Discovery of novel cardiac troponin activators using fluorescence polarization-based high throughput screening assays

Figures

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  Figure 1. High throughput screens for cardiac troponin modulators. (a) Schematic illustration of the calcium-dependent thin filament activation pathway. In the thin filament OFF state (left), myosin binding sites on actin (light gray) are blocked by tropomyosin (Tm; dark gray). Ca2+ binding to troponin C (cTnC; red) allows switch peptide (SP; blue) interaction with the regulatory N-lobe of cTnC (NcTnC; pink). Removal of the C-terminal domain of cTnI (CTD) allows Tm to move azimuthally around the thin filament, exposing myosin binding sites on actin. The C-lobe cTnC (CCTnC; red) is constitutively bound to divalent cations and the anchoring region of cTnI (purple). (b) Atomic structure of the human cardiac troponin core complex (PDB 1J1D). The N- and C-lobe of cTnC are shown in surface representation in red and pink, respectively. Cardiac troponin I (cTnI) and troponin T (cTnT) are shown in purple and orange, respectively. The switch region of cTnI is shown in blue. Molecular interactions between cTnC and cTnI are highlighted by yellow dashed ovals. (c) Cartoon representation of the developed fluorescence polarization (FP)-based HTS that target cTnC-cTnI interactions. Top: FP-based screen…

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  Figure 2. Validation of hit compounds from high throughput screens. (a) Dose-dependent effect of selected hit compounds on cTnC-cTnISP interactions monitored by fluorescence polarization. (b) Dose-dependent effect of hit compounds from the cTnC-cTnISP HTS on the ATPase activity of isolated bovine myofibrils at submaximal calcium activation (~ 30% of maximum activation). (c) Dose–response curves for selected hit compounds from the cTnC-cTnIAR HTS. (d) Dose-dependent effect of hit compounds from the cTnC-cTnIAR HTS on the ATPase activity of isolated bovine myofibrils at submaximal calcium activation (~ 70% of maximal activation). (e) Chemical structures of NS5806, Furamidine and Claramine. Means ± SEM, n = 3–7. Statistical significance of values vs control were assessed with a one-way ANOVA followed by Tukey’s multiple comparison test: *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 3. Biophysical characterization of NS5806-cTnC interaction. (a) NS5806 binding to Ca2+-bound NcTnC (left), Ca2+-free cChimera (middle) and Ca2+-bound cChimera monitored by isothermal titration calorimetry (ITC). (b) Top: Part of 2D-1H-15N HSQC spectrum of cTnC in the absence (blue) and in the presence (purple) of NS5806 at roughly 1:1 stoichiometry. Middle: Plot of chemical shift perturbations (∆δ) of cTnC plotted against the amino acid sequence after addition of NS5806. Thresholds for different multiples of the standard deviation (SD) are shown in red. Bottom: Chemical shift perturbations mapped onto cTnC structure. Ile36 and Val72 are labelled accordingly. (c) ITC binding isotherm for Ca2+ titrated into cChimera in the absence (left) and in the presence (right) of NS5806. Means ± SEM, n = 4–5. Statistical significance between control and drug treatment was assessed with an unpaired two-tailed student’s t-test.

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  Figure 4. Biophysical characterization of Furamidine-cTnC interaction. (a) ITC for titration of Furamidine into cTnC. Insert: Measured steady-state dissociation constants. (b) ITC titration of unmodified cTnIAR into cTnC in the absence (left) and in the presence of Furamidine (middle). Measured steady-state dissociation constants are shown on the right. (c) Top: Part of 2D-1H-15N HSQC spectrum of cTnC in the absence (blue) and in the presence (purple) of Furamidine. Bottom: Plot of chemical shift perturbations (∆δ) of cTnC after addition of excess Furamidine. (d) Top: Chemical shift perturbations mapped onto cTnC structure with the same colour coding as shown in (c). Bottom: Docking pose for Furamidine (green) binding to CcTnC (grey, surface representation) using flexible docking in AutoDock Vina. The position of cTnIAR (cyan) in the cardiac troponin complex (PDB 1J1D) was superimposed onto the structure. Means ± SEM, n=3. Statistical significance between control and drug treatment was assessed with an unpaired two-tailed student’s t-test: **p<0.01.

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  Figure 5. Biophysical characterization of Claramine-cTnC interaction. (a) ITC for titration of Claramine into cTnC. Insert: Measured steady-state dissociation constants. (b) Left: Part of 2D-1H-15N HSQC spectrum of cTnC in the absence (blue) and in the presence (purple) of Claramine. Right: Plot of chemical shift perturbations (∆δ) of cTnC after addition of excess Furamidine. Chemical shift perturbations mapped onto cTnC structure. (c) ITC titration of unmodified cTnIAR into cTnC in the presence of Claramine (middle). Please note that Claramine had no effect on the cTnC-cTnIAR interaction.

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  Figure 6. Functional effects of hit compounds in demembranated human myocardium. (a) Force-pCa relations of demembranated human myocardium in the absence and in the presence of 100 μmol/L NS5806 (left), Furamidine (middle) and Claramine (right). (b) Effects of hit compounds on calcium sensitivity (pCa50) and cooperativity (nH) of force development of demembranated human myocardium. Means ± SEM, n = 5–9. Statistical significance of differences between before and after drug treatment were analyzed by a two-way ANOVA followed by Sidak’s multiple comparison test: *p < 0.05, ****p < 0.0001, ns—not significant.