High content screening to identify therapeutics for multiple sclerosis

描述(申请人提供)：多发性硬化症(MS)是最常见的中枢神经系统(CNS)炎症性脱髓鞘疾病，也是北美年轻人非创伤性神经残疾的主要原因。虽然MS的病因尚不清楚，但其发病特点是免疫细胞浸润，少突胶质细胞和髓鞘破坏。5-15年后，大多数患者进入轴突变性和逐渐的、不可逆转的神经衰退阶段。目前还没有治愈多发性硬化症的方法，目前的抗炎或免疫调节疗法在延缓疾病方面部分有效，但这些疗法不能保证功能恢复。少突胶质细胞的替代及其随后脱髓鞘轴突的重新髓鞘化可以阻止和逆转轴突丢失和神经功能衰退。增加髓鞘再生的一种方法是通过内源性修复的治疗性刺激。这种方法是可行的，因为在疾病的早期阶段，许多MS病变确实会产生新的少突胶质细胞和自发的髓鞘再分化，甚至在一些慢性病变中也是如此，尽管内源性的髓鞘再分化最终失败。这一建议的假设是，选择的小分子可以用作再髓鞘治疗药物，以刺激少突胶质细胞的分化和再髓鞘形成。最近的两项技术发展使这种小分子的鉴定和评估成为可能。首先，优化了一种从小鼠中获得高纯度的原代少突胶质前体细胞(OPC)的方法，该OPC可以用作筛选的起始材料。第二，一种高含量、基于细胞的表型筛选方法，以中等吞吐量的方式自动对分化的少突胶质细胞进行评分。这一创新平台将用于筛选小分子文库，以识别可以促进OPC分化的命中。然后，来自主屏幕的假定点击量将 评估其激活少突胶质细胞特异性标志物表达的能力。还将评估调节少突胶质细胞生成的效力、细胞毒性、细胞类型选择性以及与已知信号通路的相互作用。这一第一阶段项目的目标是确定能够促进OPC分化的最佳小分子候选药物，以用于后续第二阶段的研究和开发。未来的第二阶段项目将包括对先导化合物的详细毒理学研究，以及在小鼠脱髓鞘模型中的体内测试。该项目的长期目标是开发治疗药物，以促进髓鞘再生，扭转多发性硬化症患者的神经功能衰退。

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is the most common inflammatory demyelinating disease of the central nervous system (CNS) and is the major cause of non-traumatic neurological disability in young adults in North America. Although the etiology of MS is not clear, its onset is characterized by infiltration of immune cells and the destruction of oligodendrocytes and myelin. After 5-15 years, most patients enter a phase of axonal degeneration and gradual, irreversible neurological decline. There is no cure for MS. Current anti- inflammatory or immunomodulatory therapies are partially effective in delaying the disease, but these therapies do not guarantee functional recovery. Replacement of oligodendrocytes and their subsequent remyelination of demyelinated axons could halt and reverse axonal loss and neurological decline. One approach to increase remyelination is through therapeutic stimulation of endogenous repair. This approach is feasible since generation of new oligodendrocytes and spontaneous remyelination do occur in many MS lesions during early stages of the disease, and even in some chronic lesions, although endogenous remyelination eventually fails. The hypothesis of this proposal is that selected small molecules can be used as remyelination therapeutics to stimulate differentiation of oligodendrocytes and remyelination. Two recent technological developments enable the identification and evaluation of such small molecules. First, an optimized method to produce a highly pure population of primary oligodendrocyte progenitor cells (OPCs) from the mouse that can be used as starting material for screening. Second, a high-content, cell-based phenotypic screening method to automatically score differentiated oligodendrocytes in a medium-throughput manner. This innovative platform will be used to screen a small molecule library to identify hits that can promote OPC differentiation. Putative hits from the primary screen will then be evaluated for their ability to activate the expression of oligodendrocyte-specific markers. Potency, cytotoxicity, cell-type selectivity and interaction with known signaling pathways that regulate oligodendrocyte generation will also be evaluated. The goal of this Phase I project is to identify the best small molecule drug candidate that can promote differentiation of OPCs for subsequent Phase II studies and development. Future Phase II projects will include detailed toxicological studies of the lead compound and in vivo testing in the mouse cuprizone model of demyelination. The long-term goal of this project is to develop therapeutic agents that will promote remyelination to reverse neurological decline in MS patients.