Structural and functional analysis of GCN2 for drug discovery

综合应激反应（ISR）是药物发现的一个日益增长的研究课题，因为该途径对适当的细胞功能至关重要。ISR通过一组四种激酶感知环境应激，所述激酶通过真核起始因子eIF 2a的磷酸化终止全局翻译。对氨基酸饥饿应激的反应由一般对照非去阻遏2（GCN 2）激酶促进-这是针对GCN 2的癌症治疗剂的许多临床试验。通常，总的和活性/磷酸化的GCN 2的水平在癌细胞中升高。缺乏关于GCN 2如何感知压力的信息，特别是关于结构数据的信息。本项目旨在阐明GCN 2的非活性和活性构象。将通过采取综合结构方法观察每个状态/复合物的结构和构象变化。具体而言，每种构象的新结构将通过低温电子显微镜（cryoEM）获得，因为目前只有通过X射线晶体学获得的高度截短的GCN 2结构可用。除了3D结构，GCN 2的动力学数据将使用氢氘交换质谱（HDX-MS）获得。其中氘摄取水平指示蛋白质运动并且可以随时间给出肽水平结构变化。cryoEM和HDX-MS的组合提供了结构验证，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GCN 2上的可药用位点，并因此确定配体设计和药物开发的位点。

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.