Coupled-enzyme assay for MTAP activity in biological samples

5'-Methylthioadenosine phosphorylase (MTAP) catalyzes the reversible phosphorolysis of 5'-methylthioadenosine (MTA), a product of polyamine synthesis. The resulting products, adenine and 5-methylthio-α-d-ribose 1-phosphate (MTR-1-P), are salvaged to the adenylate and methionine pathways. Homozygous deletion of MTAP occurs in approximately 15% of all human cancers due to its chromosomal proximity to the CDKN2A/B locus. Loss of MTAP creates a metabolic vulnerability to PRMT5 and MAT2A inhibition, resulting in a synthetic lethal interaction. Consequently, MTAP has emerged as a target for enzymatic inhibition to enable synthetic lethal strategies in the 85% of cancers that are MTAP^+/+. Analysis of MTAP inhibitors in biological samples necessitates a robust assay for MTAP activity. Previous assays for MTAP activity have relied on radiolabeled substrates and chromatography, methods that are cumbersome for routine testing. We report a continuous assay suitable for biological samples that quantifies MTAP activity by coupling adenine deaminase, xanthine oxidase, and horseradish peroxidase. The method is validated using whole blood and by analyzing kinetic and inhibition constants with transition-state analogs, methylthio-DADMe-Immucillin-A (MTDIA) and para-chloro-phenylthio-DADMe-Immucillin-A (pClPhTDIA). MTAP activity was also measured across a panel of cancer cell lines to determine intracellular MTAP levels and to complement whole-blood measurements. This approach enables real-time evaluation of MTAP function and drug response in μL volumes of lysate, providing a direct biological or clinical platform for tracking targeted MTAP therapies.