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 IC(50) 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.

Figure 1. Inhibition of 3-D matrigel invasion by MDA MB 231 cells following MRCK and ROCK inhibition.

(A) Knock-down of MRCKα and MRCKβ individually or in combination. Double knockdown was achieved either by combining separate siRNAs (MRCKα+β) or single siRNA duplexes that target both kinases (MRCKα/β). NTC = non-targeting control. (B) Optical slices were obtained every 10 µm by confocal imaging. (C) Invasion 40 µm above the transwell filter surface was normalized to non-targeting control (NTC) siRNA transfected cells. Significant differences between groups of columns indicated. (Average ± SEM, n = 3). MRCKα/β knockdown or ROCK inhibition with Y-27632 (Y) significantly decreased invasion, with the combination of MRCKα/β knockdown and ROCK inhibition resulting in significantly more inhibition. (D) Effectiveness and specificity of ROCK1, ROCK2 and MRCKαβ knockdowns. (E) The combination of MRCKα/β with ROCK1 and ROCK2 knockdown individually or in combinations were tested for their effects on 3-D matrigel invasion. ROCK1, ROCK2 or ROCK1+ROCK2 combination were able to significantly inhibit invasion. However, additional MRCKα/β knockdown significantly increased the inhibition of invasion in each instance. (Average ± SEM, n = 3).

Figure 2. Inhibition of MRCK activity by kinase inhibitors.

A collection of 159 kinase inhibitors were tested for their ability to inhibit MRCKβ activity in vitro at (A) 30 µM and (B) 3 µM. Pie charts represent the proportion inhibiting >80% at each concentration. Inhibition of (C) MRCKα or (D) MRCKβ activity by Y-27632, TPCA-1 and Fasudil. Both kinases were inhibited by these compounds, although some differences in sensitivity were apparent.

Figure 3. Structure of dimeric MRCKβ.

(A) Overall structure of the dimeric MRCKβ shows the interactions of the two monomers at the dimerization domain. The four Fasudil molecules observed per asymmetric unit are also shown, two bound to the surface of the protein (central) and one bound to each of the ATP-binding sites (lateral). (B) A close-up of one monomer reveals a typical two-lobed kinase structure, with both the N-terminus (orange) and the C-terminus (purple; disorganized loop shown as a dashed line) forming the dimerization domain. The glycine-rich loop (blue) and activation loop (red) are fully ordered, and the αEF/αF-loop is also indicated (yellow). Fasudil is shown bound in the ATP-binding site.

Figure 4. Detailed views of intramolecular interations.

(A) Interactions involved in the small antiparallel β-sheet formed from a part of the activation loop (red) and a part of the αEF/αF loop (yellow). (B) Hydrogen bonding interactions involved in the stabilization of the hydrophobic motif within the dimerization domain.

Figure 5. Positions of Fasudil and TPCA-1 in MRCKβ active site.

(A) Structure of Fasudil bound to the active site of MRCKβ. Hydrogen bonds are marked with yellow dashed lines. The Fo–Fc omit map is shown contoured at 2σ, with the inhibitor modeled in the density. In the topology diagram, surface contour is shown with a gray dashed line and hydrogen bonds with blue dashed lines. Hydrophobic residues lining the cavity are shown in light green circles, and polar residues with light magenta. Blue shading indicates solvent exposed ligand groups. (B) Structure of TPCA-1 bound to the active site of MRCKβ. (C) Overlay of the structures of Fasudil and TPCA-1 bound to the active site of MRCKβ.

Figure 6. Sequence alignment of MRCKβ and the most closely related AGC kinases.

Sequences have been truncated after the C-terminal lobe. The conserved HRD and DFG motifs have been highlighted with red arrows, and the conserved salt bridge at the active site with green arrows. Predicted phosphorylation sites have been indicated with magenta arrows, and the residues involved in hydrogen bonds between the C- and N-termini at the dimerization domain are indicated by orange arrows.

Figure 7. An overlay of MRCKβ (green) with ROCK2 (A; cyan) and ROCK1 (B; magenta) highlights the very subtle structural differences that could be exploited in developing specific inhibitors.

The surface shown is the nucleotide binding cavity of MRCKβ, with Fasudil bound at the ATP binding site. Apart from the highlighted residues, the ATP binding sites of the three kinases are identical at sequence level.