In-cell click labelling of small molecules to determine subcellular localisation

Area of Science:

• Medicinal Chemistry
• Virology
• Molecular Biology

Background:

• Hepatitis C virus (HCV) infection is a major global health concern.
• The NS5A protein is a key target for antiviral therapies.
• Understanding drug distribution is crucial for developing effective treatments.

Purpose of the Study:

• To develop and validate small molecule probes for studying the subcellular distribution of the HCV NS5A protein.
• To investigate the antiviral mode of action of a specific chemotype targeting NS5A.

Main Methods:

• Development of small molecule fluorometric boron dipyrromethene probes.
• Utilizing 'silent' azide reporters for improved localization studies.
• Application of click chemistry to append fluorophores and confirm probe-target interaction.
• Co-localization studies with NS5A in replicon cells.

Main Results:

• Initial probes did not co-localize with NS5A, hindering distribution studies.
• Azide-tagged probes, when functionalized via click chemistry, successfully co-localized with NS5A.
• This confirmed the intended binding and distribution of the small molecules.

Conclusions:

• Click chemistry is a valuable tool for validating the localization of small molecule probes.
• The validated probes provide insights into the antiviral mechanism of action against HCV NS5A.
• This approach aids in the rational design of future antiviral agents.