Structural and functional analysis of GCN2 for drug discovery

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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.