Fluorescence-based biochemical high throughput dose response assay for activators of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF)
Name: Fluorescence-based biochemical high throughput dose response assay for activators of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF). ..more
BioActive Compounds: 76
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_ACT_FLINT_1536_3XEC50 DRUN SENS
Name: Fluorescence-based biochemical high throughput dose response assay for activators of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF).
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).
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. 101-142, 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.
RTF, regulated thin filament, troponin C type I, TNC, TNNC, CMD1Z, CMH13, TNNC1, muscle, cardiac, contraction, contractile, biochemical, calcium, sensitization, sensitizer, fluorescence, fluorophore, IANBD, FLINT, activate, activator, activation, modulate, modulator, 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.
The purpose of this assay is to determine dose response curves for compounds that confirmed activity in a previous set of experiments entitled, "Fluorescence-based biochemical high throughput confirmation assay for activators of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF)" (AID 504383), and that were inactive in a set of experiments entitled, "Counterscreen for activators of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF): Fluorescence-based biochemical assay to identify fluorescence artifacts" (AID 504382).
This biochemical assay monitors the calcium sensitivity of regulated thin filaments (RTF) of cardiac muscle. The assay employs the visible fluorophore, IANBD (4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole) (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, an increase 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. Compounds are tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 72 uM at a calcium concentration corresponding to the EC20 (0.472 mM).
Prior to the start of the assay, 4 uL of RTF Assay Buffer (3mM 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 dichoic mirror.
The percent activation for each compound was calculated as follows:
%_Activation = ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) ) * 100
Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO and 0.472 mM Ca2+ (EC20).
High_Control is defined as wells containing DMSO and 2.9 mM Ca2+ (EC100).
For each test compound, percent activation 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 EC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation 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% activation, the EC50 was determined manually as greater than 71.5 uM.
PubChem Activity Outcome and Score:
Compounds with an EC50 greater than 10 uM were considered inactive. Compounds with an EC50 equal to or less than 10 uM were considered active.
Any compound with a percent activity value <5 0% 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 100-78, and for inactive compounds 74-0.
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)
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. In order to retain possible partial activators and avoid the influence of highly fluorescent artifacts, the hit cutoff for this assay was determined as Avg(Median_Low_Control) +/- 3 * StdDev(Median_Low_Control). The MLSMR was not able to provide all compounds selected for testing.
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.
Assay: Dictionary: Version: 0.1
Assay: CurveFit : Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
* Activity Concentration. ** Test Concentration.
Data Table (Concise)