Small molecules to block measles spreading in the central nervous system

麻疹病毒（MV）是发展中国家儿童死亡的主要原因， 减毒活疫苗已有40多年历史。免疫力严重受损的人特别容易患上 MV. MV感染最严重的表现，包括脑炎，发生在受损的人， 细胞免疫MV在多达一半的常规病例中影响中枢神经系统（CNS）; 足够的细胞免疫力，感染被根除，但细胞免疫力受损的人， 一个劣势 在南非最近的MV爆发中，有几人死于MV CNS感染。我们分析了 从这些病人身上提取病毒，发现MV融合机制的特异性宿主内进化-- 受体结合蛋白（H）+融合蛋白（F）--已经发生。通常，MV F被合成， 维持在融合前状态，直到它到达细胞表面，并且这种融合前状态本质上是 在需要来自H的信号的过程中，不稳定且被驱动到融合后状态 H与受体的相互作用。然而，在“CNS适应”病毒的情况下，F 允许其促进融合，而较少依赖于H与两种已知MV细胞的相互作用， 受体;该F独立于H或受体被激活。 CNS分离物F代表了鉴定阻断MV传播的小分子的理想靶标 在CNS中通过使F蛋白不稳定，从而促进F过早折叠到其融合后 状态此类化合物将有效地减少可用于介导细胞凋亡的融合前F的量。 细胞融合，最终阻止MV在CNS中的传播。我们已经调整并验证了一个基于细胞的 基于生物发光的高通量筛选（HTS）测定。哥伦比亚大学医学中心 HTS设施文库将筛选有效阻断F介导的融合的小分子。 中枢神经系统分离株 目的1：进行小分子文库的初级HTS。在细胞系中进行正交筛选， 人类神经元将证实对抗活病毒的功效。 目的2：使用特定的功能性方法评估所选小分子的作用机制。 测定。离体功效研究将评估小分子在相关组织中的抗病毒活性。病毒 在小分子的选择压力下进行的进化研究将决定出现的潜力 抵抗病毒的能力。

Measles virus (MV) is a leading cause of child mortality in developing countries despite the availability of a live attenuated vaccine for over 40 years. Severely immune-compromised people are particularly at risk for MV. The most serious manifestations of MV infection, including encephalitis, occur in people with impaired cellular immunity. MV affects the central nervous system (CNS) in up to half of routine cases; with adequate cellular immunity the infection is eradicated, but individuals with impaired cellular immunity are at a disadvantage. In a recent MV outbreak in South Africa several people died of MV CNS infection. We analyzed the viruses from these patients and found that specific intra-host evolution of the MV fusion machinery -- receptor binding protein (H) + fusion protein (F) -- had occurred. Normally, the MV F is synthesized and maintained in a pre-fusion state until it reaches the cell surface, and this pre-fusion state is intrinsically unstable and thermodynamically driven to the post-fusion state in a process that requires a signal from H upon H's interaction with receptor. However in the case of the “CNS-adapted” viruses, a mutation in F allows it to promote fusion with less dependence on interaction of H with the two known MV cellular receptors; this F is activated independently of H or receptor. The CNS isolate F represents an ideal target for identifying small molecules that block the spread of MV in the CNS by destabilizing the F protein, and thereby promoting premature folding of F to its post-fusion state. Such compounds will effectively decrease the amount of pre-fusion F that is available to mediate cell- to-cell fusion, ultimately halting spread of MV in the CNS. We have adapted and validated a cell based bioluminescence based High Throughput Screen (HTS) assay. The Columbia University Medical Center HTS facility library will be screened for small molecules that efficiently block the fusion mediated by the F from the CNS isolate. Aim 1: Primary HTS of small molecule libraries will be performed. Orthogonal screenings in cell lines and human neurons will confirm efficacy against live virus. Aim 2: The mechanism of action of selected small molecules will be assessed using specific functional assays. Ex vivo efficacy studies will assess the antiviral activity of small molecules in relevant tissues. Viral evolution studies under the selective pressure of small molecules will determine the potential for emergence of viral resistance.