Abstract
<jats:title>Abstract</jats:title> <jats:p> APOBEC3A and APOBEC3B are antiviral cytidine deaminases found to drive cancer-associated mutagenesis, contributing to tumor evolution and therapeutic resistance across multiple cancer types. Inhibiting these enzymes holds promise for prolonging response to a wide range of cancer therapies by delaying development of resistance. However, APOBEC3A and APOBEC3B remain challenging drug targets, with no potent and selective small molecule inhibitors reported. Here, we use a fluorescence polarization-based assay to identify small molecules inhibitors of the APOBEC3A-single-stranded DNA interaction. From a library of 2,400 disulfide compounds, we identified 64 hits (mean polarization +/- 3 sigma, hit rate of 2.7%). Intact protein mass spectrometry revealed that a subset of compounds covalently engages A3A at cysteine 64, including Compounds 1 and 2. Compounds 1 and 2 disrupt APOBEC3A/APOBEC3B-single-stranded DNA interactions and inhibit APOBEC3A/APOBEC3B deaminase activity in a dose-dependent manner, with micromolar IC <jats:sub>50</jats:sub> . Surprisingly, inhibition of APOBEC3A/APOBEC3B by Compounds 1 and 2 is independent of covalent tethering to cysteine, suggesting a predominantly non-covalent mode of binding. Together, these studies establish an integrated workflow for APOBEC ligand discovery and identify Compounds 1 and 2 as starting points for developing chemical probes to investigate APOBEC-driven mutagenesis and therapeutic resistance. </jats:p>