Underlying both cardiomyopathies and heart failure is the alteration of the contractile profile of the myocardium leading to impairments in force generation (i.e., systolic function) or relaxation kinetics (i.e., diastolic function) of the sarcomere. Fundamental studies of the sarcomeric unit of striated muscle as well as significant genetic evidence from hereditary cardiomyopathies have implicated the thin filament complex as a key multi-protein complex tightly regulating contraction and relaxation kinetics in cardiac muscle. Utilizing affinity selection mass spectrometry high-throughput screening, we set out to identify small-molecules that bind to the cardiac thin filament and result in calcium desensitization in reconstituted biochemical and tissue-isolated myofibril systems. Through chemical optimization to improve potency and solubility, we further developed a series and were able to confirm target engagement with intact reconstituted cardiac thin filaments using isothermal calorimetry. Optimization of several absorption, distribution, metabolism, and excretion properties provided exemplar compounds that in matrixed tension assays using mouse skinned fibers showed diverse antagonist and desensitization profiles. Two of these compounds with mixed desensitization-antagonist profiles were further tested in an engineered heart tissue model of hypertrophic cardiomyopathy and were confirmed to demonstrate mixed pharmacology with impacts on max force contraction and relaxation kinetics from the contracted to relaxed state. Together these studies provide a starting point for future efforts to develop selective, thin filament-targeted small molecules that potentially provide a new mechanism for treatment of heart failure.

Sirtuins catalyze NAD+-dependent protein deacetylation and are critical regulators of transcription, apoptosis, metabolism, and aging. There are seven human sirtuins (SIRT1–7), and SIRT1 has been implicated as a key mediator of the pathways downstream of calorie restriction that have been shown to delay the onset and reduce the incidence of age-related diseases such as type 2 diabetes. Increasing SIRT1 activity, either by transgenic overexpression of the Sirt1 gene in mice or by pharmacological activation by small molecule activators resveratrol and SRT1720, has shown beneficial effects in rodent models of type 2 diabetes, indicating that SIRT1 may represent an attractive therapeutic target. Herein, we have assessed purported SIRT1 activators by employing biochemical assays utilizing native substrates, including a p53-derived peptide substrate lacking a fluorophore as well as the purified native full-length protein substrates p53 and acetyl-CoA synthetase1. SRT1720, its structurally related compounds SRT2183 and SRT1460, and resveratrol do not lead to apparent activation of SIRT1 with native peptide or full-length protein substrates, whereas they do activate SIRT1 with peptide substrate containing a covalently attached fluorophore. Employing NMR, surface plasmon resonance, and isothermal calorimetry techniques, we provide evidence that these compounds directly interact with fluorophore-containing peptide substrates. Furthermore, we demonstrate that SRT1720 neither lowers plasma glucose nor improves mitochondrial capacity in mice fed a high fat diet. SRT1720, SRT2183, SRT1460, and resveratrol exhibit multiple off-target activities against receptors, enzymes, transporters, and ion channels. Taken together, we conclude that SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1.