Coupled-enzyme assay for MTAP activity in biological samples.

Area of Science:

• Biochemistry
• Enzymology
• Cancer Biology

Background:

• 5'-Methylthioadenosine phosphorylase (MTAP) is crucial for salvaging products of polyamine synthesis.
• Homozygous MTAP deletion in ~15% of human cancers creates a synthetic lethal vulnerability.
• Targeting MTAP offers synthetic lethal strategies for MTAP-proficient cancers.

Purpose of the Study:

• To develop a robust, continuous assay for quantifying MTAP activity in biological samples.
• To provide a reliable method for evaluating MTAP inhibitors and drug response.
• To enable real-time monitoring of MTAP function in cancer research and clinical settings.

Main Methods:

• A continuous assay was developed by coupling adenine deaminase, xanthine oxidase, and horseradish peroxidase.
• The assay was validated using whole blood and analyzed with transition-state analogs (MTDIA, pClPhTDIA).
• MTAP activity was measured in cancer cell lines to assess intracellular levels and complement whole-blood data.

Main Results:

• A continuous, coupled enzymatic assay for MTAP activity was successfully established.
• The assay demonstrated reliability in whole blood and cancer cell line lysates.
• Kinetic and inhibition constants were determined, validating the assay's performance.

Conclusions:

• The developed assay provides a sensitive and efficient platform for measuring MTAP activity.
• This method facilitates the study of MTAP inhibitors and the development of synthetic lethal cancer therapies.
• It enables direct biological or clinical tracking of targeted MTAP therapies in small sample volumes.