Discovery of a small-molecule inhibitor that traps Polθ on DNA and synergizes with PARP inhibitors

The DNA damage response (DDR) protein DNA Polymerase theta (Pol theta) is synthetic lethal with homologous recombination (HR) factors and is therefore a promising drug target in BRCA1/2 mutant cancers. We discover an allosteric Pol theta inhibitor (Pol theta i) class with 4-6 nM IC50 that selectively kills HR-deficient cells and acts synergistically with PARP inhibitors (PARPi) in multiple genetic backgrounds. X-ray crystallography and biochemistry reveal that Pol theta i selectively inhibits Pol theta polymerase (Pol theta-pol) in the closed conformation on B-form DNA/DNA via an induced fit mechanism. In contrast, Pol theta i fails to inhibit Pol theta-pol catalytic activity on A-form DNA/RNA in which the enzyme binds in the open configuration. Remarkably, Pol theta i binding to the Pol theta-pol:DNA/DNA closed complex traps the polymerase on DNA for more than forty minutes which elucidates the inhibitory mechanism of action. These data reveal a unique small-molecule DNA polymerase:DNA trapping mechanism that induces synthetic lethality in HR-deficient cells and potentiates the activity of PARPi. Here the authors discover a small-molecule that inhibits DNA polymerase theta by trapping the enzyme on DNA in the closed conformation. The inhibitor selectively kills BRCA-mutant cells and exhibits strong synergistic activity with PARP inhibitors.