Assay provider mechanism-of-action assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF): biochemical assay to identify inhibitors of the Ca2+-sensitivity of cardiac muscle contraction/tension
Name: Assay provider mechanism-of-action assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF): biochemical assay to identify inhibitors of the Ca2+-sensitivity of cardiac muscle contraction/tension ..more
BioActive Compound: 1
Depositor Specified Assays
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: FIBER-TENSION_INH_FORCE_CLIPS_1X-FOLD-CHANGE MCSRUN DESENS
Name: Assay provider mechanism-of-action assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF): biochemical assay to identify inhibitors of the Ca2+-sensitivity of cardiac muscle contraction/tension
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.
fiber tension, 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, desensitization, desensitizer, fluorescence, fluorophore, IANBD, FLINT, inhibit, modulate, modulator, inhibitor, inhibition, 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 desensitizer (inhibitor)probe candidates act to decrease the Ca2+ sensitivity of permeabilized cardiac muscle fiber contraction. The permeabilized muscle fiber is tethered by a clip at one end and the tension that is generated by the contracting fiber is measured by a force transducer attached at the other end. In this assay, tension is acquired as the permeabilized fiber contracts in response to 6 different solutions that vary in free [Ca2+] and contain a fixed amount of compound. The results are depicted as sigmoid curves with tension (y-axis) represented as a function of the pCa (-log (free [Ca2+]) (x-axis). Fitting the curve will calculate the pCa50 (i.e. -log (free [Ca2+]) that yields 50% of the maximum response). As designed, compounds that are calcium desensitizers in this assay will interact with the RTF to decrease the Ca2+ sensitivity (i.e., pCa50) of muscle fiber tension and shift the sigmoid curve and pCa50 righttward (i.e. increase the EC50 free [Ca2+]). The effects on contraction arising from compounds are considered a high content screen of which numerous parameters not listed here may be affected. Compounds were tested at a nominal maximum concentration of 10 uM.
Porcine papillary muscle fibers (~2 mM in length and 0.150 mM in diameter) were mounted using stainless steel clips to a force transducer and immersed in a relaxation solution (pCa 8.0) containing 10-8 M free [Ca2+]. The fiber slack was removed until tension developed, and then the fiber was stretched 1.2 X its length. This results in estimated sarcomere lengths between 2.2 and 2.3 um. The following six pCa solutions were prepared: 8.0, 6.2, 6.0, 5.8, 5.6 and 4.5 which contained 10e-8, 10e-6.2, 10e-6.0, 10e-5.8, 10e-5.6 and 10e-4.5 M free [Ca2+], respectively. All solutions contained 1.0 mM free [Mg2+], 7 mM EGTA, 20 mM MOPS, 2.5 mM [Mg-ATP2-], 15 mM creatine phosphate, 15 units/ml phosphocreatine kinase, pH 7.0, and between 33.6 and 47.9 mM KPr to achieve a constant ionic strength of 150 mM in all solutions. Once mounted, the fiber was chemically permeabilized by submerging it into pCa 8.0 solution containing 1.0% (v/v) Triton-X 100 for 30 min. After permeabilization, the fiber was rinsed 3x (5 min) in pCa 8.0. The mounted fiber was submerged into the various pCa solutions in sequential order (i.e. from lowest to highest free Ca2+ concentration) containing 0.1% v/v DMSO until the generated force reached a plateau. Thereafter, the fiber was rinsed 3x in pCa 8.0 solution and submerged sequentially (as above) into the various pCa solutions containing 0.1% v/v DMSO and 10 uM of the test compound. The data were fit to a sigmoid Hill equation where, tension and free [Ca2+] were plotted along the y- and x-axis, respectively; and the slope and KD (i.e. pCa50) of skinned cardiac muscle contraction were calculated.
Tension/force was measured using a Guth Force Transducer.
Force = (Maximum _Force * ( free[Ca2+] ^ Hill_Slope ) ) / ( ( KD ^ Hill_Slope ) + ( free[Ca2+] ^ Hill_Slope ) )
pCa = -log(free[Ca2+])
pCa50 = -log(KD)
Delta(pCa50) = [pCa50 of fiber in the presence of compound] - [pCa50 of fiber in the presence of DMSO]
Fold_Change_KD = [KD of fiber in the presence of compound] / [KD of fiber in the presence of DMSO]
Maximum_Force = force generated in the presence of 10e-4.5 free Ca2+ (pCa 4.5).
Hill_Slope = slope of the sigmoid curve.
KD = the concentration of free Ca2+ in which 50% of the maximum response is observed
pCa50 = -log(KD)
For each test compound, force was plotted against the free [Ca2+]. A 3 parameter equation describing a sigmoidal dose-response curve was then fitted using the SigmPlot 11 software suite. The reported KD and pCa50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value.
PubChem Activity Outcome and Score:
Compounds that were capable of decreasing the KD greater than 10% were considered active. Compounds that decreased KD equal to or less than 10% were considered inactive.
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. There are no inactive compounds.
List of Reagents:
Left ventricle papillary muscle fibers isolated from pig.
This assay was performed by the assay provider. Compounds were provided by Kansas University Specialized Chemistry Center.
BAO: version: 1.4b1090
BAO: bioassay specification: assay stage: secondary: mmoa characterization
BAO: bioassay specification: assay biosafety level: bsl1
BAO: assay format: biochemical format: protein format: protein complex format
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: meta target: molecular target: protein target: enzyme: transferase: generic transferase
BAO: meta target: biological process target: regulation of molecular function
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: assay design: enzyme reporter: substrate coupled enzyme: atp coupled enzyme
BAO: detection technology: spectrophotometry: absorbance
BAO: bioassay specification: bioassay type: functional: enzyme activity
BAO: bioassay specification: assay footprint: microplate: 96 well plate
BAO: bioassay specification: assay measurement throughput quality: single concentration multiple replicates
** Test Concentration.
Data Table (Concise)