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BioAssay: AID 602246

Assay provider mechanism-of-action assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF): absorbance-based biochemical assay to identify desensitizers of the Ca2+ sensitivity of cardiac myofibrillar ATPase

Name: Assay provider mechanism-of-action assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF): absorbance-based biochemical assay to identify desensitizers of the Ca2+ sensitivity of cardiac myofibrillar ATPase. ..more
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 Tested Compounds
 Tested Compounds
All(5)
 
 
Active(1)
 
 
Inactive(4)
 
 
 Tested Substances
 Tested Substances
All(5)
 
 
Active(1)
 
 
Inactive(4)
 
 
AID: 602246
Data Source: The Scripps Research Institute Molecular Screening Center (ATPASE_INH_ABS_0096_1X-FOLD-CHANGE MCSRUN DESENS)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2012-02-03
Hold-until Date: 2013-02-03
Modify Date: 2013-02-04

Data Table ( Complete ):           View Active Data    View All Data
Targets
BioActive Compound: 1
Related Experiments
AIDNameTypeComment
493244Fluorescence-based biochemical primary high throughput screening assay to identify inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF)Screeningdepositor-specified cross reference: Primary screen (RTF inhibitors in singlicate)
504337Summary of the probe development efforts to identify inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF)Summarydepositor-specified cross reference: Summary (RTF inhibitors)
504635Fluorescence-based biochemical high throughput confirmation assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF)Screeningdepositor-specified cross reference: Confirmation screen (RTF inhibitors in triplicate)
504742Fluorescence-based biochemical high throughput dose response assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF)Confirmatorydepositor-specified cross reference: Dose Response (RTF inhibitors in triplicate)
602245Assay provider mechanism-of-action assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF): biochemical assay to identify inhibitors of the Ca2+-sensitivity of cardiac muscle contraction/tensionOthersame project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: James D. Potter, University of Miami School of Medicine
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R21 NS064821-01
Grant Proposal PI: James D. Potter, University of Miami School of Medicine
External Assay ID: ATPASE_INH_ABS_0096_1X-FOLD-CHANGE MCSRUN DESENS

Name: Assay provider mechanism-of-action assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF): absorbance-based biochemical assay to identify desensitizers of the Ca2+ sensitivity of cardiac myofibrillar ATPase.

Description:

Cardiomyopathies are myocardial diseases that often lead to cardiac remodeling to compensate for deficiencies in cardiac output (1). Cardiomyopathies are characterized as having systolic dysfunctions (i.e. reduced ejection fraction) in dilated cardiomyopathy or diastolic dysfunctions (i.e. impaired relaxation) in hypertrophic and restrictive cardiomyopathies (2). The regulated thin filament (RTF) is a multi-protein complex responsible for switching cardiac muscle contraction on and off in a calcium dependent manner. Mutations in the genes encoding RTF subunits are often the etiological agents for dilated, hypertrophic and restrictive cardiomyopathies. The RTF is comprised of troponin C (TnC), troponin I (TnI), troponin T (TnT), tropomyosin (Tm) and F-actin. Notably, a hallmark of RTF subunit gene mutations in cardiomyopathies is their ability to alter the calcium sensitivity of cardiac muscle contraction and the morphology of the heart (3). Since multiple forms of cardiomyopathies exist, the identification of new drugs that sensitize (+) or desensitize (-) the calcium sensitivity could potentially reverse these aberrant changes. Moreover, there are no calcium desensitizers in clinical use today. As a result of this HTS campaign, the identification of RTF calcium sensitivity modulators may serve as useful tools for elucidating the roles of these proteins in cardiac muscle contraction and disease (4).

References:

1. Fatkin D, Graham RM. Physiol Rev. 2002 Oct;82(4):945-80. Molecular mechanisms of inherited cardiomyopathies.
2. Griffin, B. P., and Topol, E. J. (2004) Manual of Cardiovascular Medicine, 2nd Ed., pp. 101142, Lippincott Williams and Wilkins, Philadelphia.
3. Dweck, D., Hus, N., and Potter, J. D. (2008) Challenging current paradigms related to cardiomyopathies. Are changes in the Ca2+ sensitivity of myofilaments containing cardiac troponin C mutations (G159D and L29Q) good predictors of the phenotypic outcomes? J Biol Chem 283, 33119-28.
4. Ingraham, R. H., and Swenson, C. A. (1984) Binary interactions of troponin subunits. J Biol Chem 259, 9544-8.

