Small molecule targeting an epigenetic regulator for the treatment of osteoarthritis

该项目的目标是通过靶向USP16来开发一种治疗骨关节炎(OA)的疾病修正疗法。 一种染色质修饰物，参与调节衰老和干细胞自我更新。治疗将包括 在中度骨性关节炎患者的关节内注射抑制Usp16的小分子。USP16是一种 脱泛素酶(DUB)，从赖氨酸119上的组蛋白H2A中去除泛素，这是 多个体细胞组织的维持。有趣的是，Usp16的三重与唐氏综合症有关 (DS)，一种以21号染色体三倍体(HSA21)为特征的先天性疾病。DS患者表现出 衰老加速的迹象，包括早发性阿尔茨海默氏症、免疫功能障碍和骨质疏松症。我们有 先前表明，降低DS小鼠模型或人类组织中Usp16的水平会导致改善 躯体干细胞的功能和减少衰老，从而缓解与 DS.这一证据使USP16成为改善一些与衰老相关的病理的有吸引力的靶点。 值得注意的是，Usp16在骨性关节炎软骨细胞和滑膜组织中的表达高度上调。此外， Usp16基因座包含一个与家族性骨性关节炎密切相关的SNP。我们发现基因 患者来源的关节软骨细胞中Usp16的下调促进细胞和线粒体健康 并减少衰老相关标志物，如SA-ꞵ-GAL和p16INK4a。此外，骨性关节炎的基因芯片分析 靶向Usp16的siRNA处理软骨细胞后，其增殖和胶原标记物增加 沉积，以及细胞凋亡和分解代谢标志物的减少。此外，人类Usp16基因敲除 骨髓间充质干细胞促进向成熟软骨细胞分化 基质沉积。用生化方法检测重组人Usp16的酶活性，我们 确定了小分子抑制剂。我们验证了低IC50的19次命中，并选择了小分子支架 IC50介于0.06μM和9.4μM之间。在项目的第一阶段，我们将对它们进行优化以提高效率。 专一性和溶解性。在第二阶段，我们将把两个最好的化合物系列转移到铅优化， 其次是在OA模型中的临床前疗效和两种最好的先导化合物的早期毒性测试。我们会 评估它们的安全性、药代动力学(PK)、结合靶点和调节染色质的能力 药效学(PD)测量和预防或逆转手术大鼠骨性关节炎软骨丢失的效果 模型，目标是建立靶向时间、剂量和疗效之间的关系。我们还将研究 Rs6516886 SNP的作用，并了解该基因组座位的特定变异是否与差异有关 Usp16的表达和对治疗的不同反应，可能导致目标患者的分层 人口。拟议的研究将为后续的GLP研究提供信息，以支持IND和临床试验。

The goal of the project is to develop a disease-modifying treatment for Osteoarthritis (OA) by targeting USP16, a chromatin modifier involved in regulation of senescence and stem cell self-renewal. The treatment will consist of an intra-articular injection of a small molecule inhibiting USP16 in patients with moderate OA. USP16 is a deubiquitinase (DUB) enzyme that removes ubiquitin from histone H2A on lysine 119, a critical mark for the maintenance of multiple somatic tissues. Interestingly, triplication of USP16 is associated with Down Syndrome (DS), a congenital disorder characterized by triplication of chromosome 21 (HSA21). Patients with DS show signs of accelerated aging, including early-onset Alzheimer’s, immune dysfunction and osteoporosis. We have previously shown that reducing the levels of USP16 in DS mouse models or human tissues results in improved function of somatic stem cells and reduction in senescence, therefore alleviating the conditions associated with DS. This evidence makes USP16 an attractive target to ameliorate some of the aging-related pathologies. Notably, USP16 expression is highly upregulated in OA chondrocytes and synovial tissues. Moreover, the genetic locus of USP16 contains a SNP strongly associated with familial OA. We found that genetic downregulation of USP16 in patient-derived articular chondrocytes promotes cellular and mitochondrial health and reduces senescence associated markers, like SA-ꞵ-gal and p16Ink4a. Moreover, microarray analyses of OA chondrocytes treated with siRNA targeting USP16 showed an increase in markers of proliferation and collagen deposition, and a reduction of apoptosis and catabolism markers. Furthermore, USP16 knockdown in human bone marrow-derived mesenchymal stem cells promoted differentiation into mature chondrocytes and increased matrix deposition. Using a biochemical assay testing the enzymatic activity of recombinant human USP16, we identified small molecule inhibitors. We validated 19 hits with low IC50 and chose small molecule scaffolds with IC50 between 0.06 μM and 9.4 μM. During Phase I of the project we will optimize them to increase potency, specificity and solubility. During Phase II we will move the two best compound series into Lead Optimization, followed by preclinical efficacy in OA models and early toxicity testing of the two best lead compounds. We will assess their safety, pharmacokinetics (PK), ability to engage target and modulate chromatin as a pharmacodynamic (PD) measure, and efficacy in preventing or reversing loss of cartilage in a surgical rat OA model, with the goal of establishing a relationship between time on target, dose and efficacy. We will also study the role of the rs6516886 SNP and understand if specific variants of this genomic locus are linked to differential expression of USP16 and different responses to treatment, potentially leading to stratification of the target patient population. The proposed studies will inform subsequent GLP studies to support an IND and clinical trial.