At nanomolar concentrations, a PERK inhibitor (PERKi) saturates its primary target and inhibits the ISR, as anticipated 5 , 6 , 7 . At concentrations oversaturating the primary target PERK, binding to the off-target kinase GCN2 is enabled. Binding of the inhibitor to the ATP-binding site of one protomer of the GCN2 dimer, increases the affinity for ATP of the second protomer leading to GCN2 activation. This in turn results in functional ISR induction in absence of specific kinase activating stressors. Saturation of both PERK and GCN2 with complete inhibition of the two kinases is achieved at higher concentrations of the inhibitors. Note that GCN2 and the ISR can be activated by the PKR inhibitor C16 by a similar mechanism.
