Assay provider mechanism-of-action assay for activators of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF): absorbance-based biochemical assay to identify sensitizers of the Ca2+ sensitivity of cardiac myofibrillar ATPase
Name: Assay provider mechanism-of-action assay for activators of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF): absorbance-based biochemical assay to identify sensitizers of the Ca2+ sensitivity of cardiac myofibrillar ATPase. ..more
BioActive Compounds: 2
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: ATPASE_ACT_ABS_0096_1X-FOLD-CHANGE MCSRUN SENS
Name: Assay provider mechanism-of-action assay for activators of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF): absorbance-based biochemical assay to identify sensitizers of the Ca2+ sensitivity of cardiac myofibrillar ATPase.
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.
ATPase, hydrolysis, assay provider, powders, purchased, synthesized, late stage, RTF, regulated thin filament, troponin C type I, TNC, TNNC, CMD1Z, CMH13, TNNC1, muscle, cardiac, contraction, contractile, calcium, sensitization, sensitizer,abs, absorbance, modulate, modulator,activate, activator, activation, singlicate, Kansas, Kansas Specialized Chemistry Center, 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 whether powder samples of compounds identified as possible RTF activator probe candidates act to increase the CMF ATPase activity. In addition to the RTF proteins, the CMF contains the motor protein myosin, which catalyzes the hydrolysis of ATP. The energy from the hydrolysis of ATP is used to power the stroke of contraction. In this biochemical assay, compounds are incubated in a suspension of CMFs. As designed, compounds that are calcium sensitizers (i.e. activators) will interact with the RTF to increase the CMF ATPase activity in an EC50 calcium concentration. The amount of hydrolyzed ATP is directly proportional to the amount of inorganic phosphate released which is detected colorimetrically by well absorbance (660 nm) using the Fiske-Subbarow reagent. Compounds were tested at a nominal maximum concentration of 10 uM.
Prior to the start of the assay, 5 mg of porcine cardiac myofibrils (PCMF) were previously prepared and stored at -20 C in 20 mM MOPS, 40 mM KCl and pH 7.0 (50% v/v glycerol). PCMF were resuspended in 10X the starting volume in pCa buffer (2.083 mM EGTA, 3.125 mM NTA, 4.390 mM MgCl2, 20.833 mM MOPS, 1.523 mM CaCl2, 20.427 mM KCl and 1mM DTT [pH 7.0]) followed by centrifugation at 750 x g (4 C) for 7 min. The supernatent was discarded and the pellet was resuspended in pCa buffer using 20-25X the pellet volume. Centrifugation and resuspension were repeated two more times as above. A final centrifugation was followed by resuspending the myofibril pellet to a final concentration of 0.521 g/L. 96e-6 L (50e-6 g) of PCMF were aliquoted into 96 well plates using a multi-channel pipettor. Compounds (in 100% DMSO) were added to the wells in quintuplicate to achieve a final concentration of 10e-6 M. Control wells received DMSO (0.004 % v/v ). The plates were incubated at 25 C while shaking gently for 5 min. To start the ATPase reaction, 4e-6 L of ATP was added to each well (final [ATP] = 3.02 mM) and the plates were incubated at 25 C while shaking gently. The reaction was stopped 7 min later by the addition of 4 % TCA. The plates were centrifuged at 4000 x g for 7 min (4 C) and the supernatants were collected in order to colorimetrically measure the amount of liberated phosphate using the Fiske-Subbarow reducing agent according to the manufacturer's guidelines (Sigma).
Absorbance was read using a plate reader (Biotek) with a 660 nm filter.
The normalized ATPase activity for each compound was calculated as follows:
Normalized_ATPase_Activity = (Mean_Test_Compound / Mean_Control)
Mean_Test_compound is defined as the combined average values of wells containing test compound.
Mean_Control is defined as the combined average values of wells containing DMSO.
The effect of each compound on the Normalized ATPase activity was compared to the control experiments. Any compound that could increase the ATPase activity > 10 % percent warranted further investigation in more a more complex physiological system, such as in skinned muscle fibers.
PubChem Activity Outcome and Score:
Compounds that were significantly different (Student's t-test) from the control and increased the ATPase activity > 10 % were considered active.
Active compounds were given a score of 100, and inactive compounds were given a score of 0.
The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
List of Reagents:
Cardiac myofibrils (CMF) prepared from porcine left ventricles.
This assay was run by the assay provider. Compounds were provided by Kansas University Specialized Chemistry Center. 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 absorbance.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay design: enzyme reporter: substrate coupled enzyme: atp coupled enzyme
BAO: assay format: biochemical format: protein format: protein complex format
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay footprint: microplate: 96 well plate
BAO: bioassay specification: assay measurement throughput quality: single concentration multiple replicates
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: bioassay specification: assay stage: secondary: mmoa characterization
BAO: bioassay specification: bioassay type: functional: enzyme activity
BAO: detection technology: spectrophotometry: absorbance
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: meta target: biological process target: regulation of molecular function
BAO: meta target: molecular target: protein target: enzyme: transferase: generic transferase
BAO: version: 1.4b1090
Assay Format: Biochemical
** Test Concentration.
Data Table (Concise)