Discovery, Characterization, and Structure-Based Optimization of Small-Molecule In Vitro and In Vivo Probes for Human DNA Polymerase Theta
Human DNA polymerase theta (Polθ), crucial for microhomology-mediated DNA double-strand break repair, has emerged as a promising target for treating BRCA-deficient and other DNA repair pathway-deficient cancers. We recently identified the first selective small molecule Polθ probes: 22 (ART558), an in vitro probe that mimics the phenotype of Polθ loss, and 43 (ART812), an in vivo probe effective in a PARP inhibitor-resistant TNBC model. This paper details the discovery, biochemical, and biophysical characterization of these probes, including co-crystal structures of small molecule ligands with Polθ. The crystallographic data elucidates the unique inhibition mechanism of these compounds, which rely on stabilizing a “closed” enzyme conformation. Furthermore, this structural biology platform has enabled rational optimization, primarily through reducing ligand conformational flexibility.