Comparative HTS to identify small molecule effectors of cell fate specification

描述（申请人提供）：复杂组织和器官的发育需要特化细胞从多能祖细胞中分化出来。最初的细胞命运规范发生在胚胎发生的早期。然而，许多成人组织维持一个介导损伤修复的祖细胞生态位。越来越清楚的是，rna结合蛋白在调节分化的决定中起着重要作用。这在早期胚胎中尤其正确，因为新生转录尚未开始，而在大脑中，高度分支的细胞形态需要在远离细胞核的距离上进行调节。在本提案中，我们描述了一种内聚的HTS检测开发策略，以识别调节细胞命运的rna结合蛋白的小分子抑制剂。所描述的测定包括鉴定阻断rna结合活性的分子的体外测定和鉴定影响分化状态的化合物的基于细胞的测定。一旦hts准备就绪和技术可行性确定，这些检测将提交给NIH MLPCN进行高通量筛选和探针鉴定。所鉴定的化合物将用于研究细胞和动物模型以及提取物中细胞命运规范的分子机制。一个独立的结果将是直接比较三种不同的RNA结合蛋白家族对小分子的敏感性。这种分析有助于制定更有效的目标选择和图书馆设计策略。最后，这里要筛选的靶点与人类疾病有关，包括脑癌、多发性硬化症和寄生虫感染。因此，通过HTS确定的探针最终有可能发展成治疗方法。

DESCRIPTION (provided by applicant): Development of complex tissues and organs requires specialized cells to differentiate from pluripotent progenitors. Initial cell fate specification ocurs early in embryogenesis. However, many adult tissues maintain a niche of progenitor cells that mediate repair upon damage. It is becoming increasingly clear that RNA-binding proteins play an important role in regulating the decision to differentiate. This is particularly true in early embryos, where nascent transcription has not yet begun, and in the brain, where the highly branched cellular morphology necessitates regulation at a distance from the nucleus. In this proposal, we describe a cohesive HTS assay development strategy to identify small molecule inhibitors of the RNA-binding proteins that regulate cell fate. The assays described include in vitro assays to identify molecules that block RNA-binding activity and cell-based assays to identify compounds that effect differentiation state. Once HTS-readiness and technical feasibility are established, these assays will be submitted to the NIH MLPCN for high throughput screening and probe identification. The compounds identified will be used to study the molecular mechanisms of cell fate specification in cell and animal models and in extracts. An independent outcome will be a direct comparison of the susceptibility of three different RNA- binding protein families to small molecules. This analysis may lead to development of more efficient target selection and library design strategy. Finally, the targets to be screened here are relevant to human diseases including brain cancer, multiple sclerosis, and parasitic infection. As such, it is possible that probes identified through HTS could eventually be developed into therapies.