Discovery and optimization of 1,3,5-trisubstituted pyrazolines as potent and highly selective allosteric inhibitors of protein kinase C-ζ.

There is increasing evidence that the atypical protein kinase C, PKCζ, might be a therapeutic target in pulmonary and hepatic inflammatory diseases. However, targeting the highly conserved ATP-binding pocket in the catalytic domain held little promise to achieve selective inhibition. In the present study, we introduce 1,3,5-trisubstituted pyrazolines as potent and selective allosteric PKCζ inhibitors. The rigid scaffold offered many sites for modification, all acting as hot spots for improving activity, and gave rise to sharp structure-activity relationships. Targeting of PKCζ in cells was confirmed by reporter gene assay, transfection assays, and Western blotting. The strongly reduced cell-free and cellular activities toward a PIF-pocket mutant of PKCζ suggested that the inhibitors most likely bound to the PIF-pocket on the kinase catalytic domain. Thus, using a rigidification strategy and by establishing and optimizing multiple molecular interactions with the binding site, we were able to significantly improve the potency of the previously reported PKCζ inhibitors.