Evaluation of USP30 small molecule inhibitors in models relevant to Cardiac Aging

ABSTRACT Vincere Biosciences has proprietary USP30 inhibitor small molecules being optimized in vivo towards development candidates for heart failure (HF) and cardiac aging. HF is an age-related disorder mechanistically rooted in the decline of mitochondrial quality control mechanisms in myocardial cells which leads to increased inflammation and senescence with decreased myocardial cell turnover. Despite strong rationale for the involvement of mitochondrial dysfunction and mitophagy, there have been no efforts in clinical trials to enhance mitophagy as a therapeutic approach for cardiac aging. Enhancement of mitophagy via knockdown of USP30 or increase in parkin reduces myocardial cell senescence in an aging model of heart disease both in primary cardiomyocytes and in adult rat and mouse myocardial cells in vivo. In light of this compelling evidence that increased mitophagy may provide benefit for cardiac aging, we are compelled to test our USP30 inhibitor compounds in relevant models. Our preliminary data demonstrate that we have developed proprietary compounds with low nanomolar in vitro potency for USP30 inhibition as measured using two orthogonal methods. The compounds are cell-penetrant and enhance mitophagy in the presence of antimycin/oligomycin (A/O) in human cells with endogenous expression of USP30, parkin, and substrates. Importantly, compounds do not damage or depolarize healthy mitochondria as measured by TMRE. Candidate lead-like compounds are highly selective when tested against a panel of over 40 deubiquitinating enzymes using two orthogonal assays. We have profiled compounds for various ADME properties and successfully optimized properties including solubility, permeability, microsomal stability, and plasma protein binding. Our lead compound has an excellent PK profile in rat and mouse and penetrates the heart at sustained levels well above 2x the compounds’ low nM IC50. The following Aims test the hypothesis that our selective USP30 inhibitors can reduce senescence in cell and animal models of cardiac aging. Aim 1: Determine whether USP30 inhibitors can reduce cell senescence in primary myocardial cells We will treat neonatal mouse myocardial cells with USP30 inhibitor/vehicle control in a D-Gal model to determine whether the small molecule inhibitors can reduce cellular senescence in culture. Aim 2: Assess the effects of USP30 inhibition on D-gal induced myocardial cell senescence in vivo . We will evaluate whether inhibition of USP30 can reduce the deficits induced by D-gal, including mitochondrial dysfunction, cellular senescence, and inflammatory markers. We will treat mice with daily D-gal injections, with or without co-administration of USP30 inhibitor for 8 weeks to induce senescence. We will evaluate the effect of compound on multiple markers of mitochondrial function, senescence, and inflammation.