Fluorescence-based biochemical high throughput dose response assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF)
BioActive Compounds: 14
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).
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).
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 ) )
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.