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Updated: Aug 21, 2026
A Fluorescence-based Protocol for Preliminary Screening of Protein Synthesis Inhibitors from Natural Sources
Published on: January 27, 2026
Fluorescence polarization-based fragment screen identifies inhibitors of APOBEC3A and APOBEC3B
Abstract:
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 50 . 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.
Insights
Researchers identified novel small molecule inhibitors targeting APOBEC3A/APOBEC3B enzymes, which drive cancer mutagenesis. These compounds offer a promising starting point for developing new cancer therapies to overcome drug resistance.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- APOBEC3A and APOBEC3B are cytidine deaminases implicated in cancer mutagenesis and therapeutic resistance.
- Targeting these enzymes is a promising strategy to enhance cancer therapy response.
- Developing potent and selective small molecule inhibitors for APOBEC3A/APOBEC3B remains a significant challenge.
Purpose of the Study:
- To identify small molecule inhibitors of the APOBEC3A-single-stranded DNA interaction.
- To establish an integrated workflow for APOBEC ligand discovery.
- To identify starting points for developing chemical probes against APOBEC-driven mutagenesis.
Main Methods:
- Utilized a fluorescence polarization-based assay to screen a library of 2,400 disulfide compounds.
- Employed intact protein mass spectrometry to identify covalent engagement with APOBEC3A.
- Assessed inhibition of APOBEC3A/APOBEC3B deaminase activity and disruption of DNA interactions.
Main Results:
- Identified 64 hit compounds (2.7% hit rate) that inhibit APOBEC3A-single-stranded DNA interaction.
- Compounds 1 and 2 were found to covalently engage APOBEC3A at cysteine 64.
- Compounds 1 and 2 demonstrated dose-dependent inhibition of deaminase activity (micromolar IC50) and disrupted DNA interactions, surprisingly via a non-covalent mechanism.
Conclusions:
- Established a robust workflow for APOBEC ligand discovery.
- Identified Compounds 1 and 2 as promising chemical starting points for further development.
- These compounds can be used to investigate APOBEC-driven mutagenesis and therapeutic resistance in cancer.
