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 targeting the interaction between NcTnC and the switch region of cTnI. Bottom: FP-based screen targeting the interaction between CcTnC and the anchoring region of cTnI. (d) HTS of three commercially available compound libraries targeting the interaction between cTnC and switch peptide (top) or the anchoring region of cTnI (bottom). Hits were defined by compounds that change the FP more than five times the standard deviation (5xSD) away from the average value (indicated by red lines).
