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BioAssay: AID 504742

Fluorescence-based biochemical high throughput dose response assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF)

Name: Fluorescence-based biochemical high throughput dose response assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF). ..more
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 Tested Compounds
 Tested Compounds
All(32)
 
 
Active(14)
 
 
Inactive(18)
 
 
 Tested Substances
 Tested Substances
All(32)
 
 
Active(14)
 
 
Inactive(18)
 
 
AID: 504742
Data Source: The Scripps Research Institute Molecular Screening Center (RTF_INH_FLINT_1536_3XIC50 DRUN DESENS)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-05-10

Data Table ( Complete ):           View Active Data    View All Data
Targets
BioActive Compounds: 14
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)
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/tensionOtherdepositor-specified cross reference
602246Assay 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 ATPaseOtherdepositor-specified cross reference
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: RTF_INH_FLINT_1536_3XIC50 DRUN DESENS

Name: Fluorescence-based biochemical high throughput dose response assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF).

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:

RTF, regulated thin filament, troponin C type I, TNC, TNNC, CMD1Z, CMH13, TNNC1, muscle, cardiac, contraction, contractile, calcium, desensitization, desensitizer, fluorescence, fluorophore, IANBD, FLINT, inhibit, modulate, modulator, inhibitor, inhibition, dose response, triplicate, uHTS, HTS, high throughput screen, 1536, 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 dose response curves for compounds that confirmed activity in a set of previous experiments entitled, "Fluorescence-based biochemical high throughput confirmation assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF)" (AID 504635). This biochemical assay employs the visible fluorophore, IANBD (Ex ~ 485 nm; Em ~ 535 nm) attached to troponin C (TnC, the Ca2+ binding subunit of the RTF) to monitor the Ca2+ dependent changes in fluorescence arising from IANBD-labeled RTF. As designed, a decrease in the RTF fluorescence intensity at a fixed calcium concentration and wavelength in response to a test compound will indicate that a change in the RTF calcium sensitivity has occurred. This assay employs an EC70 calcium concentration (1.6 mM). Compounds are tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 71.5 uM.

Protocol Summary:

Prior to the start of the assay, 4 uL of RTF Assay Buffer (3 mM EGTA, 4 mM NTA, 1.5 mM MgCl2, 60 mM KCl, 190 mM MOPS, 1 mM DTT, 0.02% IGEPAL CA630, pH 7.20) containing 1.25X (0.0313 g/L) of RTF protein were dispensed into 1536 microtiter plates. Next, 1 uL of the 5X EC20 activation mix (2.36 mM Ca2+) in Assay Buffer was added to all wells. Plates were centrifuged and 36 nL of test compound in DMSO or DMSO alone (0.7% final concentration) were added to the appropriate wells and incubated for 15 minutes at 25 C.

Fluorescence was read using the ViewLux plate reader (Perkin Elmer) using an excitation filter of 480/20 nm and emission filter of 540/25 nm and a dichroic mirror.

The percent inhibition for each compound was calculated as follows:

%_Inhibition = 100 * ( ( Test_Compound - Median_High_Control ) / ( Median_Low_Control - Median_High_Control ) )

Where:

Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing DMSO and 14.3 uM Ca2+ (EC0).
Low_Control is defined as wells containing DMSO and 1.6 mM Ca2+ (EC70).

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 71.5 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 71.5 uM.

PubChem Activity Outcome and Score:

Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.

Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-83, and for inactive compounds 77-0.

List of Reagents:

