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

Abstract

BackgroundCompromised 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 CLR01 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 CLR01 in cardiomyocytes undergoing proteotoxic stress have not been explored. Here we assess whether CLR01 can decrease cardiac protein aggregation catalyzed by cardiomyocyte‐specific expression of mutated αB‐crystallin (CryABR 120G).Methods and ResultsA proteotoxic model of desmin‐related cardiomyopathy caused by cardiomyocyte‐specific expression of CryABR 120G was used to test the efficacy of CLR01 therapy in the heart. Neonatal rat cardiomyocytes were infected with adenovirus expressing either wild‐type CryAB or CryABR 120G. Subsequently, the cells were treated with different doses of CLR01 or a closely related but inactive derivative, CLR03. CLR01 decreased aggregate accumulation and attenuated cytotoxicity caused by CryABR 120G expression in a dose‐dependent manner, whereas CLR03 had no effect. Ubiquitin‐proteasome system function was analyzed using a ubiquitin‐proteasome system reporter protein consisting of a short degron, CL1, fused to the COOH‐terminus of green fluorescent protein. CLR01 improved proteasomal function in CryABR 120G cardiomyocytes but did not alter autophagic flux. In vivo, CLR01 administration also resulted in reduced protein aggregates in CryABR 120G transgenic mice.Conclusions CLR01 can inhibit CryABR 120G aggregate formation and decrease cytotoxicity in cardiomyocytes undergoing proteotoxic stress, presumably through clearance of the misfolded protein via increased proteasomal function. CLR01 or related compounds may be therapeutically useful in treating the pathogenic sequelae resulting from proteotoxic heart disease.

The molecular tweezer CLR01 improves behavioral deficits and reduces tau pathology in P301S-tau transgenic mice

“…Experimental design and safety assessment of CLR01 treatment Previously, CLR01 was used to treat different animal models of proteinopathy using intracerebroventribular (32,36) or subcutaneous administration. In the latter route, the compound was administered either via osmotic minipumps (20,25,36,37), as was done here, or by daily injection, 2-7 days a week (38)(39)(40)(41). Here, we chose to use the less labor-intensive osmotic-minipump subcutaneous administration to answer the speci c question whether CLR01 treatment affected tau directly.…”

“…Finally, the delivery of the drug via osmotic pumps, which was chosen for the same reasons, limits translatability to humans. As discussed above, recent studies have demonstrated that continuous administration is unnecessary and subcutaneous administration of CLR01 daily (38,40), or even twice a week can result in a robust therapeutic effect (41).…”

The molecular tweezer CLR01 inhibits aberrant superoxide dismutase 1 (SOD1) self-assembly in vitro and in the G93A-SOD1 mouse model of ALS

“…CLR01 has been shown to have beneficial therapeutic effects in multiple animal models of various proteinopathies, including rodent models of AD (18,24), PD (25), transthyretin amyloidosis (26), and desmin-related cardiomyopathy (27). However, to date, MTs have not been explored in the context of ALS in general or for inhibition of the aggregation and toxicity of SOD1 in particular.…”

“…As the G93A-SOD1 mice tend to develop disease at 110 -120 days of age and die at 130 -140 days of age, using the same pumps for this study was impractical. Therefore, we decided to administer CLR01 by a s.c. daily injection, as was done in a recent study using a desmin-related cardiomyopathy mouse model (27). In using this method of administration, we took into account that the plasma half-life of CLR01, 2-3 h, and its first-pass blood-brain barrier penetration, 2-3% (35), may limit the exposure of the target, G93A-SOD1, to the compound.…”

Reductive Stress-Induced Mitochondrial Dysfunction and Cardiomyopathy

“…It is reported that during aging and during the progression of cardiomyopathy, both CryAB and its phosphorylation are elevated [118,119]. The molecular tweezer CLR01 protects against CryAB R120G -induced cytotoxicity, hampers CryAB R120G -induced protein aggregation, and alleviates proteotoxicity in cardiomyocytes [120]. Besides CLR01 treatment, other recent studies state that activation of transcriptional factor EB (TFEB) by intermittent fasting also can be utilized as a treatment of CryAB R120G -induced cardiomyopathy [121].…”