Small molecule targeting an epigenetic regulator for the treatment of osteoarthritis

该项目的目标是通过靶向Usp16，一种参与调节衰老和干细胞自我更新的染色质修饰物，开发一种治疗骨关节炎(OA)的疾病修正疗法。治疗将包括关节内注射一种抑制Usp16的小分子，用于中度OA患者。USP16是一种脱泛素酶(DUB)，它从赖氨酸119上的组蛋白H2A中去除泛素，这是维持多个体细胞组织的关键标志。有趣的是，Usp16的三倍体与唐氏综合征(DS)有关，这是一种以21号染色体三倍体(HSA21)为特征的先天性疾病。DS患者表现出加速衰老的迹象，包括早发性阿尔茨海默氏症、免疫功能障碍和骨质疏松。我们之前已经证明，降低DS小鼠模型或人类组织中Usp16的水平可以改善体细胞的功能，减少衰老，从而缓解与DS相关的条件。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的遗传位点包含一个与家族性骨性关节炎密切相关的SNP。我们发现，患者来源的关节软骨细胞中USP16的基因下调促进了细胞和线粒体的健康，并减少了衰老相关标记物，如SA-ꞵ-GAL和p16INK4a。此外，针对Usp16的siRNA处理的OA软骨细胞的微阵列分析显示，增殖和胶原沉积的标志物增加，而凋亡和分解代谢的标志物减少。此外，人骨髓间充质干细胞中的Usp16基因敲除促进了向成熟软骨细胞的分化，并增加了基质沉积。通过生化方法检测重组人Usp16的酶活性，我们鉴定了小分子抑制物。我们验证了19个IC50值较低的HITS，并选择了IC50值在0.06μM和9.4μM之间的小分子支架。在项目的第一阶段，我们将对它们进行优化，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，随后是在骨性关节炎模型中的临床前疗效和两个最好的铅化合物的早期毒性测试。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的能力作为药效学(PD)指标，以及预防或逆转手术大鼠骨性关节炎模型中软骨丢失的有效性，目的是建立靶向时间、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Usp16的差异表达和对治疗的不同反应有关，从而潜在地导致目标患者群体的分层。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.