Allosteric activation of the protein kinase PDK1 with low molecular weight compounds

Abstract

Organisms rely heavily on protein phosphorylation to transduce intracellular signals. The phosphorylation of a protein often induces conformational changes, which are responsible for triggering downstream cellular events. Protein kinases are themselves frequently regulated by phosphorylation. Recently, we and others proposed the molecular mechanism by which phosphorylation at a hydrophobic motif (HM) regulates the conformation and activity of many members of the AGC group of protein kinases. Here we have developed specific, low molecular weight compounds, which target the HM/PIF-pocket and have the ability to allosterically activate phosphoinositidedependent protein kinase 1 (PDK1) by modulating the phosphorylation-dependent conformational transition. The mechanism of action of these compounds was characterized by mutagenesis of PDK1, synthesis of compound analogs, interaction-displacement studies and isothermal titration calorimetry experiments. Our results raise the possibility of developing drugs that target the AGC kinases via a novel mode of action and may inspire future rational development of compounds with the ability to modulate phosphorylation-dependent conformational transitions in other proteins.

Discovery of PDK1 Kinase Inhibitors with a Novel Mechanism of Action by Ultrahigh Throughput Screening

“…Although the most likely reason is the relatively low affinity of these unoptimized scaffolds, one cannot exclude the possibility that these chemotypes do not modulate the PDK1 functional activity despite their specific binding. At the present time, because of the low affinity of the allosteric ligands, both those discovered in this work and those described in the literature (15,16,37,38), any meaningful modulation studies in intact cells are deferred until more potent ligands are developed from these initial leads in the future.…”

“…Several independent studies demonstrated that the PIF pocket is amenable to small molecule binding (15,16,37,38). Using a virtual screening approach of a ϳ60,000 compound chemical library, Engel et al (15) identified several compounds that increased the activity of ⌬PH-PDK1.…”

“…Also, the side chain of Arg-131 shifted ϳ3.5 Å to accommodate a network of ionic and hydrogen bonds involving Thr-148, Arg-131, Lys-76, and Gln-150 with the carboxylate moiety of PS48. Hindie and co-workers (15,37) highlighted the downstream effects these local conformational changes have on the overall allostery of PDK1 structure and thus the activation mechanism seen with PS48 and similar analogs. The alkaloid compounds identified in this study do not contain carboxylate moiety; thus, it may be reasonable to hypothesize that the binding mode may differ, especially the region around Phe-157 and Arg-131, from that observed with PS48.…”

“…Conjugating the PIF-tide with the T-loop peptide from AKT-(Thr-308)-tide resulted in a prevailing model PDK1 substrate, termed the "PDK1-tide," that reportedly undergoes phosphorylation at a rate 100-fold greater than the AKT(Thr-308)-tide (9). More recently, small molecule PIF pocket binders were reported (15,16), supporting the feasibility of developing allosteric modulators of PDK1 by interrogating this site.…”

Regulation of the Interaction between Protein Kinase C-related Protein Kinase 2 (PRK2) and Its Upstream Kinase, 3-Phosphoinositide-dependent Protein Kinase 1 (PDK1)

“…The results were similar with proteins expressed and purified in parallel in at least two separate occasions. PDK1 activity assays were performed essentially as previously described (26), in the presence of 150 -300 ng PDK1 and T308tide (100 M) as the substrate. PDK1 activity was also measured on the GST-CT-PRK2 pull-down.…”