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Successful Identification of Cardiac Troponin Calcium Sensitizers Using a Combination of Virtual Screening and ROC Analysis of Known Troponin C Binders.

ABSTRACT: Calcium-dependent cardiac muscle contraction is regulated by the protein complex troponin. Calcium binds to the N-terminal domain of troponin C (cNTnC) which initiates the process of contraction. Heart failure is a consequence of a disruption of this process. With the prevalence of this condition, a strong need exists to find novel compounds to increase the calcium sensitivity of cNTnC. Desirable are small chemical molecules that bind to the interface between cTnC and the cTnI switch peptide and exhibit calcium sensitizing properties by possibly stabilizing cTnC in an open conformation. To identify novel drug candidates, we employed a structure-based drug discovery protocol that incorporated the use of a relaxed complex scheme (RCS). In preparation for the virtual screening, cNTnC conformations were identified based on their ability to correctly predict known cNTnC binders using a receiver operating characteristics analysis. Following a virtual screen of the National Cancer Institute's Developmental Therapeutic Program database, a small number of molecules were experimentally tested using stopped-flow kinetics and steady-state fluorescence titrations. We identified two novel compounds, 3-(4-methoxyphenyl)-6,7-chromanediol (NSC600285) and 3-(4-methylphenyl)-7,8-chromanediol (NSC611817), that show increased calcium sensitivity of cTnC in the presence of the regulatory domain of cTnI. The effects of NSC600285 and NSC611817 on the calcium dissociation rate was stronger than that of the known calcium sensitizer bepridil. Thus, we identified a 3-phenylchromane group as a possible key pharmacophore in the sensitization of cardiac muscle contraction. Building on this finding is of interest to researchers working on development of drugs for calcium sensitization.

SUBMITTER: Aprahamian ML

PROVIDER: S-EPMC5743627 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Publications

Successful Identification of Cardiac Troponin Calcium Sensitizers Using a Combination of Virtual Screening and ROC Analysis of Known Troponin C Binders.

Aprahamian Melanie L ML Tikunova Svetlana B SB Price Morgan V MV Cuesta Andres F AF Davis Jonathan P JP Lindert Steffen S

Journal of chemical information and modeling 20171116 12

Calcium-dependent cardiac muscle contraction is regulated by the protein complex troponin. Calcium binds to the N-terminal domain of troponin C (cNTnC) which initiates the process of contraction. Heart failure is a consequence of a disruption of this process. With the prevalence of this condition, a strong need exists to find novel compounds to increase the calcium sensitivity of cNTnC. Desirable are small chemical molecules that bind to the interface between cTnC and the cTnI switch peptide and ...[more]

PMID: 29144742

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Project description:ObjectiveCardiac troponin I (cTnI) is an essential physiological and pathological regulator of cardiac relaxation. Significant to this regulation, the post-translational modification of cTnI through phosphorylation functions as a key mechanism to accelerate myofibril relaxation. Similar to phosphorylation, post-translational modification by acetylation alters amino acid charge and protein function. Recent studies have demonstrated that the acetylation of cardiac myofibril proteins accelerates relaxation and that cTnI is acetylated in the heart. These findings highlight the potential significance of myofilament acetylation; however, it is not known if site-specific acetylation of cTnI can lead to changes in myofilament, myofibril, and/or cellular mechanics. The objective of this study was to determine the effects of mimicking acetylation at a single site of cTnI (lysine-132; K132) on myofilament, myofibril, and cellular mechanics and elucidate its influence on molecular function.MethodsTo determine if pseudo-acetylation of cTnI at 132 modulates thin filament regulation of the acto-myosin interaction, we reconstituted thin filaments containing WT or K132Q (to mimic acetylation) cTnI and assessed in vitro motility. To test if mimicking acetylation at K132 alters cellular relaxation, adult rat ventricular cardiomyocytes were infected with adenoviral constructs expressing either cTnI K132Q or K132 replaced with arginine (K132R; to prevent acetylation) and cell shortening and isolated myofibril mechanics were measured. Finally, to confirm that changes in cell shortening and myofibril mechanics were directly due to pseudo-acetylation of cTnI at K132, we exchanged troponin containing WT or K132Q cTnI into isolated myofibrils and measured myofibril mechanical properties.ResultsReconstituted thin filaments containing K132Q cTnI exhibited decreased calcium sensitivity compared to thin filaments reconstituted with WT cTnI. Cardiomyocytes expressing K132Q cTnI had faster relengthening and myofibrils isolated from these cells had faster relaxation along with decreased calcium sensitivity compared to cardiomyocytes expressing WT or K132R cTnI. Myofibrils exchanged with K132Q cTnI ex vivo demonstrated faster relaxation and decreased calcium sensitivity.ConclusionsOur results indicate for the first time that mimicking acetylation of a specific cTnI lysine accelerates myofilament, myofibril, and myocyte relaxation. This work underscores the importance of understanding how acetylation of specific sarcomeric proteins affects cardiac homeostasis and disease and suggests that modulation of myofilament lysine acetylation may represent a novel therapeutic target to alter cardiac relaxation.

Dataset Information

Successful Identification of Cardiac Troponin Calcium Sensitizers Using a Combination of Virtual Screening and ROC Analysis of Known Troponin C Binders.

Publications

Successful Identification of Cardiac Troponin Calcium Sensitizers Using a Combination of Virtual Screening and ROC Analysis of Known Troponin C Binders.

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