High-throughput analysis of CLIC5 interactants using a thermal-stability assay

Area of Science:

• Biochemistry
• Molecular Biology
• Ion Channel Research

Background:

• Chloride intracellular channel (CLIC) proteins are unique metamorphic proteins with roles in various physiological and pathological processes.
• CLIC5, a member of this family, is implicated in hereditary deafness and cancer, but its natural interactants remain unknown.

Purpose of the Study:

• To identify ligands that modulate the biochemical properties and activity of CLIC5.
• To explore potential therapeutic avenues for CLIC5-associated conditions by discovering its interactants.

Main Methods:

• Overexpression and purification of CLIC5 in Escherichia coli.
• Establishment of a high-throughput differential scanning fluorimetry thermal shift assay (TSA).
• Screening of approximately 500 natural compounds for CLIC5 interaction using TSA.

Main Results:

• A TSA-based screening method was developed, enabling parallel evaluation of ~100 compounds per hour.
• Eleven potential CLIC5-interacting compounds were identified based on thermal stability.
• Curcumin was identified as a specific CLIC5 ligand through dose-dependence analysis.

Conclusions:

• The developed TSA approach efficiently screens small molecules for novel CLIC5 interactants.
• Identifying CLIC5 ligands can provide tools to modulate its activity.
• Modulating CLIC5 activity may offer therapeutic strategies for CLIC5-related pathologies.