Hit finding and assay enablement for VRK1, a paralog synthetic lethal kinase target in VRK2-deficient cancers

Vaccinia-related kinases (VRKs) are a family of serine/threonine kinases that regulate diverse cellular processes, including transcription factor activity, chromatin remodeling, nuclear envelope formation, and cell-cycle progression. Among them, VRK1 has emerged as a paralog-selective synthetic lethal target in cancers with low VRK2 expression, encompassing nearly all neuroblastomas and over 60% of glioblastomas, with potential for therapeutic expansion into additional tumor types. Here, we describe the biochemical, biophysical, and cellular assays that enabled the discovery and optimization of novel inhibitors of VRK1 activity found through variety of binding screens, activity-based screens, and rational design. High-resolution crystal structures of both VRK1 and VRK2 in complex with inhibitors revealed their binding poses, identified key interactions, and confirmed orthosteric binding. Medicinal chemistry optimization yielded chemical series capable of achieving >4000X selectivity over the VRK1 paralog VRK2 in a biochemical assay conducted with a physiologically relevant ATP concentration, and >70X selectivity for VRK2-deficient cells in cellular viability assays. Notably, strong correlations between biochemical potency, cellular target engagement, pharmacodynamic response, and functional viability supported both the specificity of the chemical matter and the robustness of the assay platform. Together, these findings establish VRK1 as a tractable and structurally enabled target and demonstrate that high paralog selectivity in cells can be achieved through orthosteric inhibition. This work provides the strong foundation necessary for structure-guided optimization of VRK1 inhibitors with potential therapeutic applications in cancers with low VRK2 expression, such as glioblastoma and neuroblastoma.