Joint Screening and Identification of Potential Targets of Nitazoxanide by Affinity Chromatography and Label-Free Techniques

Background: Nitazoxanide not only exhibits a broad spectrum of activities against various pathogens infecting animals and humans but also induces cellular autophagy. Currently, the pattern of action and subcellular targets of nitazoxanide-induced cellular autophagy are still unclear.

Methods: To identify potential targets of nitazoxanide in mammalian cells, we developed an af-finity chromatography system using tizoxanide, a deacetyl derivative of nitazoxanide, as a ligand. Affinity chromatography was performed using VERO cell extracts on tizoxanide-biotin, and the isolated binding proteins were identified by mass spectrometry. Candidate target proteins ob-tained using affinity chromatography were co-analysed with the drug affinity response target sta-bility method. Fluorescent probes obtained by coupling rhodamine B to nitazoxanide were used for intracellular localisation of the binding targets. Solvent-induced protein precipitation profiling and thermal proteome profiling were used to further validate the binding proteins.

Results: The joint analysis of the drug affinity response target stability method and affinity chro-matography resulted in the screening of six possible candidate target proteins. Fluorescent probes localised the nitazoxanide-binding protein around the nuclear membrane. Molecular docking re-vealed that the binding proteins mainly formed hydrogen bonds with the nitro group of nitazoxa-nide. Solvent-induced protein precipitation profiling and thermal proteome profiling further vali-dated SEC61A, PSMD12, and PRKAG1 as potential target proteins of nitazoxanide.

Conclusion: The data supports the idea that nitazoxanide is a multifunctional compound with multiple targets.