A Nonradioactive Fluorimetric SPE-Based Ceramide Kinase Assay Using NBD-C(6)-Ceramide

Area of Science:

• Biochemistry
• Enzymology
• Cellular Biology

Background:

• Ceramide kinase (CERK) plays a crucial role in cellular processes including inflammation and apoptosis.
• Traditional CERK activity assays rely on radioactive labeling, posing safety and disposal challenges.

Purpose of the Study:

• To develop a non-radioactive, fluorescence-based assay for measuring ceramide kinase activity.
• To establish a sensitive and high-throughput screening (HTS) compatible method for CERK activity assessment.

Main Methods:

• A fluorescence-based assay was developed using N-[7-(4-nitrobenz-2-oxa-1,3-diazole)]-6-aminohexanoyl-sphingenine (NBD-C(6)-ceramide) as a substrate.
• The fluorescent product, NBD-C(6)-ceramide-1-phosphate, was quantified using solid-phase extraction on an aminopropyl phase.
• The assay was adapted for a 96-well format to facilitate high-throughput screening.

Main Results:

• The developed assay demonstrated a comparable K(m) value to existing methods using N-hexanoyl-sphingenine (C(6)-ceramide).
• The assay provides a fast and sensitive measurement of CERK activity.
• A limited screen identified protein kinase inhibitors (e.g., U-0126) and ceramide analogues (e.g., fenretinide, AMG-9810) as modulators of CERK activity in vitro.

Conclusions:

• A novel, non-radioactive fluorescence-based assay offers a sensitive and efficient method for CERK activity measurement.
• This assay is suitable for high-throughput screening to discover novel CERK modulators.
• The findings highlight the potential of specific kinase inhibitors and ceramide analogues in modulating CERK.