Development of Simplified Assay for the Detection of Agmatine to Assess Arginine Decarboxylase Activity

Abstract

Arginine deprivation therapy represents a promising strategy for targeting arginine-auxotrophic cancers, exploiting the metabolic vulnerability of tumor cells deficient in argininosuccinate synthetase (ASS1) and argininosuccinate lyase (ASL). Among arginine-depleting enzymes, arginine decarboxylase (RDC) offers a unique advantage due to its pH-dependent activation in acidic tumor microenvironments. However, the relatively low enzymatic activity of RDC compared to other arginine-depleting enzymes limits its therapeutic potential. Directed evolution has shown promise in enhancing enzyme activity, but its application to RDC has been constrained by the lack of a robust, high-throughput screening (HTS) assay tailored to measure RDC activity. This study addresses this limitation by developing an innovative enzymatic cascade assay that uses diamine oxidase (DAO) and horseradish peroxidase (HRP) in a cascade reaction with TMB as the substrate. This system allows for rapid, sensitive, and high-throughput quantification of RDC activity. The assay was successfully validated with RDC wild-type (RDC-WT) and mutant (RDC-T39W) forms, demonstrating its ability to detect activity differences in both purified enzymes and cell lysates. By eliminating separation steps and streamlining the workflow, this mix-and-measure assay significantly simplifies RDC activity screening and paves the way for directed evolution to optimize RDC functionality. The establishment of this HTS-compatible assay marks a critical step toward enhancing RDC's therapeutic potential as a tumor-targeted arginine depletion enzyme. These findings contribute to the broader development of innovative enzymatic therapies for cancer treatment.