Co-crystal structures of inhibitors with MRCKß, a key regulator of tumor cell invasion.

MRCKα and MRCKß (myotonic dystrophy kinase-related Cdc42-binding kinases) belong to a subfamily of Rho GTPase activated serine/threonine kinases within the AGC-family that regulate the actomyosin cytoskeleton. Reflecting their roles in myosin light chain (MLC) phosphorylation, MRCKα and MRCKß influence cell shape and motility. We report further evidence for MRCKα and MRCKß contributions to the invasion of cancer cells in 3-dimensional matrix invasion assays. In particular, our results indicate that the combined inhibition of MRCKα and MRCKß together with inhibition of ROCK kinases results in significantly greater effects on reducing cancer cell invasion than blocking either MRCK or ROCK kinases alone. To probe the kinase ligand pocket, we screened 159 kinase inhibitors in an in vitro MRCKß kinase assay and found 11 compounds that inhibited enzyme activity >80% at 3 µM. Further analysis of three hits, Y-27632, Fasudil and TPCA-1, revealed low micromolar IC(50) values for MRCKα and MRCKß. We also describe the crystal structure of MRCKß in complex with inhibitors Fasudil and TPCA-1 bound to the active site of the kinase. These high-resolution structures reveal a highly conserved AGC kinase fold in a typical dimeric arrangement. The kinase domain is in an active conformation with a fully-ordered and correctly positioned αC helix and catalytic residues in a conformation competent for catalysis. Together, these results provide further validation for MRCK involvement in regulation of cancer cell invasion and present a valuable starting point for future structure-based drug discovery efforts.

Characterization of the biological effects of a novel protein kinase D inhibitor in endothelial cells.

VEGF (vascular endothelial growth factor) plays an essential role in angiogenesis during development and in disease largely mediated by signalling events initiated by binding of VEGF to its receptor, VEGFR2 (VEGF receptor 2)/KDR (kinase insert domain receptor). Recent studies indicate that VEGF activates PKD (protein kinase D) in endothelial cells to regulate a variety of cellular functions, including signalling events, proliferation, migration and angiogenesis. To better understand the role of PKD in VEGF-mediated endothelial function, we characterized the effects of a novel pyrazine benzamide PKD inhibitor CRT5 in HUVECs (human umbilical vein endothelial cells). The activity of the isoforms PKD1 and PKD2 were blocked by this inhibitor as indicated by reduced phosphorylation, at Ser916 and Ser876 respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB (cAMP-response-element-binding protein) and HSP27 (heat-shock protein 27) at Ser82, was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 Ser82 phosphorylation. Furthermore, phosphorylation of HSP27 at Ser78, which occurs solely via the p38 MAPK (mitogen-activated protein kinase) pathway, was also unaffected by CRT5. In vitro kinase assays show that CRT5 did not significantly inhibit several PKC isoforms expressed in endothelial cells. CRT5 also decreased VEGF-induced endothelial migration, proliferation and tubulogenesis, similar to effects seen when the cells were transfected with PKD siRNA (small interfering RNA). CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.

A novel small-molecule inhibitor of protein kinase D blocks pancreatic cancer growth in vitro and in vivo.

Protein kinase D (PKD) family members are increasingly implicated in multiple normal and abnormal biological functions, including signaling pathways that promote mitogenesis in pancreatic cancer. However, nothing is known about the effects of targeting PKD in pancreatic cancer. Our PKD inhibitor discovery program identified CRT0066101 as a specific inhibitor of all PKD isoforms. The aim of our study was to determine the effects of CRT0066101 in pancreatic cancer. Initially, we showed that autophosphorylated PKD1 and PKD2 (activated PKD1/2) are significantly upregulated in pancreatic cancer and that PKD1/2 are expressed in multiple pancreatic cancer cell lines. Using Panc-1 as a model system, we showed that CRT0066101 reduced bromodeoxyuridine incorporation; increased apoptosis; blocked neurotensin-induced PKD1/2 activation; reduced neurotensin-induced, PKD-mediated Hsp27 phosphorylation; attenuated PKD1-mediated NF-kappaB activation; and abrogated the expression of NF-kappaB-dependent proliferative and prosurvival proteins. We showed that CRT0066101 given orally (80 mg/kg/d) for 24 days significantly abrogated pancreatic cancer growth in Panc-1 subcutaneous xenograft model. Activated PKD1/2 expression in the treated tumor explants was significantly inhibited with peak tumor concentration (12 micromol/L) of CRT0066101 achieved within 2 hours after oral administration. Further, we showed that CRT0066101 given orally (80 mg/kg/d) for 21 days in Panc-1 orthotopic model potently blocked tumor growth in vivo. CRT0066101 significantly reduced Ki-67-positive proliferation index (P < 0.01), increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic cells (P < 0.05), and abrogated the expression of NF-kappaB-dependent proteins including cyclin D1, survivin, and cIAP-1. Our results show for the first time that a PKD-specific small-molecule inhibitor CRT0066101 blocks pancreatic cancer growth in vivo and show that PKD is a novel therapeutic target in pancreatic cancer.