Protein phosphorylation transduces a large set of intracellular signals. One mechanism by which phosphorylation mediates signal transduction is by prompting conformational changes in the target protein or interacting proteins. Previous work described an allosteric site mediating phosphorylation-dependent activation of AGC kinases. The AGC kinase PDK1 is activated by the docking of a phosphorylated motif from substrates. Here we present the crystallography of PDK1 bound to a rationally developed low-molecular-weight activator and describe the conformational changes induced by small compounds in the crystal and in solution using a fluorescence-based assay and deuterium exchange experiments. Our results indicate that the binding of the compound produces local changes at the target site, the PIF binding pocket, and also allosteric changes at the ATP binding site and the activation loop. Altogether, we present molecular details of the allosteric changes induced by small compounds that trigger the activation of PDK1 through mimicry of phosphorylation-dependent conformational changes.

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.

The PIF-pocket of AGC protein kinases participates in the physiologic mechanism of regulation by acting as a docking site for substrates and as a switch for the transduction of the conformational changes needed for activation or inhibition. We describe the effects of compounds that bind to the PIF-pocket of PDK1. In vitro, PS210 is a potent activator of PDK1, and the crystal structure of the PDK1-ATP-PS210 complex shows that PS210 stimulates the closure of the kinase domain. However, in cells, the prodrug of PS210 (PS423) acts as a substrate-selective inhibitor of PDK1, inhibiting the phosphorylation and activation of S6K, which requires docking to the PIF-pocket, but not affecting PKB/Akt. This work describes a tool to study the dynamics of PDK1 activity and a potential approach for drug discovery.

Protein kinases are key mediators of cellular signaling, and therefore, their activities are tightly controlled. AGC kinases are regulated by phosphorylation and by N- and C-terminal regions. Here, we studied the molecular mechanism of inhibition of atypical PKCζ and found that the inhibition by the N-terminal region cannot be explained by a simple pseudosubstrate inhibitory mechanism. Notably, we found that the C1 domain allosterically inhibits PKCζ activity and verified an allosteric communication between the PIF-pocket of atypical PKCs and the binding site of the C1 domain. Finally, we developed low-molecular-weight compounds that bind to the PIF-pocket and allosterically inhibit PKCζ activity. This work establishes a central role for the PIF-pocket on the regulation of PKCζ and allows us to envisage development of drugs targeting the PIF-pocket that can either activate or inhibit AGC kinases.

The modulation of protein kinase activities by low molecular weight compounds is a major goal of current pharmaceutical developments. In this line, important efforts are directed to the development of drugs targeting the conserved ATP binding site. However, there is very little experience on targeting allosteric, regulatory sites, different from the ATP binding site, in protein kinases. Here we describe the synthesis, cell-free activation potency, and calorimetric binding analysis of 3,5-diphenylpent-2-enoic acids and derivatives as allosteric modulators of the phosphoinositide-dependent kinase-1 (PDK1) catalytic activity. Our SAR results combined with thermodynamic binding analyses revealed both favorable binding enthalpy and entropy and confirmed the PIF-binding pocket of PDK1 as a druggable site. In conclusion, we defined the minimal structural requirements for compounds to bind to the PIF-binding pocket and to act as allosteric modulators and identified two new lead structures (12Z and 13Z) with predominating binding enthalpy.