Discovery of RP-37 analogues as potent, selective irreversible inhibitors targeting DNA polymerase theta (POLθ)

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 a potent and selective irreversible inhibitor targeting the POLθ polymerase domain. It demonstrated potent enzymatic inhibition with an 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, k _inact/K _i determination and molecular docking studies confirmed the irreversible binding mechanism and sustained target occupancy of B2.