Structural characterization of human Vaccinia-Related Kinases (VRK) bound to small-molecule inhibitors identifies different P-loop conformations

Abstract

The human genome encodes two active Vaccinia-related protein kinases (VRK), VRK1 and VRK2. These proteins have been implicated in a number of cellular processes and linked to a variety of tumors. However, understanding the cellular role of VRKs and establishing their potential use as targets for therapeutic intervention has been limited by the lack of tool compounds that can specifically modulate the activity of these kinases in cells. Here we identified BI-D1870, a dihydropteridine inhibitor of RSK kinases, as a promising starting point for the development of chemical probes targeting the active VRKs. We solved co-crystal structures of both VRK1 and VRK2 bound to BI-D1870 and of VRK1 bound to two broad-spectrum inhibitors. These structures revealed that both VRKs can adopt a P-loop folded conformation, which is stabilized by different mechanisms on each protein. Based on these structures, we suggest modifications to the dihydropteridine scaffold that can be explored to produce potent and specific inhibitors towards VRK1 and VRK2.

Crystal structure of human vaccinia‐related kinase 1 in complex with AMP‐PNP, a non‐hydrolyzable ATP analog

“…(B)], though this needs further investigation. It was concluded that the P‐loop is a highly flexible region in all VRK1 crystal structures and could be traced in only one of chains in the inhibitor complex (Chain A of PDB ID 5UKF) . Nonetheless, our NMR titration shows that the residues in the P‐loop (Gln45, Gly45, Gly46, and Gly49) are affected upon AMP‐PNP binding, validating their interaction.…”

“…Although the comparison provides a structural perspective, in future, it would be necessary to study the enzyme kinetics of these inhibitors and AMP‐PNP, lacking till‐date, to further enhance our understanding into VRK1 activity and specificity. In conclusion, as VRK1 is regarded as an important cancer drug target due to its enhanced expression levels in many cancer tissues, the molecular insights on the VRK1 interaction with AMP‐PNP presented here, along with the earlier inhibitor complexes, could be used to develop a pharmacophore model followed by fragment screening, toward identifying effective VRK1‐specific inhibitors.…”

“…The superposition of VRK1–AMP‐PNP complex on the apo structure (PDB ID 2LAV) and VRK1–inhibitor complex (PDB ID 5UKF) revealed a root‐mean‐square deviation (RMSD) of 1.70 Å (270 equivalent C α atoms) and 0.56 Å (243 equivalent C α atoms), respectively, indicating that the ligand bound structures are similar to each other compared to the apo form (Fig. S2).…”

“…(A) and (B)]. A Sulfur‐Π interaction with Phe48 has been reported to stabilize this region in one of the chains in the inhibitor bound structures . The nitrogen atom in thiazole ring forms a hydrogen bond with a water molecule, but not with any neighboring residues [Fig.…”

“…Recent studies led to the discovery of small molecule VRK1 inhibitors from natural products, such as luteolin, brazilin, and ursolic acid . An extensive thermal shift screening using a well‐known kinase library, followed by crystal structure determination, revealed the first molecular basis of the VRK1 inhibitors, ASC24 (PDBID 3OP5), BI‐D1870 (PDBID 5UVF), and GW297361X (PDBID 5UKF), providing cues toward possible VRK1 selective inhibitor design . While these inhibitors were shown to recognize the ATP binding pocket, their specificity as ATP‐competitive or ATP‐mimetic VRK1 inhibitors remains elusive.…”