Cholesterol Unbound RORγt Protein Enables a Sensitive Inverse Agonist Screening

Area of Science:

• Molecular Biology
• Pharmacology
• Immunology

Background:

• Retinoic acid-related orphan receptor gamma T (RORγt) is crucial for Th17 cell function and implicated in autoimmune diseases.
• RORγt activity is physiologically modulated by cholesterol derivatives, suggesting a therapeutic target.
• RORγt inverse agonists are potential treatments for Th17-mediated autoimmune conditions like rheumatoid arthritis, asthma, IBD, and psoriasis.

Purpose of the Study:

• To identify novel RORγt inverse agonists through a high-throughput screening campaign.
• To evaluate the suitability of apo-RORγt protein for sensitive screening.
• To characterize the mechanism of action of newly identified inverse agonists.

Main Methods:

• Comparative analysis of apo-RORγt and cholesterol-bound RORγt using known inverse agonist TO901317.
• High-throughput screening using a fluorescence-based cholesterol binding assay with apo-RORγt.
• In vitro assays to assess compound 1's inhibition of RORγt-cholesterol interaction, coactivator recruitment, and transcriptional activity.
• Cell-based reporter gene assays, including lipid depletion treatment.

Main Results:

• The known RORγt inverse agonist TO901317 exhibited higher potency against apo-RORγt compared to cholesterol-bound RORγt.
• High-throughput screening identified compound 1 as a novel cholesterol-competitive RORγt inverse agonist.
• Compound 1 effectively inhibited RORγt-cholesterol binding, coactivator recruitment, and transcriptional activity.
• Cell-based assays confirmed compound 1's enhanced potency following lipid depletion.

Conclusions:

• Apo-RORγt protein is a suitable and sensitive target for high-throughput screening of RORγt inverse agonists.
• Compound 1 represents a promising novel RORγt inverse agonist with potential therapeutic applications in autoimmune diseases.
• The findings support a strategy of targeting cholesterol-regulated RORγt for drug discovery.