High-throughput discovery of novel small-molecule inhibitors of acid Ceramidase

Area of Science:

• Biochemistry and Molecular Biology
• Drug Discovery and Development
• Cancer Research

Background:

• Ceramide metabolism is critical in regulating cellular senescence and apoptosis.
• Acid ceramidase (AC) lowers ceramide levels and is abnormally expressed in various cancers.
• Targeting AC with inhibitors is a promising strategy for cancer treatment, but hindered by screening limitations.

Purpose of the Study:

• To develop a miniaturized, high-throughput screening assay for acid ceramidase (AC) inhibitors.
• To identify novel AC inhibitors with potential therapeutic applications in cancer.

Main Methods:

• Adapted a fluorogenic assay to a 384-well plate format for miniaturized screening.
• Screened a library of 4100 diverse chemical compounds using the optimized assay.
• Evaluated inhibitor potency, selectivity against neutral ceramidase, and cellular activity.

Main Results:

• The miniaturized assay demonstrated suitability for large-scale screening.
• Identified several potent AC inhibitors (2–25 µM) from different chemical classes.
• Selected inhibitors exhibited selectivity over neutral ceramidase and retained activity in cellular models.

Conclusions:

• The developed 384-well plate assay is effective for identifying AC inhibitors.
• The identified hit compounds serve as a valuable starting point for further chemical optimization.
• This work facilitates the discovery of novel therapeutics targeting AC in cancer.