Fluorescence-based biochemical primary high throughput screening assay to identify activators of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF)
Name: Fluorescence-based biochemical primary high throughput screening assay to identify activators of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF). ..more
BioActive Compounds: 801
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_1X%ACT PRUN SENS
Name: Fluorescence-based biochemical primary high throughput screening assay to identify 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. PMID: 12270949.
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. PMID: 6204984.
RTF, regulated thin filament, troponin C type I, TNC, TNNC, CMD1Z, CMH13, TNNC1, muscle, cardiac, contraction, contractile, calcium, sensitization, sensitizer, fluorescence, fluorophore, IANBD, FLINT, activate, activator, activation, modulate, modulator, primary, primary screen, 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 identify compounds that increase the calcium sensitivity of regulated thin filaments (RTF) of cardiac muscle. This biochemical 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 singlicate at a final nominal concentration of 7.2 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 (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 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).
A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % activation than the cutoff parameter was declared active.
PubChem Activity Outcome and Score:
The reported PubChem Activity Score has been normalized to 100% observed primary activation. Negative % activation values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-0, and for inactive compounds 0-0.
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.
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.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)