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.