence of autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Agonist_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
651816Cholera Quorum: HTS for inducers of light production in the absence of autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Agonist_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
651841Quorum CqsS/ LuxQ: Assay for inducers of light production in the absence of autoinducers using WN1103 (delta luxQ) Measured in Microorganism System Using Plate Reader - 2132-05_Agonist_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651847Quorum LuxO: Assay for inducers of light production in the absence of autoinducers using BH1651 (luxOD47E) Measured in Microorganism System Using Plate Reader - 2132-03_Agonist_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
651854Cholera Quorum: HTS for inducers of light production in the absence of autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Agonist_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651864Cytotoxicity at 24 hours in dose with HeLa cells Measured in Cell-Based System Using Plate Reader - 2132-02_Agonist_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
652219Quorum LuxO: Assay for change of GFP production in the absence of autoinducers using SL353 (luxOD47E, qrr4-gp) Measured in Microorganism System Using Plate Reader - 2132-06_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
652239Cholera Quorum: HTS for inducers of light production in the absence of autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
686928Cytotoxicity at 24 hours in dose with HeLa cells Measured in Cell-Based System Using Plate Reader - 2132-02_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
686929Cholera Quorum: HTS for inducers of light production in the absence of autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
686930Quorum CqsS/ LuxQ: Assay for inducers of light production in the absence of autoinducers using WN1103 (delta luxQ) Measured in Microorganism System Using Plate Reader - 2132-05_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
686931Quorum CqsS/ LuxQ: Assay for inducers of light production in the absence of autoinducers using DH231 (delta cqsS) Measured in Microorganism System Using Plate Reader - 2132-04_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
686936In vitro LuxO ATPase Assay Measured in Biochemical System Using Plate Reader - 2132-11_Inhibitor_Dose_DryPowder_ActivityOther same project related to Summary assay
686939Counterscreen for activators of the function of SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2 (SMARCA2, BRM): Luminescence-based cell-based high throughput screening assay to identify non-selective compounds using the VP16 reporter assayScreening same project related to Summary assay
686942Quorum LuxO: Assay for change of GFP production in the absence of autoinducers using SL353 (luxOD47E, qrr4-gp) Measured in Microorganism System Using Plate Reader - 2132-06_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
686945Broad Institute Quorum Sensing in Vibrio cholera: LuxO Inhibitor Probe ProjectSummary same project related to Summary assay
Description:
Keywords: kinase, CqsS, quorum, cholera, in vitro


Assay Overview:

In order to investigate how CAI-1 binding regulates quorum sensing signaling events, we used a biochemical approach to reconstitute the CqsS->LuxU->LuxO circuit in vitro. We cloned and over-expressed the CqsS receptor in recombinant E. coli, and examined in vitro auto- phosphorylation of CqsS using inverted membrane vesicles and [gamma-32P] ATP. In this assay, wild type CqsS exhibits auto-phosphorylation similar to what happens in cells. Compounds were tested for their ability to inhibit CqsS auto-phosphorylation. Reaction products are terminated and run out by SDS-PAGE. Gels are dried and exposed to a phosphoscreen. From epistatic studies in a number of different bacterial strains, we know that our lead compounds act upon CqsS or require CqsS for activity because they are inactive in the absence of CqsS. It is likely that agonism occurs by preventing the auto-phosphorylation or preventing a change in the protein that results from auto-phosphorylation. A probe candidate that inhibits auto-phosphorylation is acceptable but so is a compound that works by some other method, such as allosteric modification.

Expected Outcome: A decrease in phosphorylation will coincide with a less radiolabel incorporation in the gel band (corresponding with CqsS) and suggest that a compound is inhibiting cqsS auto-phosphorylation. No activity in this assay is also acceptable if the compound works some other mechanism.
Protocol
Preparation of inverted membranes
E. coli BL21 (DE3) harboring plasmids encoding wild type CqsS were grown with shaking in LB with 100 mug/mL kanamycin at 37 degrees C. Overnight bacterial cultures were diluted 1:100 into fresh LB medium with kanamycin. After growth with aeration for 3 h at 37 degrees C, protein production was induced with 1 mM IPTG. The cultures were shifted to room temperature and grown for an additional 5 h with shaking. Cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris pH 8.0, 200 mM NaCl, 5 mM beta-mercaptoethanol, 5 mM MgCl2 and 20 mM imidazole), and lysed under 15,000 psi. Cell lysates were centrifuged at 9,300 x g for 30 min and the cleared fluids were harvested and subjected to ultra-centrifugation at 180,000 x g for 1 h. Membrane pellets were resuspended in kinase buffer (50 mM Tris pH 8.0, 100 mM KCl, 5 mM MgCl, and 10% (v/v) glycerol) and quantified by SDS-PAGE and Western blot using CqsS purified in detergent as the standard.
Phosphorylation assays
Phosphorylation assays were performed with inverted membranes containing 2 muM wild type CqsS protein. In assays containing LuxU and/or LuxO, LuxU and LuxO were supplied at 10 muM. Reactions were carried out in phosphorylation buffer (50 mM Tris pH 8.0, 100 mM KCl, 5 mM MgCl, and 10% (v/v) glycerol), and were initiated with the addition of 100 muM ATP and 2 muCi [gamma-32P]-ATP (from a stock of 3000 Ci/mmol: Perkin Elmer). For experiments with CAI-1 and analogs, compounds were added 10 min prior to the initiation of the reactions. For the reconstitution of the complete CAI-1/CqsS, LuxU, LuxO circuit, CAI-1 was supplied at 500 muM. Regulation of histidine phosphorylation by CAI-1 was assayed with 500 muM CAI-1. Agonism was tested at 100 muM. Antagonism was tested with 10 muM agonist and 100 muM. Reactions were incubated at room temperature and terminated with SDS-PAGE loading buffer. Reaction products were separated using SDS- PAGE. Gels were dried at 80 degrees C on filter paper under vacuum, exposed to a phosphoscreen overnight, and subsequently analyzed using a Typhoon 9400 scanner and ImageQuant software.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Assay Test Type: In vitro
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPLICATE_A_ACTIVITY_SCORE_100uM_(%) Float%
2REPLICATE_B_ACTIVITY_SCORE_100uM_(%) .Float%
Additional Information
Grant Number: R03 MH094166-01

Data Table (Concise)
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