Abstact

DNA polymerase theta (Poltheta) is a polymerase-helicase fusion protein that is synthetically lethal with homologous recombination (HR) factors, such as BRCA1/2, and confers resistance to PARP inhibitors (PARPi) and other genotoxic cancer therapies. Previously developed Poltheta polymerase (Poltheta-pol) inhibitors (Polthetai) exhibited limited pharmacological activity and metabolic stability, warranting the development of a Polthetai with improved drug-like properties. Here, we developed RTx-303, a selective allosteric small-molecule Poltheta-pol inhibitor that exhibits 5.1 nM IC(50), 88% oral bioavailability, and a prolonged half-life along with its equipotent metabolite. X-ray crystallography highlights the development of a solvent-exposed side-chain that is essential for the optimal drug-like properties of RTx-303. Notably, RTx-303 exhibits significantly higher cellular potency than previously developed Poltheta-pol inhibitors and strongly potentiates PARPi in BRCA1/2 mutant cells and patient-derived xenograft models. The superior potency, robust pharmacological activity, and high tolerability of RTx-303 warrant further development as a Poltheta-pol inhibitor drug candidate.