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.

VRK1 is a Paralog Synthetic Lethal Target in VRK2-methylated Glioblastoma

“…We believe that selectively targeting VRK1 over VRK2 may be possible as the kinase domains of VRK1 and VRK2 have approximately 80% protein sequence identity. Recent structural biology analysis revealed differential mechanisms for stabilization of an ATP-competitive inhibitor in the binding pocket of VRK1 compared to VRK2 (44,45), further supporting the possibility for development of a paralog-selective VRK1 kinase inhibitor.…”

Development of Pyridine-based Inhibitors for the Human Vaccinia-related Kinases 1 and 2

“…Previously, our analysis of the Published Kinase Inhibitor Set (PKIS) 14 revealed that VRK1 shared a similar activity profile to a small number of kinases, including members of the kinase family Ste20, such as MINK1 and TNIK. 15 The Ste20 kinase family also includes MAP4K4, whose phosphate-binding loop (P-loop, also referred to as glycine-rich loop) can adopt an unusual conformation in which this conserved structural motif folds over the ligand. It has been suggested that this folded Ploop conformation might be targeted to obtain potent and selective kinase inhibitors.…”

“…16 Cocrystal structures of VRK1 and VRK2 bound to the p90 RSK (ribosomal S6 kinase) inhibitor BI-D1870 (1, Figure 1A) revealed that both kinases can also adopt the folded P-loop conformation, further suggesting that MAP4K4 and these two VRKs have similar topography and structural features in their ATP-binding sites. 15 BI-D1870 displays a highly developed pteridine scaffold, and recent selectivity data revealed this compound to be quite promiscuous. 17 These observations motivated us to search for MAP4K4 ligands known to induce a folded P-loop conformation, and that could be used as starting points to develop potent and selective inhibitors against VRK1 and VRK2.…”

“…The cocrystal structures of VRK1 and VRK2 bound to 1 (BI-D1870) also revealed that the ligand difluorophenol moiety promoted favorable polar interactions to structurally conserved residues within the VRK ATP-binding site. 15 Thus, our design strategy to develop new small molecule inhibitors for VRK1 and VRK2 was to generate hybrid ligands by fusing the aminopyridine core with the difluorophenol group.…”

Development of Pyridine-Based Inhibitors for the Human Vaccinia-Related Kinases 1 and 2

“…The cocrystal structures of VRK1 and VRK2 bound to 1 (BI-D1870) also revealed that the ligand difluorophenol moiety promoted favorable polar interactions to structurally conserved residues within the VRK ATP-binding site. 15 Thus, our design strategy to develop new small molecule inhibitors for VRK1 and VRK2 was to generate hybrid ligands by fusing the aminopyridine core with the difluorophenol group. Synthesis of aminopyridine derivatives was performed using a rapid and efficient two-step Suzuki−Miyaura coupling (Figure 1B) with yields ranging between 18% and 89%.…”