Allosteric regulation of protein kinase PKCζ by the N-terminal C1 domain and small compounds to the PIF-pocket.

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.

4-benzimidazolyl-3-phenylbutanoic acids as novel PIF-pocket-targeting allosteric inhibitors of protein kinase PKCζ.

Protein kinase inhibitors with an allosteric mode of action are expected to reach, in many cases, higher selectivity for the target enzyme than ATP-competitive compounds. Therefore, basic research is aiming at identifying and establishing novel sites on the catalytic domain of protein kinases which might be targeted by allosteric inhibitors. We previously published the first structure-activity relationships (SARs) for allosteric activators of protein kinase PDK1. Here, we present the design, synthesis, and SAR data on a series of novel compounds, 4-benzimidazolyl-3-phenylbutanoic acids, that inhibit the atypical protein kinace C (PKC) ζ via binding to the PIF-pocket. Key positions were identified in the compounds that can be modified to increase potency and selectivity. Some congeners showed a high selectivity toward PKCζ, lacking inhibition of the most closely related isoform, PKCι, and of further AGC kinases. Furthermore, evidence is provided that these compounds are also active toward cellular PKCζ without loss of potency compared to the cell-free assay.