Keywords:

ATPase, hydrolysis, assay provider, powders, purchased, synthesized, late stage, RTF, regulated thin filament, troponin C type I, TNC, TNNC, CMD1Z, CMH13, TNNC1, muscle, cardiac, contraction, contractile, calcium, desensitization, desensitizer,abs, absorbance, modulate, modulator, inhibit, inhibitor, inhibition, singlicate, Kansas, Kansas Specialized Chemistry Center, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:

The purpose of this assay is to determine whether powder samples of compounds identified as possible RTF inhibitor (desensitizer) probe candidates act to decrease the CMF ATPase activity. In addition to the RTF proteins, the CMF contains the motor protein, myosin which catalyzes the hydrolysis of ATP. The energy from the hydrolysis of ATP is used to power the stroke of contraction. In this biochemical assay, compounds are incubated in a suspension of CMFs. As designed, compounds that are calcium desensitizers in this assay will interact with the RTF to decrease the CMF ATPase activity in an EC50 calcium concentration. The amount of hydrolyzed ATP is directly proportional to the amount of inorganic phosphate released which is detected colorimetrically by well absorbance (660 nm) using the Fiske-Subbarow reagent. Compounds were tested at a nominal maximum concentration of 10 uM.

Protocol Summary:

Prior to the start of the assay, 5 mg of porcine cardiac myofibrils (PCMF) were previously prepared and stored at -20 C in 20 mM MOPS, 40 mM KCl and pH 7.0 (50% v/v glycerol). PCMF were resuspended in 10X the starting volume in pCa buffer (2.083 mM EGTA, 3.125 mM NTA, 4.390 mM MgCl2, 20.833 mM MOPS, 1.523 mM CaCl2, 20.427 mM KCl and 1mM DTT [pH 7.0]) followed by centrifugation at 750 x g (4 C) for 7 min. The supernatent was discarded and the pellet was resuspended in pCa buffer using 20-25X the pellet volume. Centrifugation and resuspension were repeated two more times as above. A final centrifugation was followed by resuspending the myofibril pellet to a final concentration of 0.521 g/L. 96e-6 L (50e-6 g) of PCMF were aliquoted into 96 well plates using a multi-channel pipettor. Compounds (in 100% DMSO) were added to the wells in quintuplicate to achieve a final concentration of 10e-6 M. Control wells only received DMSO (0.004 % v/v ). The plates were incubated at 25 C while shaking gently for 5 min. To start the ATPase reaction, 4e-6 L of ATP was added to each well (final [ATP] = 3.02 mM) and the plates were incubated at 25 C while shaking gently. The reaction was stopped 7 min later by the addition of 4 % TCA. The plates were centrifuged at 4000 x g for 7 min (4 C) and the supernatants were collected in order to colorimetrically measure the amount of liberated phosphate using the Fiske-Subbarow reducing agent according to the manufacturer's guidelines (Sigma).

Absorbance was read using a plate reader (Biotek) with a 660 nm filter.

The percent activity for each compound was calculated as follows:

Normalized ATPase Activity = Mean_Test_Compound / Mean_Control

Where:

Mean_Test_Compound is defined as the combined average value of all wells containing test compound.
Mean_Control is defined as the combined averaged value of all wells containing DMSO.

The effect of each compound on the Normalized ATPase activity was compared to the control experiments. Any compound that could decrease the ATPase activity greater than or equal to 10 % percent warranted further investigation in more a more complex physiological system, such as in skinned muscle fibers.

PubChem Activity Outcome and Score:

Compounds that were significantly different (Student's t-test) from the control and decreased the ATPase activity greater than or equal to 10 % were considered active.

Active compounds were given a score of 100, and inactive compounds were given a score of 0.

The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.

List of Reagents:

Cardiac myofibrils (CMF) prepared from porcine left ventricles.
Comment
This assay was run by the assay provider. Compounds were provided by Kansas University Specialized Chemistry Center. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well absorbance.
Categorized Comment
BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: secondary: mmoa characterization

BAO: bioassay specification: assay biosafety level: bsl1

BAO: assay format: biochemical format: protein format: protein complex format

BAO: bioassay specification: assay measurement type: endpoint assay

BAO: bioassay specification: assay readout content: assay readout method: regular screening

BAO: bioassay specification: assay readout content: content readout type: single readout

BAO: meta target: molecular target: protein target: enzyme: transferase: generic transferase

BAO: meta target: biological process target: regulation of molecular function

BAO: meta target detail: binding reporter specification: interaction: protein-small molecule

BAO: assay design: enzyme reporter: substrate coupled enzyme: atp coupled enzyme

BAO: detection technology: spectrophotometry: absorbance

BAO: bioassay specification: bioassay type: functional: enzyme activity

BAO: bioassay specification: assay footprint: microplate: 96 well plate

BAO: bioassay specification: assay measurement throughput quality: single concentration multiple replicates

Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Normalized ATPase ActivityThe activity of cardiac myofibrils (CMF) ATPase to hydrolyse ATP, normlized to the ATPase activity of CMF in wells treated with DMSO.Floatratio
2Standard DeviationStandard deviation derived from the normalized percent inhibition of the replicate data for each compound.Float
3Qualifier [p value]Qualifier identifies if the p value was less than or greater than 0.05.String
4p valueStatistical parameter describing the significance of the change in ATPase activity following treatment with test compound. Values less than 0.05 are considered statistically different from the DMSO control group.Float
Additional Information
Grant Number: 1 R21 NS064821-01

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