Paradoxical Activation of GCN2 by ATP-competitive inhibitors via allosteric activation and autophosphorylation

Recently it has been found that General Control Non-derepressible 2 (GCN2) can be activated by an array of small molecule ATP-competitive inhibitors, including clinically relevant compounds such as Ponatinib, and compounds specifically designed to be GCN2 inhibitors, such as GCN2iB. Furthermore, we recently showed that GCN2 can be activated in cells by clinically approved small molecule RAF inhibitors. GCN2 is a drug target, specifically in cancers such as mesothelioma, and a better understanding of this paradoxical activation is required to develop drugs which truly inhibit the enzyme. Using biochemical assays and structural mass spectrometry, we present a model for how GCN2 is activated by these compounds by promoting an active conformation in the HisRS domain while competitively inhibiting the kinase domain. This conformation promotes activating phosphorylation of GCN2, potentially through phosphorylation of other activated GCN2 molecules which are not bound to compound. Together this model suggests that efforts to inhibit GCN2 would benefit from exploring allosteric routes rather than targeting the ATP-binding pocket of the kinase domain.