Discovery of RP-37 Analogues as Potent, Selective Irreversible Inhibitors Targeting DNA Polymerase Theta (POLθ)

Abstract

DNA polymerase theta (POLθ) is a promising synthetic lethal target for BRCA-deficient cancers, while reversible POLθ-pol inhibitors exhibit limited cellular efficacy. Here, we report the structure-guided discovery of RP-37 analogue B2 as potent and selective irreversible inhibitor targeting POLθ polymerase domain. It demonstrated potent enzymatic inhibition with IC50 value of 1.54 nM and significantly enhanced antiproliferative activity against BRCA2-deficient DLD-1 cells (IC50 = 1.16 μM), which was about 5-fold more potent than the reversible analog C10 (IC50 = 5.36 μM). Further time-dependent enzymatic inhibition, washout experiments, kinact/Ki determination and molecular docking studies confirmed the irreversible binding mechanism and sustained target occupancy of B2.