A rapid and affordable screening platform for membrane protein trafficking

Area of Science:

• Membrane protein research
• Drug discovery and development
• Biochemical screening technologies

Background:

• Membrane proteins are crucial drug targets, but screening is resource-intensive.
• Limited chemical space exploration and screening resources hinder drug discovery.
• A universally applicable and cost-effective screen for membrane protein trafficking was developed.

Purpose of the Study:

• To develop a high-throughput screening (HTS) platform for membrane protein trafficking.
• To enable low-cost, rapid, and quantitative assessment of membrane protein cell surface expression.
• To facilitate drug discovery and repurposing for membrane protein targets, particularly Lgr5.

Main Methods:

• Utilized the MarsCy1-fluorogen activating protein and near-infrared fluorogen SCi1 for detection.
• Developed the IRFAP-HTS assay for cell surface expression visualization on a standard infrared western-blotting scanner.
• Screened 11,258 FDA-approved and drug-like small molecules for modulators of Lgr5 internalization.

Main Results:

• Validated IRFAP-HTS by confirming trafficking of vasopressin-2 and dopamine receptor-2.
• Identified glucocorticoids as potent modulators that increase Lgr5 expression at the plasma membrane.
• Achieved screening of 5,000 compounds per hour with robust and quantitative results.

Conclusions:

• IRFAP-HTS offers a versatile, user-friendly, and cost-effective solution for membrane protein screening.
• The platform democratizes access to advanced screening for smaller laboratories by utilizing standard equipment.
• IRFAP-HTS accelerates drug discovery and repurposing, exemplified by the identification of Lgr5 modulators.