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BioAssay: AID 686936

In vitro LuxO ATPase Assay Measured in Biochemical System Using Plate Reader - 2132-11_Inhibitor_Dose_DryPowder_Activity

A modified coupled-enzyme assay measured the rate of ATP hydrolysis by LuxOD47E. Briefly, ADP released from ATP by LuxOD47E is reacted with phosphoenolpyruvate (PEP) to form pyruvate using pyruvate kinase (PK). Pyruvate is reacted with NADH to form NAD and lactate using lactate dehydrogenase (LDH). The rate of NAD production is followed at 340 nm using a spectrophotometer. ATP hydrolysis rates more ..
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 Tested Compounds
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Active(2)
 
 
 Tested Substances
 Tested Substances
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Active(2)
 
 
AID: 686936
Data Source: Broad Institute (2132-11_Inhibitor_Dose_DryPowder_Activity)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2013-04-13
Hold-until Date: 2013-07-17
Modify Date: 2013-07-17

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2
Related Experiments
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AIDNameTypeProbeComment
588521Broad Institute Quorum Sensing in Vibrio cholera: CqsS/LuxQ Agonist Probe ProjectSummary1 depositor-specified cross reference: Summary
686945Broad Institute Quorum Sensing in Vibrio cholera: LuxO Inhibitor Probe ProjectSummary depositor-specified cross reference
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
651905CqsS In vitro Kinase Assay Measured in Biochemical System Using Imaging - 2132-10_Agonist_Dose_DryPowder_ActivityOther 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
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
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
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
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
652289Quorum LuxO: Assay for inducers of light production in the absence of autoinducers using BH1651 (luxOD47E) Measured in Microorganism System Using Plate Reader - 2132-03_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
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
Description:
Keywords: LuxO, cholera, ATPase, kinase

Assay Overview:

A modified coupled-enzyme assay measured the rate of ATP hydrolysis by LuxOD47E. Briefly, ADP released from ATP by LuxOD47E is reacted with phosphoenolpyruvate (PEP) to form pyruvate using pyruvate kinase (PK). Pyruvate is reacted with NADH to form NAD and lactate using lactate dehydrogenase (LDH). The rate of NAD production is followed at 340 nm using a spectrophotometer. ATP hydrolysis rates were inferred from the absorbance change observed (aNADH,340 -a-NAD,340 = 6220 M-1 cm-1 for NADH). The rates of ATP hydrolysis by LuxOD47E were measured in reactions containing 100 mM sodium phosphate buffer pH 7.4, 5 mM MgCl2, 0.2 mM NADH, 1 mM PEP, 5-20 units of PK/LDH mix (Sigma), and 10 mM LuxOD47E. ATP and inhibitors were added to the reactions at indicated concentrations. The rate of ATP hydrolysis was monitored for 5 minutes. Data were fitted using GraphPad Prism to obtain the kinetic parameters. Percent ATPase inhibition was calculated.


Expected Outcome: A compound that inhibits ATPase activity of LuxO will lead to a decrease in ATP hydrolysis, measured as a lower OD value.
Protocol
A modified coupled-enzyme assay was used to measure the rate of ATP hydrolysis by LuxOD47E. Briefly, ADP released from ATP by LuxO D47E is reacted with phosphoenolpyruvate (PEP) to form pyruvate using pyruvate kinase (PK). Pyruvate is reacted with NADH to form NAD and lactate using lactate dehydrogenase (LDH). The rate of NAD production is followed at 340 nm using a spectrophotometer. The rates of ATP hydrolysis by LuxOD47E were measured in reactions containing 100 mM sodium phosphate buffer pH 7.4, 5 mM MgCl2, 0.2 mM NADH, 1 mM PEP, 5-20 units of PK/LDH mix (Sigma), and 10 microM LuxOD47E. ATP was added at a final concentration of 2.5 mM. The rate of ATP hydrolysis was monitored for 5 minutes. The values reported are delta OD340/min.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Assay Test Type: In vitro
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AVG_DELTA_OD_AT_100um (100μM**)Float
2AVG_DELTA_OD_AT_25um (25μM**)Float
3AVG_DELTA_OD_AT_1um (1μM**)Float
4DELTA_OD_AT_100um_TEST1 (100μM**)Float
5DELTA_OD_AT_25um_TEST1 (25μM**)Float
6DELTA_OD_AT_1um_TEST1 (1μM**)Float
7DELTA_OD_AT_100um_TEST2 (100μM**)Float
8DELTA_OD_AT_25um_TEST2 (25μM**)Float
9DELTA_OD_AT_1um_TEST2 (1μM**)Float
10DELTA_OD_AT_100um_TEST3 (100μM**)Float
11DELTA_OD_AT_25um_TEST3 (25μM**)Float
12DELTA_OD_AT_1um_TEST3 (1μM**)Float
13DELTA_OD_AT_100um_TEST4 (100μM**)Float

** Test Concentration.
Additional Information
Grant Number: R03 MH094166-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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