Structural and functional analysis of GCN2 for drug discovery

整合应激反应(ISR)是药物开发中一个日益重要的研究课题，因为该途径对细胞的正常功能至关重要。ISR通过一组四种蛋白来感知环境压力，这些蛋白通过真核生物起始因子eIF2a的磷酸化来终止全球翻译。对氨基酸饥饿应激的反应是由一般控制的不可降压2(GCN2)激酶促进的--有许多针对GCN2的癌症治疗的临床试验。通常，癌细胞中总GCN2和活性/磷酸化的GCN2水平升高。缺乏关于GCN2如何感知压力的信息，特别是关于结构性数据。本项目旨在阐明GCN2的非活性构象和活性构象。每个态/络合物的结构和构象变化将通过采取综合的结构方法来观察。具体地说，每种构象的新结构将通过低温电子显微镜(Cryo-Electron Microscope，CryoEM)获得，因为目前只有通过X射线结晶学获得的高度截断的GCN2结构可用。除了三维结构外，GCN2的动力学数据还将通过氢-氚交换质谱学(HDX-MS)获得。其中，氚的摄取水平可以指示蛋白质的运动，并可以随着时间的推移而产生多肽水平的结构变化。低温电子显微镜和HDX-MS的结合提供了结构验证以及蛋白质运动的可视化。随着对蛋白质结构的了解，我们可以通过使用纳米抗体来评估GCN2上的可用药部位，从而确定配基设计和药物开发的部位。

The Integrated Stress Response (ISR) is a growing research topic for drug discovery, as the pathway is vital for proper cellular function. The ISR senses environmental stresses by a set of four kinases which terminate global translation by phosphorylation of the eukaryotic initiation factor eIF2a. The response to amino acid starvation stress is facilitated by the general control non-derepressible 2 (GCN2) kinase - the are a number of clinical trials for cancer therapeutics targeting GCN2. Typically, levels of total and active/phosphorylated GCN2 are elevated in cancer cells. There is a lack of information regarding how GCN2 senses stress, especially with regards to structural data. This project aims to elucidate the inactive and active conformations of GCN2. The structures of each state/complex and conformation shifts will be observed by taking an integrative structural approach. Specifically, novel structures of each conformation will be obtained by Cryo- Electron Microscopy (cryoEM), as currently only highly truncated GCN2 structures obtained by X-ray Crystallography are available. In addition to 3D structures, dynamics data of GCN2 will be obtained using Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS). Where deuterium uptake levels are indicative of protein motion and can give peptide-level structural changes over time. The combination of cryoEM and HDX-MS provides structural validation, as well as visualization of protein motion. With understanding of protein structure, we can evaluate druggable sites on GCN2 by the use of nanobodies, and as such identify the sites for ligand design and drug development.