(1149-B) A NanoBRET™ Target Engagement Assay for Querying Domain Selectivity at Full-Length Polymerase Theta in Live Cells
Monday, February 5, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: In HR-deficient cells, Polymerase Theta (Pol-theta) serves to repair double-strand breaks and has emerged as a therapeutic target in HR-deficient cancers. Encoded by the POLQ gene, Pol-theta is a large (290 Kda) protein with two distinct functional domains (polymerase and helicase), separated by an unstructured region. Each domain of Pol-theta has proven vulnerable to small molecule inhibitors, with a number of molecules undergoing clinical evaluation. Unlike the helicase domain, target engagement at the polymerase domain requires cooperativity with DNA fragments. This study describes a novel NanoBRET™ Target Engagement assay that enables domain-specific analysis of small molecule occupancy in live cells. To observe the high affinity-state of small molecule inhibitors to the polymerase domain, we have developed a cell-permeable pol domain NanoBRET™ probe, along with a novel work flow to introduce small DNA fragments into live cells. Together these components form a nucleoprotein complex with full length polymerase theta. This assay method can be used to quantify and rank-order engagement to the polymerase domain in live cells. To enable domain-selectivity analysis, we have developed a second NanoBRET™ probe directed to the helicase domain. Together these two NanoBRET™ probes allow for mechanism of action studies at Pol-theta in living cells.