A direct fluorescence assay for quantitative analysis of ornithine decarboxylase activity and inhibitor screening

Ornithine decarboxylase (ODC) catalyzes the first committed step in polyamine biosynthesis and plays a central role in cellular growth and proliferation. Quantitative analysis of ODC activity has traditionally relied on radiometric or coupled-enzyme assays, which limit scalability and accessibility. Here, we report on the development of 1,2-diacetylbenzene (DAB)-ODC, a fluorescence-based assay that enables direct, sensitive, and high-throughput quantification of ODC activity by detecting putrescine through its reaction with DAB. Using purified recombinant ODC from Saccharomyces cerevisiae and humans, we show that DAB-ODC supports measurement of enzyme activity and accurate determination of steady-state kinetic parameters. Inhibition studies with the inhibitor DL-α-difluoromethylornithine and three recently reported DL-α-difluoromethylornithine analogs yielded IC₅₀ values consistent with those obtained using established orthogonal assays. All enzymatic measurements were validated by thin-layer chromatography. Assay performance metrics, including CV, Z' factor, and signal-to-background ratios, demonstrate compatibility with large-scale chemical screening. Together, these results establish DAB-ODC as a versatile platform for ODC enzymology, inhibitor profiling, and high-throughput interrogation of polyamine metabolism.