Enhanced Therapeutic Potential of Arginine Decarboxylase through the Introduction of Aromatic Amino Acids and Human Serum Albumin

Abstract

The formidable challenges posed by cancer's high mortality and invasiveness necessitate innovative therapeutic approaches. Targeting nutrient dependence within cancer cells has emerged as a promising strategy. This study focuses on demonstrating the potential of arginine depletion for cancer therapy, with a particular emphasis on arginine decarboxylase (RDC), a pH-dependent enzyme expected to exhibit enhanced activity within the weakly acidic microenvironment of tumors. The study successfully increased the therapeutic potential of RDC by introducing aromatic amino acids to the multimer-forming surface to improve pH-dependent activity and human serum albumin (HSA) to extend its half-life. Aromatic amino acid substitutions were explored at the multimer-forming interface, specifically at position 39. Phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), frTet (a phenylalanine analog), and p-azido-phenylalanine (AzF) were introduced at position 39, with alanine (Ala) and glycine (Gly) as controls. The RDC variants were successfully expressed, purified, and characterized. Enzyme activity assays demonstrated a significant increase at pH 6.4, crucial for tumor-specific therapy, with aromatic substitutions showing higher agmatine production than wild-type RDC. Cell assays using HeLa cells validated the antitumor efficacy of the engineered RDC variants: the selected variant RDC-W showed noticeable growth inhibition at low concentrations, consistent with increased enzymatic activity observed in vitro. To address the short serum half-life of therapeutic proteins such as RDCs, a novel strategy was proposed to site-specifically conjugate human serum albumin (HSA) to genetically engineered RDCs. Computational analysis guided the selection of optimal sites to introduce the fast-reacting tetrazine (Tz) nonnatural amino acid. RDC-W-K522Tz emerged as the optimal variant, and the successful synthesis of RDC-W-HSA was confirmed by size exclusion chromatography (SEC), demonstrating site-specific conjugation without compromising enzyme activity. This two-pronged strategy offers a promising approach to addressing the short serum half-life in clinical applications and enhancing the therapeutic potential of RDC variants. Future studies will investigate the in vitro and in vivo antitumor effects of RDC-W-HSA to solidify the potential impact of this comprehensive approach.