PROJECT SUMMARY CK2α’, one of two catalytic subunits of human protein kinase CK2 holoenzyme, is inappropriately upregulated in cellular and animal models of Huntington’s disease (HD), and in human patients with HD. There are currently no selective inhibitors for CK2α’ available. Our work shows that CK2α’ is involved in the hyperphosphorylation and degradation of the stress protective Heat Shock transcription Factor 1 (HSF1). HSF1 has several protective roles in vivo, including regulation of stress protective chaperones and synaptic proteins, and energy metabolism. HSF1 levels are increased in an HD mouse model lacking one allele of CK2α’ (zQ175 HD), leading to increased chaperone expression and excitatory synapse density, decreased HTT aggregates and inflammation, and improved motor behavior. Given these exciting and promising results, we are initiating a program to identify selective allosteric inhibitors of CK2α’ that can serve as chemical probes for in vitro and in vivo target validation studies. The single specific aim of this exploratory project is to identify and characterize allosteric inhibitors of CK2α’ that can serve as leads for selective probe development. Herein, we propose to employ an ADP-GloTM luminescence high-throughput screen of the ChemDiv Allosteric Kinase Inhibitor (CDAKI) Library, increasing the likelihood that we will discover a small-molecule that binds allosterically to CK2α’. Active compounds will be further characterized by isothermal titration calorimetry (ITC) and x-ray crystallography. Confirmed active compounds will be validated using SAR (structure-activity relationship) by commerce. Our working hypothesis is that this library of known allosteric kinase inhibitors will generate excellent starting points for structurally-enabled compound development leading to selective allosteric probes. The potential impact of this project on human health is considerable. There is an unmet medical need for therapeutic agents that can halt or reverse the cognitive and motor decline associated with HD. This work will have a positive impact on the field as it will provide a path toward chemical probes for the validation of a new target for therapeutic development. The eventual development of a selective allosteric inhibitor of CK2α’ would address this unmet medical need and represent a significant advancement in the field of HD.