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

摘要 Biosciences拥有专有的USP 30抑制剂小分子，正在体内优化， 心力衰竭（HF）和心脏老化的发展候选人。HF是一种与年龄相关的疾病， 根源于心肌细胞中线粒体质量控制机制的下降， 炎症和衰老，心肌细胞更新减少。尽管有很强的理由， 线粒体功能障碍和线粒体自噬的参与，在临床试验中没有努力提高 线粒体自噬作为心脏衰老的治疗方法。通过敲低USP 30或 在原发性和非原发性心脏病衰老模型中，帕金增加减少心肌细胞衰老 在成年大鼠和小鼠体内心肌细胞中。鉴于这一令人信服的证据， 增加线粒体自噬可能对心脏衰老有益，我们不得不测试我们的USP 30抑制剂 相关模型中的化合物。 我们的初步数据表明，我们已经开发出具有低纳摩尔浓度的专利化合物， 使用两种正交方法测定的USP 30抑制效价。这些化合物能渗透细胞 并在抗霉素/寡霉素（A/O）存在下增强人细胞中具有内源性 USP 30、parkin和底物的表达。重要的是，化合物不会损害或破坏健康 通过TMRE测量的线粒体。候选的类铅化合物在测试时具有高度的选择性， 使用两个正交测定的40多种去泛素化酶的组。我们分析了化合物 各种ADME特性和成功优化的特性，包括溶解性、渗透性、微粒体 稳定性和血浆蛋白结合。我们的先导化合物在大鼠和小鼠中具有优异的PK特征， 以远高于化合物的低nM IC 50的2倍的持续水平穿透心脏。以下目标测试 假设我们的选择性USP 30抑制剂可以减少心脏病细胞和动物模型中的衰老， 衰老 目的1：确定USP 30抑制剂是否可以减少原代心肌细胞的细胞衰老 我们将在D-Gal模型中用USP 30抑制剂/溶剂对照处理新生小鼠心肌细胞，以确定 小分子抑制剂是否可以减少培养物中的细胞衰老。 目的2：研究USP 30抑制剂对D-半乳糖诱导的心肌细胞衰老的影响。 我们将评估USP 30的抑制是否可以减少D-半乳糖诱导的缺陷，包括线粒体 功能障碍、细胞衰老和炎症标志物。我们将每天注射D-半乳糖， 或不共同施用USP 30抑制剂8周以诱导衰老。我们将评估 化合物对线粒体功能、衰老和炎症的多个标志物的影响。

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.