DN-F01 is a potent cardiac sarcomere inhibitor, DNF01 targeting the cardiac muscle thin filament protein troponin complex. Functional studies show that DN-F01 has a strong inhibitory calcium-dependent effect on cardiac myofibrillar ATPase activity with an IC50 value of 11 ± 4 nmol/L. DN-F01 is shown to bind to a cardiac troponin C?troponin I chimera (cChimera) with a KD of ~50 nM using fluorescence spectroscopy, indicating that troponin is the likely target for DN-F01. NMR titrations of DNF01 to C35S and A-Cys cChimera show covalent and noncovalent binding of DN-F01 bound to the calcium-saturated cChimera.