Mitchison, Timothy J.Crowley-Dolen, Emma Kathryn2026-06-0920262026-05-122026Crowley-Dolen, Emma Kathryn. 2026. Targeting VRK1: Selective Inhibitors, Chemical Probes, and Biochemical Insights. Doctoral Dissertation, Harvard University Graduate School of Arts and Sciences.32699720https://dash.harvard.edu/handle/1/42740229Vaccinia-related kinase 1 (VRK1) is a promising target in gliomas and glioblastomas where VRK2 is silenced by promoter methylation, rendering VRK1 essential for accurate nuclear envelope reassembly following mitosis. Small-molecule ATP-site drug discovery for VRK1 has been hindered by the absence of robust and reproducible biochemical assays. Through virtual screening, we identified previously unreported VRK1-binding scaffolds and validated them in biochemical kinase assays, yielding an 82 nM inhibitor with high selectivity for VRK1 over VRK2. During characterization of this compound, we found that a commonly used commercial time-resolved fluorescence energy transfer (TR-FRET) VRK1 activity assay is dependent on purification tag-mediated VRK1 dimerization. Leveraging the new inhibitor, we developed fluorogenic tool compounds that increase in fluorescence intensity upon binding to the active site of VRK1, and do not require artificial dimerization of VRK1. The top probe exhibits a Kd of 180 nM and is useful for liganddisplacement assays using both fluorescence enhancement and TR-FRET readouts. Serine/threonine kinases are often regulated by multiple mechanisms in cells. VRK1 is not known to be regulated by activation loop phosphorylation or by additional binding partners. Here, we characterize the in vitro activity of VRK1 and report on a previously unreported interaction between VRK1 and PARP1. We found that PARP1 potently inhibits VRK1-mediated phosphorylation of commercial peptide ULight-histone H3, and that this interaction is weakened by the presence of DNA. This thesis introduces new chemical scaffolds for targeting VRK1, defines an assay artifact that has complicated VRK1 inhibitor discovery, and delivers fluorogenic tool compounds for high-throughput screening of ATP-site inhibitors. Furthermore, we characterize new regulatory mechanisms of VRK1. Together, these results enabling future drug discovery efforts against this emerging cancer vulnerability.application/pdfenAssay developmentCancer biologyEnzymologySynthetic lethalityTR-FRETVRK1BiochemistryBiologyChemistryThesis or Dissertation2026-06-090000-0002-4653-4054