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

摘要 Vincere Biosciences拥有专利的USP30抑制剂，小分子正在体内优化，以达到 开发出心力衰竭(HF)和心脏老化的候选药物。心力衰竭是一种与年龄相关的机制障碍 根源于心肌细胞线粒体质量控制机制的下降，导致心肌细胞内线粒体质量控制机制的下降 炎症和衰老，心肌细胞周转率降低。尽管有很强的理由支持 参与线粒体功能障碍和有丝分裂，目前还没有在临床试验中努力增强 线粒体吞噬作为心脏老化的一种治疗方法。通过敲除USP30基因增强有丝分裂吞噬作用 在心脏病老化模型中，Parkin的增加减少了心肌细胞的衰老 心肌细胞以及成年大鼠和小鼠的在体心肌细胞。鉴于这一令人信服的证据， 丝裂原吞噬增加可能有利于心脏老化，我们被迫测试我们的USP30抑制剂 相关模型中的化合物。 我们的初步数据表明，我们已经在体外开发出了低纳摩尔的专利化合物。 用两个正交法测定对USP30抑制的效力。这些化合物是细胞渗透性的。 并在抗霉素/寡霉素(A/O)存在下增强内源性细胞的有丝分裂吞噬作用 USP30、parkin和底物的表达。重要的是，化合物不会损害或去极化健康 TMRE测量线粒体。候选类铅化合物在测试时具有很高的选择性 一个由40多种去泛素酶组成的小组，使用两种正交试验。我们已经对化合物进行了分析 多种ADME性质和成功优化的性质，包括溶解性、渗透性、微粒体 稳定性好，与血浆蛋白结合。我们的先导化合物在大鼠和小鼠身上有很好的PK图谱 以远高于化合物低NM IC50 2倍的持续水平渗透到心脏。以下目标将考验 假设我们的选择性USP30抑制剂可以延缓心脏细胞和动物模型的衰老 衰老。 目的1：确定USP30抑制剂是否能延缓原代心肌细胞衰老 我们将在D-Gal模型中用USP30抑制剂/载体对照处理新生小鼠心肌细胞以确定 小分子抑制剂能否延缓培养中的细胞衰老。 目的：观察USP30抑制对D-半乳糖诱导的在体心肌细胞衰老的影响。 我们将评估抑制USP30是否可以减少D-半乳糖诱导的缺陷，包括线粒体 功能障碍、细胞衰老和炎症标记物。我们将每天给小鼠注射D-半乳糖， 或不联合应用USP30抑制剂8周诱导衰老。我们将评估这些措施的效果 线粒体功能、衰老和炎症的多个标志物上的化合物。

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.