Characterisation of Small-Molecule GCN2 Modulators

Diversely derived ATP-competitive inhibitors have shown the ability to paradoxically activate the kinase GCN2. This project investigates clinically approved BRAF inhibitors dabrafenib and encorafenib, that are used as chemotherapeutic treatments for V600E mutant BRAF tumours. Collaborators determined these RAFi compounds elicited a GCN2dependent activation of the integrated stress response (ISR) in cell lines. Here, I expressed and purified GCN2 from Sf9 cells, and subsequently conducted ADP-Glo kinase assays that showed an inhibiting effect on ATPhydrolysis by the ATP-competitive inhibitors. However, using a phosphorylation specific antibody for threonine 899 in the activation loop of GCN2, I demonstrated that the compounds activated GCN2 in a concentration dependent bell-shaped curve. Furthermore, eIF2α phosphorylation by GCN2 was found induced by the compounds although this was determined to have an ATP concentration dependence. Hydrogendeuterium exchange mass spectrometry (HDX-MS) was then used to determine that the compounds bind in the kinase active site, detecting no alternative binding sites. Further afield allosteric effects of the compounds in the HisRS-like domain of GCN2 was also detected. Evidence is found for activation loop phosphorylations of GCN2 occurring in an inter-dimer trans phosphorylation event with an experiment involving the use of kinase dead K619R GCN2. Furthermore, the related eIF2α kinase HRI was found to have ability to induce activation loop phosphorylations of GCN2. This work contradicts a published theory of paradoxical activation of GCN2. A new theory is proposed, involving compound binding in an active-like conformation that allosterically conforms the activation loop so that it can be phosphorylated. This differs significantly from the proposed theory of paradoxical activation that has been published in recent years. A systematic literature review was carried out regarding the transcription factor ATF4 where evidence was found for a cell-cycle inhibiting effect of ATF4 and factors relating to the induction of apoptosis. Furthermore, a role of ATF4 in the inhibition of activity in the brain is presented, with ATF4 theorised to play a protective role although inhibiting synaptic plasticity.