Recombinant RTF complex (supplied by Assay Provider)
RTF Assay Buffer (supplied by Assay Provider)
Ca2+ stocks (supplied by Assay Provider)
IGEPAL CA630 (Sigma, part 542334)
DTT (Invitrogen, part 15508-013)
1536-well plates (Corning, part 7261)
Comment
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. 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 fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. This assay monitors the activity of recombinant RTF, which is a complex of 5 proteins. Protein and gene identifiers for one of the components (rabbit fast skeletal alpha actin), was not available in the NCBI database. The MLSMR was unable to provide all samples requested for testing.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
Assay: CurveFit [1]: Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: Dictionary: Version: 0.1
From ChEMBL:
Assay Type: Binding
Assay Cell Type: NULL
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentrationFloat
4Maximal ResponseThe maximal or asymptotic response above the baseline as concentration increases without bound.Float
5Baseline ResponseAdjustable baseline of the curve fit, minimal response value.Float
6Inflection Point ConcentrationThe concentration value for the inflection point of the curve.FloatμM
7Hill SlopeThe variable 'Hill Slope' describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
8Hill S0Y-min of the curve.Float
9Hill SinfY-max of the curve.Float
10Hill dSThe range of Y.Float
11Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
12RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
13Number of DataPointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
14Inhibition at 0.004 uM [1] (0.004μM**)Value of % inhibition at 0.004 uM concentration; replicate [1]Float%
15Inhibition at 0.004 uM [2] (0.004μM**)Value of % inhibition at 0.004 uM concentration; replicate [2]Float%
16Inhibition at 0.004 uM [3] (0.004μM**)Value of % inhibition at 0.004 uM concentration; replicate [3]Float%
17Inhibition at 0.011 uM [1] (0.011μM**)Value of % inhibition at 0.011 uM concentration; replicate [1]Float%
18Inhibition at 0.011 uM [2] (0.011μM**)Value of % inhibition at 0.011 uM concentration; replicate [2]Float%
19Inhibition at 0.011 uM [3] (0.011μM**)Value of % inhibition at 0.011 uM concentration; replicate [3]Float%
20Inhibition at 0.033 uM [1] (0.033μM**)Value of % inhibition at 0.033 uM concentration; replicate [1]Float%
21Inhibition at 0.033 uM [2] (0.033μM**)Value of % inhibition at 0.033 uM concentration; replicate [2]Float%
22Inhibition at 0.033 uM [3] (0.033μM**)Value of % inhibition at 0.033 uM concentration; replicate [3]Float%
23Inhibition at 0.098 uM [1] (0.098μM**)Value of % inhibition at 0.098 uM concentration; replicate [1]Float%
24Inhibition at 0.098 uM [2] (0.098μM**)Value of % inhibition at 0.098 uM concentration; replicate [2]Float%
25Inhibition at 0.098 uM [3] (0.098μM**)Value of % inhibition at 0.098 uM concentration; replicate [3]Float%
26Inhibition at 0.294 uM [1] (0.294μM**)Value of % inhibition at 0.294 uM concentration; replicate [1]Float%
27Inhibition at 0.294 uM [2] (0.294μM**)Value of % inhibition at 0.294 uM concentration; replicate [2]Float%
28Inhibition at 0.294 uM [3] (0.294μM**)Value of % inhibition at 0.294 uM concentration; replicate [3]Float%
29Inhibition at 0.882 uM [1] (0.882μM**)Value of % inhibition at 0.882 uM concentration; replicate [1]Float%
30Inhibition at 0.882 uM [2] (0.882μM**)Value of % inhibition at 0.882 uM concentration; replicate [2]Float%
31Inhibition at 0.882 uM [3] (0.882μM**)Value of % inhibition at 0.882 uM concentration; replicate [3]Float%
32Inhibition at 2.6 uM [1] (2.6μM**)Value of % inhibition at 2.6 uM concentration; replicate [1]Float%
33Inhibition at 2.6 uM [2] (2.6μM**)Value of % inhibition at 2.6 uM concentration; replicate [2]Float%
34Inhibition at 2.6 uM [3] (2.6μM**)Value of % inhibition at 2.6 uM concentration; replicate [3]Float%
35Inhibition at 7.9 uM [1] (7.9μM**)Value of % inhibition at 7.9 uM concentration; replicate [1]Float%
36Inhibition at 7.9 uM [2] (7.9μM**)Value of % inhibition at 7.9 uM concentration; replicate [2]Float%
37Inhibition at 7.9 uM [3] (7.9μM**)Value of % inhibition at 7.9 uM concentration; replicate [3]Float%
38Inhibition at 23.8 uM [1] (23.8μM**)Value of % inhibition at 23.8 uM concentration; replicate [1]Float%
39Inhibition at 23.8 uM [2] (23.8μM**)Value of % inhibition at 23.8 uM concentration; replicate [2]Float%
40Inhibition at 23.8 uM [3] (23.8μM**)Value of % inhibition at 23.8 uM concentration; replicate [3]Float%
41Inhibition at 71.5 uM [1] (71.5μM**)Value of % inhibition at 71.5 uM concentration; replicate [1]Float%
42Inhibition at 71.5 uM [2] (71.5μM**)Value of % inhibition at 71.5 uM concentration; replicate [2]Float%
43Inhibition at 71.5 uM [3] (71.5μM**)Value of % inhibition at 71.5 uM concentration; replicate [3]Float%

* Activity Concentration. ** Test Concentration.
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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