Fluorescence-based biochemical high throughput confirmation assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF)
Name: Fluorescence-based biochemical high throughput confirmation assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF). ..more
BioActive Compounds: 30
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_3X%INH DESENS
Name: Fluorescence-based biochemical high throughput confirmation assay for inhibitors 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. 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.
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, confirmation, 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 confirm activity of compounds identified as active in a set of previous experiments entitled, Fluorescence-based biochemical primary high throughput screening assay to identify inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF) (AID 493244). 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 singlicate at a final nominal concentration of 7.2 uM.
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 inhibition for each compound was calculated as follows:
%_Inhibition = 100 * ( ( Test_Compound - Median_High_Control ) / ( Median_Low_Control - Median_High_Control ) )
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).
The average percent inhibition and standard deviation of each compound tested were calculated. Any compound that exhibited an average percent inhibition greater than the modified hit cutoff calculated for the primary screen (AID 493244) was declared active. In order to retain possible partial inhibitors and avoid the influence of highly fluorescent artifacts, the hit cutoff for this assay was determined as the PRUN Avg(Median_Low_Control) +/- 3 * StdDev(Median_Low_Control).
PubChem Activity Outcome and Score:
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-10, and for inactive compounds 9-0.
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. The MLSMR was unable to provide all samples requested for testing.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)