SA-β-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Area of Science:

• Gerontology and cellular aging research.
• Drug discovery and screening methodologies.

Background:

• Cellular senescence is a key aging hallmark negatively impacting healthspan.
• Senolytics, drugs targeting senescent cells, show promise in reducing senescent cell burden and extending healthspan.
• Existing senolytics include HSP90 inhibitors, Bcl-2 inhibitors, piperlongumine, FOXO4 inhibitory peptide, and Dasatinib/Quercetin.

Purpose of the Study:

• To develop and validate a high-throughput screening (HTS) method for identifying senotherapeutic drugs.
• To enable rapid screening of drug libraries for senolytic and senomorphic activity.
• To utilize senescence-associated β-galactosidase (SA-β-Gal) activity and cell number as key screening metrics.

Main Methods:

• Live cell measurement of SA-β-Gal activity using the C12FDG fluorescent substrate.
• Quantification of total cell number via Hoechst dye staining.
• High-content fluorescent image acquisition and analysis for HTS.
• Screening drug libraries for effects on SA-β-Gal activity, cell morphology, and cell number.

Main Results:

• Established a method for live cell detection of SA-β-Gal activity at increased lysosomal pH.
• Demonstrated the capability of HTS platform for rapid drug screening.
• Enabled simultaneous assessment of senolytic (cell killing) and senomorphic (phenotype suppression) drug effects.

Conclusions:

• The developed HTS method provides a robust platform for discovering novel senotherapeutic agents.
• This approach facilitates the identification of drugs that either eliminate senescent cells or modify their phenotype.
• Accelerates the search for interventions to improve healthy aging by targeting cellular senescence.