Small molecule targeting an epigenetic regulator for the treatment of osteoarthritis

该项目的目标是通过针对USP 16， 一种参与调节衰老和干细胞自我更新的染色质修饰剂。治疗将包括 在中度OA患者中关节内注射抑制USP 16的小分子。USP 16是一个 去泛素化酶（DUB）酶，从组蛋白H2 A的赖氨酸119上去除泛素，赖氨酸119是一个关键的标记， 维持多个躯体组织。有趣的是，USP 16的三倍与唐氏综合症有关。 (DS)是一种以21号染色体三倍化（HSA 21）为特征的先天性疾病。DS患者显示 加速老化的迹象，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小鼠模型或人体组织中USP 16的水平可改善 体干细胞的功能和减少衰老，从而缓解与 DS.这一证据使USP 16成为改善某些衰老相关病理的有吸引力的靶点。 值得注意的是，USP 16表达在OA软骨细胞和滑膜组织中高度上调。而且 USP 16基因座含有与家族性OA强相关的SNP。我们发现基因 患者来源的关节软骨细胞中USP 16的下调促进细胞和线粒体健康 并减少衰老相关的标记物，如SA-β-gal和p16 Ink 4a。此外，OA的微阵列分析 用靶向USP 16的siRNA处理的软骨细胞显示增殖和胶原蛋白标记物的增加， 沉积，以及细胞凋亡和catalysts标志物的减少。此外，USP 16在人类中的敲低 骨髓间充质干细胞促进向成熟软骨细胞分化， 基体沉积使用生化测定法检测重组人USP 16的酶活性， 鉴定了小分子抑制剂。我们验证了19个具有低IC 50的命中，并选择了具有低IC 50的小分子支架。 IC 50在0.06 μM和9.4 μM之间。在项目的第一阶段，我们将优化它们以提高效力， 特异性和溶解性。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， 随后是两种最佳先导化合物在OA模型中的临床前功效和早期毒性测试。我们将 评估它们的安全性、药代动力学（PK）、结合靶点和调节染色质作为免疫调节剂的能力。 药效学（PD）测量，以及预防或逆转外科大鼠OA中软骨损失的功效 模型，目的是建立目标时间、剂量和疗效之间的关系。我们还将研究 rs6516886 SNP的作用，并了解该基因组位点的特定变体是否与差异表达相关。 USP 16的表达和对治疗的不同反应，可能导致目标患者分层 人口拟定研究将为后续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.