Rational Design and Biological Evaluation of a Novel Polθ Polymerase Inhibitor for Synergistic Targeting of HR-Deficient Cancers

DNA
polymerase theta (Polθ) is a key enzyme in the theta-mediated
end-joining (TMEJ) and a promising target for synthetic lethality-based
cancer therapy, particularly in homologous recombination (HR)-deficient
tumors. Herein, we designed and optimized a series of Polθ polymerase
inhibitors, exploiting a previously unexplored binding pocket to enhance
potency, cellular activity, and pharmacokinetics. Compound 33 exhibited low-nanomolar Polθ inhibition, strong antiproliferative
activity in DLD1 BRCA2 KO cells, and high sensitivity to MDA-MB-436
cells. In combination with Olaparib, it significantly enhanced DNA
damage accumulation, induced γH2AX levels, and achieved 85%
tumor growth inhibition (TGI) in the MDA-MB-436 xenograft model. Pharmacokinetic
studies confirmed a favorable dose-dependent profile, and preliminary
safety evaluations indicated good tolerability. These findings establish
compound 33 as a potent Polθ inhibitor with strong
synthetic lethality effects, supporting its further preclinical development
as a novel Polθ-targeted therapeutic agent.