Structural basis for a Polθ helicase small-molecule inhibitor revealed by cryo-EM

DNA polymerase theta (Pol theta) is a DNA helicase-polymerase protein that facilitates DNA repair and is synthetic lethal with homology-directed repair (HDR) factors. Thus, Pol theta is a promising precision oncology drug-target in HDR-deficient cancers. Here, we characterize the binding and mechanism of action of a Pol theta helicase (Pol theta-hel) small-molecule inhibitor (AB25583) using cryo-EM. AB25583 exhibits 6 nM IC50 against Pol theta-hel, selectively kills BRCA1/2-deficient cells, and acts synergistically with olaparib in cancer cells harboring pathogenic BRCA1/2 mutations. Cryo-EM uncovers predominantly dimeric Pol theta-hel:AB25583 complex structures at 3.0-3.2 & Aring;. The structures reveal a binding-pocket deep inside the helicase central-channel, which underscores the high specificity and potency of AB25583. The cryo-EM structures in conjunction with biochemical data indicate that AB25583 inhibits the ATPase activity of Pol theta-hel helicase via an allosteric mechanism. These detailed structural data and insights about AB25583 inhibition pave the way for accelerating drug development targeting Pol theta-hel in HDR-deficient cancers. Pol theta helicase is crucial for DNA repair and a target for precision medicine for cancer therapy. Here, the authors reveal the mechanism of action of the small-molecule inhibitor AB25583, and show its high specificity and selective killing of BRCA1/2- cancer cells and synergy with olaparib.