A Flow Cytometry-Based High-Throughput Technique for Screening Integrin-Inhibitory Drugs.

Area of Science:

• Immunology
• Cell Biology
• Pharmacology

Background:

• β2 integrins are critical leukocyte adhesion molecules involved in immune responses and inflammatory diseases.
• Neutrophil β2 integrin activation is essential for their extravasation and plays a role in infection and inflammation.
• Targeting β2 integrin activation offers a therapeutic strategy for neutrophil-associated inflammatory conditions.

Purpose of the Study:

• To develop and validate a high-throughput screening (HTS) method for identifying small molecular antagonists of β2 integrin activation.
• To utilize conformational-change-reporting antibodies and flow cytometry for quantifying β2 integrin activation.
• To establish a protocol applicable to other antibody-based HTS assays.

Main Methods:

• Utilized a monoclonal antibody (mAb24) that recognizes the high-affinity state of β2 integrins.
• Employed high-throughput flow cytometry with automated 384-well plate processing.
• Assessed the inhibitory effects of 320 chemicals on fMLP-stimulated β2 integrin activation in primary human neutrophils within 3 hours.

Main Results:

• Successfully established a protocol for quantifying β2 integrin activation on neutrophils.
• Demonstrated the feasibility of screening a library of 320 chemicals for β2 integrin inhibitors.
• Identified potential small molecules targeting β2 integrins or their upstream signaling pathways.

Conclusions:

• The developed HTS method is effective for identifying antagonists of β2 integrin activation.
• This protocol provides a valuable tool for drug discovery targeting neutrophil-mediated inflammatory diseases.
• The methodology can be adapted for screening other cell surface receptor activation states.