Co-Crystal Structures of Inhibitors with MRCKβ, a Key Regulator of Tumor Cell Invasion

Abstract

MRCKα and MRCKβ (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 MRCKβ influence cell shape and motility. We report further evidence for MRCKα and MRCKβ 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 MRCKβ 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 MRCKβ 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 IC50 values for MRCKα and MRCKβ. We also describe the crystal structure of MRCKβ 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.

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“…Rho GTPase family proteins are key regulators of the actin cytoskeleton and, with the aid of various target proteins, maintain the tight regulation of normal cell growth and differentiation (8)(9)(10)(11). Eukaryotic cells exhibit a predisposition to rapid and uncontrollable growth following genomic alterations or carcinogenesis.…”

Targeting MRCK and LIMK kinases for cancer therapy

“…Ultimately, crystals were obtained that diffracted to 2.2 A for MRCKb-fasudil ( Fig. 2A) and 2.6 A for MRCKb-TPCA-1, and the structures determined by molecular replacement (10). These structural determinations revealed that MRCKb kinase domains are arranged in dimers with an overall active conformation in the absence of phosphorylation.…”

“…MRCKa was identified in breast cancer as part of a gene expression signature linked to poor prognosis and increased incidence of metastasis under five years (7), and one of a seven gene panel with significantly elevated expression in pancreatic adenocarcinoma compared to normal pancreas (8). Invasiveness of MDA MB 231 breast cancer cells was inhibited by knockdown of either ROCK or MRCK kinases, but the most effective treatment was combined ROCK and MRCK knockdown or inhibition (9,10). Similarly, in an organotypic invasion model, SCC12 squamous carcinoma cell collective invasion through fibroblast-initiated tracks in threedimensional protein matrices was dependent on MRCK (11).…”

“…These results suggest that MRCK is a potential anti-invasion/metastasis cancer drug target that could be used as an alternative to, or in combination with, ROCK inhibition. An in vitro high throughput screen (HTS) compatible assay was established using fluorescence polarization to monitor peptide phosphorylation by MRCKb (10). Of 159 kinase inhibitors screened, the ROCK inhibitor fasudil and the reportedly IKK2-selective In response to CDC42 activation, the MRCK kinases phosphorylate substrates that may result in their activation (grey arrow) or inactivation (black arrow).…”

“…In the absence of clear causal relationships between MRCK activation and specific cancers, further investigation will be required to identify the tumor types that would most likely benefit from MRCK inhibitor administration. The association of elevated MRCKa expression with poor prognosis and increased incidence of metastasis under five years in breast cancer patients (7), coupled with the ability of MRCK knockdown to reduce breast cancer cell line invasiveness (9,10) suggests that mammary tumors might be a strong candidate. An additional issue to be resolved is whether MRCK inhibitors would be effective as single agents, or whether their ability to block tumor cell invasiveness and distant metastasis would be dependent on the simultaneous inhibition of ROCK activity.…”