Design of a Targeted Covalent Probe to Interrogate the DNA Polymerase Activity of Polθ

Human DNA polymerase θ (Polθ) is essential for microhomology-mediated end-joining (MMEJ) and represents a therapeutic vulnerability in homologous recombination (HR)-deficient cancers. Although reversible inhibitors of Polθ have advanced into clinical development, covalent chemical probes remain unexplored. Analysis of a previously described structure of the reversible inhibitor compound 37 bound to Polθ identified Cys2411 as an accessible residue 7.4 Å adjacent to the inhibitor binding site. Guided by X-ray crystallographic studies, we designed compound 29 to reduce the separating distance between inhibitor and Cys2411 to 4.7 Å and then synthesized RP-4029 by incorporating a vinyl sulfone electrophile. Functional studies revealed efficient covalent linkage to Cys2411 ( K inact = 11.6 s –1 ), while a high-resolution (2.0 Å) cocrystal structure validated the design strategy. These findings establish Cys2411 as a privileged site for covalent inhibitor development and provide a highly potent, selective chemical probe useful for investigating Polθ biology.