ABSTRACT
Cardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac contractility. These interactions are regulated by cMyBP-C phosphorylation. Heart failure patients often have decreased cMyBP-C phosphorylation and phosphorylation in model systems appears to be cardioprotective for heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation and/or perturb its interactions with actin/myosin.
We have used a novel fluorescence lifetime-based assay to identify small-molecule inhibitors of actin-cMyBP-C binding. Actin was labeled with a fluorescent dye (Alexa Fluor 568, AF568) near its cMyBP-C binding sites. When combined with cMyBP-C N-terminal fragment, C0-C2, the fluorescence lifetime of AF568-actin decreases. Using this reduction in lifetime as a readout of actin binding, a high-throughput screen of a 1280-compound library identified 3 reproducible Hit compounds that reduced C0-C2 binding to actin in the micromolar range. Binding of phosphorylated C0-C2 was also blocked by these compounds. That they specifically block binding was confirmed by a novel actin-C0-C2 time-resolved FRET (TR-FRET) binding assay. Isothermal titration calorimetry (ITC) and transient phosphorescence anisotropy (TPA) confirmed that the Hit compounds bind to cMyBP-C but not to actin. TPA results were also consistent with these compounds inhibiting C0-C2 binding to actin. We conclude that the actin-cMyBP-C lifetime assay permits detection of pharmacologically active compounds that affect cMyBP-C’s actin binding function. TPA, TR-FRET, and ITC can then be used to understand the mechanism by which the compounds alter cMyBP-C interactions with actin.
Competing Interest Statement
Conflict of interest: D.D.T. holds equity in, and serves as President of, Photonic Pharma LLC. This relationship has been reviewed and managed by the University of Minnesota. Photonic Pharma had no role in this study, except to provide some instrumentation, as stated in Experimental Procedures. B.A.C. filed a PCT patent application based on this work (patent pending, serial no. PCT/US21/14142). The other authors declare no competing financial interests.
Abbreviations
- DCM
- dilated cardiomyopathy
- HCM
- hypertrophic cardiomyopathy
- cMyBP-C
- cardiac myosin-binding protein C
- P/A
- proline/alanine-rich linker between domains C0 and C1
- M
- M-domain, phosphorylatable linker between C1 and C2
- C0-C2
- N-terminal fragment of cMyBP-C comprised of C0-P/A-C1-M-C2 domains and linkers
- HTS
- high-throughput screen
- FLTPR
- fluorescence lifetime plate reader
- DO
- donor only
- DA
- donor plus acceptor
- LOPAC
- library of pharmacologically active compounds
- AF568
- Alexa Fluor 568
- AF546
- Alexa Fluor 546
- F-actin
- filamentous actin
- G-actin
- globular actin
- TR-F
- time-resolved fluorescence
- TR-FRET
- time-resolved fluorescence energy transfer
- ITC
- isothermal titration calorimetry
- TPA
- transient phosphorescence anisotropy
- Kd
- dissociation constant
- Bmax
- maximum molar binding ratio
- ATA
- aurintricarboxylic acid
- M-ABB
- MOPS-actin binding buffer
- DMSO
- dimethylsulphoxide
- DMF
- dimethylformamide
- BSA
- bovine serum albumin
- DTT
- dithiothreitol
- TCEP
- tris(2-carboxyethyl)phosphine
- ErIA
- erythrosine iodoacetamide phosphorescent dye
- PKA
- protein kinase A
- FMAL
- fluorescein-5-maleimide
- IAEDANS
- 5-((((2-iodoacetyl)amino)ethyl)amino)naphtalene-1-sulfonic acid
- TMR
- tetramethylrhodamine
- SD
- standard deviations
- SE
- standard errors
- DWR
- direct waveform recording