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Identification of Small-Molecule Inhibitors of Neutral Ceramidase (nCDase) via Target-Based High-Throughput Screening
Yuka Otsuka1, Michael V Airola2, Yong-Mi Choi2
1Department of Molecular Medicine, Scripps Research, The Scripps Research Molecular Screening Center, Jupiter, FL, USA.
Abstract:
There is interest in developing inhibitors of human neutral ceramidase (nCDase) because this enzyme plays a critical role in colon cancer. There are currently no potent or clinically effective inhibitors for nCDase reported to date, so we adapted a fluorescence-based enzyme activity method to a high-throughput screening format. We opted to use an assay whereby nCDase hydrolyzes the substrate RBM 14-16, and the addition of NaIO4 acts as an oxidant that releases umbelliferone, resulting in a fluorescent signal. As designed, test compounds that act as ceramidase inhibitors will prevent the hydrolysis of RBM 14-16, thereby decreasing fluorescence. This assay uses a 1536-well plate format with excitation in the blue spectrum of light energy, which could be a liability, so we incorporated a counterscreen that allows for rapid selection against fluorescence artifacts to minimize false-positive hits. The high-throughput screen of >650,000 small molecules found several lead series of hits. Multiple rounds of chemical optimization ensued with improved potency in terms of IC50 and selectivity over counterscreen assays. This study describes the first large-scale high-throughput optical screening assay for nCDase inhibitors that has resulted in leads that are now being pursued in crystal docking studies and in vitro drug metabolism and pharmacokinetics (DMPK).
Insights
Researchers developed a high-throughput screening assay to find inhibitors for human neutral ceramidase (nCDase), an enzyme crucial in colon cancer. This assay identified several promising lead compounds for further drug development.
Area of Science:
- Biochemistry and Enzymology
- Cancer Research
- Drug Discovery
Background:
- Human neutral ceramidase (nCDase) is implicated in colon cancer progression.
- A lack of potent and clinically effective nCDase inhibitors currently exists.
- Targeting nCDase presents a potential therapeutic strategy for colon cancer.
Purpose of the Study:
- To develop and validate a high-throughput screening (HTS) assay for identifying nCDase inhibitors.
- To discover novel lead compounds targeting human neutral ceramidase.
- To establish a foundation for further optimization and preclinical development of nCDase inhibitors.
Main Methods:
- Adapted a fluorescence-based enzyme activity assay using RBM 14-16 substrate and NaIO4 oxidant.
- Implemented a 1536-well plate format HTS assay with a counterscreen to mitigate fluorescence artifacts.
- Screened over 650,000 small molecules to identify potential nCDase inhibitors.
Main Results:
- Identified several lead series of compounds exhibiting inhibitory activity against nCDase.
- Optimized lead compounds demonstrated improved potency (IC50) and selectivity.
- The developed HTS assay successfully identified novel nCDase inhibitor leads.
Conclusions:
- This study reports the first large-scale HTS optical screening assay for nCDase inhibitors.
- The identified lead compounds are promising candidates for further investigation.
- Ongoing studies include crystal docking and in vitro drug metabolism and pharmacokinetics (DMPK) analysis.
