PKC isoenzymes are an important family of serine/threonine protein kinases. They contribute to many diverse cellular and tissue functions, as well as human disease pathologies including cancer development and progression. The aPKC isoforms ι and ζ play crucial roles in the formation and maintenance of cell polarity and represent attractive anti-oncogenic drug targets in Ras-dependent tumors. To date, few isoform-specific chemical biology tools are available to inhibit aPKC catalytic activity. Like many protein kinases, aPKC isoenzymes are amenable to small-molecule chemical inhibition either through their ATP-binding clefts or allosteric pocket. In this study, CRT0066854 is able to mimic both ATP and a key side chain that forms part of the aPKC nucleotide-binding cleft. CRT0066854 is a potent and selective atypical PKC isoenzymes inhibitor. It is against full-length (FL) PKCι, PKCζ, and ROCK-II kinases with IC50 values of 132 nM, 639 nM, and 620 nM, respectively.

CRT0066854 selectively inhibits PKCζ and PKCι kinase activity in vitro and in cells. IC50s for CRT0066854 against PKCζk and PKCιk are 450 nM and 86 nM respectively. CRT0066854 is a competitive ATP inhibitor, binding within the nucleotide-binding pocket of aPKC. It uses a benzyl group to displace the Ade motif from a peripheral hinge pocket adjacent to the nucleotide cleft and prevents it from forming a functional ATP-binding pocket. CRT0066854 is highly selective for aPKC. CRT0066854 displaces a crucial Asn-Phe-Asp motif. That is part of the adenosine-binding pocket and engages an acidic patch used by arginine-rich PKC substrates. In addition, CRT0066854 inhibits the LLGL2 (lethal giant larvae 2) phosphorylation in cell lines. It exhibits phenotypic effects in a range of cell-based assays.

 In summary, CRT0066854 is a potent and selective chemical tool to modulate aPKC activity both in vitro and in cells.


 Kjær S, et al. Biochem J. 2013 Apr 15;451(2):329-42.