Inhibition of Mutant αB Crystallin‐Induced Protein Aggregation by a Molecular Tweezer

Background Compromised protein quality control causes the accumulation of misfolded proteins and intracellular aggregates, contributing to cardiac disease and heart failure. The development of therapeutics directed at proteotoxicity‐based pathology in heart disease is just beginning. The molecular tweezer CLR 01 is a broad‐spectrum inhibitor of abnormal self‐assembly of amyloidogenic proteins, including amyloid β‐protein, tau, and α‐synuclein. This small molecule interferes with aggregation by binding selectively to lysine side chains, changing the charge distribution of aggregation‐prone proteins and thereby disrupting aggregate formation. However, the effects of CLR 01 in cardiomyocytes undergoing proteotoxic stress have not been explored. Here we assess whether CLR 01 can decrease cardiac protein aggregation catalyzed by cardiomyocyte‐specific expression of mutated αB‐crystallin (Cry AB R 120G ). Methods and Results A proteotoxic model of desmin‐related cardiomyopathy caused by cardiomyocyte‐specific expression of Cry AB R 120G was used to test the efficacy of CLR 01 therapy in the heart. Neonatal rat cardiomyocytes were infected with adenovirus expressing either wild‐type Cry AB or Cry AB R 120G . Subsequently, the cells were treated with different doses of CLR 01 or a closely related but inactive derivative, CLR 03. CLR 01 decreased aggregate accumulation and attenuated cytotoxicity caused by Cry AB R 120G expression in a dose‐dependent manner, whereas CLR 03 had no effect. Ubiquitin‐proteasome system function was analyzed using a ubiquitin‐proteasome system reporter protein consisting of a short degron, CL 1, fused to the COOH ‐terminus of green fluorescent protein. CLR 01 improved proteasomal function in Cry AB R 120G cardiomyocytes but did not alter autophagic flux. In vivo, CLR 01 administration also resulted in reduced protein aggregates in Cry AB R 120G transgenic mice. Conclusions CLR 01 can inhibit Cry AB R 120G aggregate formation and decrease cytotoxicity in cardiomyocytes undergoing proteotoxic stress, presumably through clearance of the misfolded protein via increased proteasomal function. CLR 01 or related compounds may be therapeutically useful in treating the pathogenic sequelae resulting from proteotoxic heart disease.