Adenosine-binding motif mimicry and cellular effects of a thieno[2,3-d]pyrimidine-based chemical inhibitor of atypical protein kinase C isoenzymes

Abstract

The aPKC [atypical PKC (protein kinase C)] 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 tumours. To date, few isoform-specific chemical biology tools are available to inhibit aPKC catalytic activity. In the present paper, we describe the identification and functional characterization of potent and selective thieno[2,3-d]pyrimidine-based chemical inhibitors of aPKCs. A crystal structure of human PKCι kinase domain bound to a representative compound, CRT0066854, reveals the basis for potent and selective chemical inhibition. Furthermore, 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. We show that CRT0066854 inhibits the LLGL2 (lethal giant larvae 2) phosphorylation in cell lines and exhibits phenotypic effects in a range of cell-based assays. We conclude that this compound can be used as a chemical tool to modulate aPKC activity in vitro and in vivo and may guide the search for further aPKC-selective inhibitors.

Regulation of polarized morphogenesis by protein kinase C iota in oncogenic epithelial spheroids

“…Furthermore, the recovery of polarity on reduction of PKCι in Ras- and ErbB2-transformed MDCK cells suggested that an overactive PKCι was responsible for this aberrant behaviour. In contrast, in the parent cell, loss of PKCι has been reported to trigger loss of polarity suggesting that in fact the PKCι dependence displays threshold behaviour (60,61,69). To test this, we manipulated gain and loss of function of PKCι in the parental MDCK cells.…”

“…To assess this threshold, predicted catalytic behaviour of PKCι, we determined the response to the catalytic inhibitors CRT0066854, a recently described thieno[2,3-d]pyrimidine-based chemical inhibitor of aPKC (69), and Gö6983 (a pan PKC inhibitor) in H-Ras MDCK spheroids. Inhibitors were added on the day of seeding a single cell suspension in Matrigel and then replenished on alternate days for 6 days.…”

“…Inhibitors were added on the day of seeding a single cell suspension in Matrigel and then replenished on alternate days for 6 days. The inhibitor doses used for compounds were determined based on their individual, previously defined cellular IC50s (69,70). Both inhibitors phenocopied the siRNA-PKCι intervention resulting in the induction of apical lumen formation in the spheroids and a reduction in spheroid size (Figure 6).…”

aPKC Inhibition by Par3 CR3 Flanking Regions Controls Substrate Access and Underpins Apical-Junctional Polarization

“…Mutagenesis of PKCι and Par3 was performed using QuikChange (Stratagene). Recombinant human PKC-iota kinase domain (PKCι KD ) was prepared using a baculovirus encoding residues 248–596 (GenBank: NM_002740.5), fused to a glutathione S-transferase (GST) tag as described previously (Kjaer et al., 2013). See Supplemental Experimental Procedures for a detailed description.…”

Targeting protein kinase C subtypes in pancreatic cancer

“…Many of the ATP competitive aPKC inhibitors such as hydroxyphenyl-1-benzopyran-4-ones [36] and PKCzI257.3 [N-(4-((dimethylamino)methyl)-benzyl)-1H-pyrrole-2-carboxamide] [37] target both PKCζ and PKCι, but also other molecules unrelated to PKC. Other inhibitors show some isoform specificity include the thieno[2,3-d]pyrimidine-based compound CRT0066854 which was reported 4-fold more potent against PKCι than PKCζ [38]; and a 2-(6-phenylindazolyl)-benzimidazole derivative that shows increased inhibitory activity for PKCζ over PKCι [39]. …”