Human DNA polymerase theta (Pol), which is necessary for microhomology-mediated DNA double strand break repair, has been proposed as an attractive target for the treatment of BRCA deficient and other DNA repair pathway defective cancers1. As previously reported2, we recently identified the first selective small molecule in vitro and in vivo probes for this enzyme which recapitulate the phenotype of Pol loss and identified PARP-resistance inducing genetic defects which further sensitise to Pol inhibition. This presentation will describe in detail the discovery, biochemical and biophysical characterization and validation of these probes including disclosure of the first small molecule ligand co-crystal structures with Polq. The crystallographic data provides a basis for understanding the unique mechanism of inhibition of these compounds which is shown to be dependent on stabilization of a “closed” conformation of the enzyme.
