The PDK1 kinase has been the focus of great attention since it was discovered over 13 years ago in the MRC Protein Phosphorylation Unit as a key enzyme that activated Akt. Work carried out in Dundee on PDK1 over these years have uncovered most of the known 23 cellular substrates of PDK1 and elucidated how PDK1 is regulated by binding to the PtdIns(3,4,5)P3 second messenger and docking to phosphorylated hydrophobic motifs of its substrates. The structure of PDK1 has also been elucidated and the key roles that PDK1 plays in cancer and insulin signalling been defined in a series of studies with PDK1 hypomorphic as well as tissue specific knockout and knock-in mice lacking the ability to interact with PtdIns(3,4,5)P3 or its AGC kinases phosphorylated at its hydrophobic motif. Importantly this work suggested that inhibitors of PDK1 should protect against cancer, as mice deficient in the PTEN tumour suppressor, expressing reduced levels of PDK1 developed far fewer tumours.
Despite all of this work described in over 70 publications, what was missing was a potent selective inhibitor of PDK1 that could be used to probe the biological roles that this enzyme plays. Over the years many inhibitors of PDK1 have been reported, but everyone we tested turned out to be insufficiently selective or potent to be able to probe the roles of PDK1 in vivo.
In a major breakthrough in this field, GlaxoSmithKline researchers including Jeffrey Axten and M. Phillip DeYoung were able to elaborate the compound GSK-2334450 which turns out to be an amazingly selective and potent (IC50 10 nM) inhibitor of PDK1 which did not inhibit significantly the activity of ~100 other kinases/lipid kinases tested. GlaxoSmithKline kindly provided us with this compound to evaluate and Ayaz Najafov with some help from Eeva Sommer (pictured), set about characterising the cellular efficacy of GSK-2334470. They found that GSK-2334470 potently suppressed activation of many PDK1 substrates including SGK, S6K and RSK, but was less efficient at inhibiting Akt, especially under conditions where large activation of the PI 3-kinase pathway is observed. Further experimentation revealed that this reduced ability to inhibit Akt was probably due to the unusually high efficiency at which Akt is activated by PDK1 on the two dimensional surface of plasma membrane. Studies with GSK-2334470 also revealed striking differences in the kinetics of T-loop dephosphorylation of diverse AGC kinase substrates. Treatment of cells with GSK-2334470 induced potent inhibition and dephosphorylation of the T-loop of Akt, S6K1, SGK isoforms within 5-30 min of GSK233440 treatment whereas equivalent levels of inactivation of RSK isoforms required ~8h.
Overall, these new data define how PDK1 inhibitors affect AGC signalling pathways and suggest that GSK-2334470 will be a useful tool for delineating roles of PDK1 in biological processes. GSK-2334470 also represents a useful addition to our ever-increasing armoury of effective signal transduction inhibitors to dissect biological roles of protein kinases.
To read a copy of our paper describing the GSK-2334470 inhibitor click here
Despite all of this work described in over 70 publications, what was missing was a potent selective inhibitor of PDK1 that could be used to probe the biological roles that this enzyme plays. Over the years many inhibitors of PDK1 have been reported, but everyone we tested turned out to be insufficiently selective or potent to be able to probe the roles of PDK1 in vivo.
In a major breakthrough in this field, GlaxoSmithKline researchers including Jeffrey Axten and M. Phillip DeYoung were able to elaborate the compound GSK-2334450 which turns out to be an amazingly selective and potent (IC50 10 nM) inhibitor of PDK1 which did not inhibit significantly the activity of ~100 other kinases/lipid kinases tested. GlaxoSmithKline kindly provided us with this compound to evaluate and Ayaz Najafov with some help from Eeva Sommer (pictured), set about characterising the cellular efficacy of GSK-2334470. They found that GSK-2334470 potently suppressed activation of many PDK1 substrates including SGK, S6K and RSK, but was less efficient at inhibiting Akt, especially under conditions where large activation of the PI 3-kinase pathway is observed. Further experimentation revealed that this reduced ability to inhibit Akt was probably due to the unusually high efficiency at which Akt is activated by PDK1 on the two dimensional surface of plasma membrane. Studies with GSK-2334470 also revealed striking differences in the kinetics of T-loop dephosphorylation of diverse AGC kinase substrates. Treatment of cells with GSK-2334470 induced potent inhibition and dephosphorylation of the T-loop of Akt, S6K1, SGK isoforms within 5-30 min of GSK233440 treatment whereas equivalent levels of inactivation of RSK isoforms required ~8h.
Overall, these new data define how PDK1 inhibitors affect AGC signalling pathways and suggest that GSK-2334470 will be a useful tool for delineating roles of PDK1 in biological processes. GSK-2334470 also represents a useful addition to our ever-increasing armoury of effective signal transduction inhibitors to dissect biological roles of protein kinases.
To read a copy of our paper describing the GSK-2334470 inhibitor click here