Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1.

PDK1 (3-phosphoinositide-dependent protein kinase 1) activates a group of protein kinases belonging to the AGC [PKA (protein kinase A)/PKG (protein kinase G)/PKC (protein kinase C)]-kinase family that play important roles in mediating diverse biological processes. Many cancer-driving mutations induce activation of PDK1 targets including Akt, S6K (p70 ribosomal S6 kinase) and SGK (serum- and glucocorticoid-induced protein kinase). In the present paper, we describe the small molecule GSK2334470, which inhibits PDK1 with an IC₅₀ of ~10 nM, but does not suppress the activity of 93 other protein kinases including 13 AGC-kinases most related to PDK1 at 500-fold higher concentrations. Addition of GSK2334470 to HEK (human embryonic kidney)-293, U87 or MEF (mouse embryonic fibroblast) cells ablated T-loop residue phosphorylation and activation of SGK isoforms and S6K1 induced by serum or IGF1 (insulin-like growth factor 1). GSK2334470 also inhibited T-loop phosphorylation and activation of Akt, but was more efficient at inhibiting Akt in response to stimuli such as serum that activated the PI3K (phosphoinositide 3-kinase) pathway weakly. GSK2334470 inhibited activation of an Akt1 mutant lacking the PH domain (pleckstrin homology domain) more potently than full-length Akt1, suggesting that GSK2334470 is more effective at inhibiting PDK1 substrates that are activated in the cytosol rather than at the plasma membrane. Consistent with this, GSK2334470 inhibited Akt activation in knock-in embryonic stem cells expressing a mutant of PDK1 that is unable to interact with phosphoinositides more potently than in wild-type cells. GSK2334470 also suppressed T-loop phosphorylation and activation of RSK2 (p90 ribosomal S6 kinase 2), another PDK1 target activated by the ERK (extracellular-signal-regulated kinase) pathway. However, prolonged treatment of cells with inhibitor was required to observe inhibition of RSK2, indicating that PDK1 substrates possess distinct T-loop dephosphorylation kinetics. Our data define how PDK1 inhibitors affect AGC signalling pathways and suggest that GSK2334470 will be a useful tool for delineating the roles of PDK1 in biological processes.