Probing the Chemical Space of Polymerase Theta with Nucleotide Analogues Bearing a Stereogenic All-Carbon Quaternary Center

We report findings on a novel family of nucleotide analogues with selective inhibitory activity against DNA polymerase theta (Polθ), a key enzyme in the theta-mediated end joining pathway and a critical player in synthetic lethality-based cancer therapies. Polθ’s intrinsically low fidelity, which contributes to genomic instability in homologous recombination-deficient tumors, was probed for selective targeting by these analogues. The newly identified compounds feature an all-carbon stereogenic quaternary center at either the C3′ or C2′ position of the furanoside ring, a structural modification that has already demonstrated potential in antiviral, anticancer, and cardioprotective applications. Biochemical assays suggest these analogues exploit Polθ’s unique active site architecture, offering a possible direction for overcoming PARP inhibitor resistance and enhancing personalized cancer treatment strategies.