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<p>Utilization of yeast cytosine deaminase (yCD) to activate the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) at the target site is an attractive strategy for overcoming the narrow therapeutic index of 5-FU. Nevertheless, protein delivery of yCD is challenging in part due to its thermal instability. Herein, we have rationally engineered a mutant yCD by replacing Met100 situated at the active site entry with the bulkier histidine to hinder potential oxidation of the active site Cys91. The engineered yCD-Met100His exhibits significantly enhanced activity and thermal stability. yCD-M100H is then genetically fused to the crystal-forming protein Cry3Aa to generate Cry3Aa-yCD-M100H fusion crystals to facilitate the enzyme’s uptake into macrophages. The resulting Cry3Aa-yCD-M100H-loaded macrophages exhibit excellent penetration into tumor spheroids and readily convert 5-FC to 5-FU leading to efficacious cancer cell killing. This study showcases a promising route for stabilizing yCD and the feasibility of enzyme-internalized macrophages to serve as tumor-specific enzyme/prodrug activators.</p><p></p>

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An engineered yeast cytosine deaminase with improved catalytic activity and stability for macrophage-mediated enzyme/prodrug therapy

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月13日

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  • Jiale Zheng,
  • Jiahao Zhou,
  • Kristen Wing Yu Yung,
  • Qipeng Hu,
  • Marianne M. Lee,
  • Michael K. Chan

摘要

Utilization of yeast cytosine deaminase (yCD) to activate the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) at the target site is an attractive strategy for overcoming the narrow therapeutic index of 5-FU. Nevertheless, protein delivery of yCD is challenging in part due to its thermal instability. Herein, we have rationally engineered a mutant yCD by replacing Met100 situated at the active site entry with the bulkier histidine to hinder potential oxidation of the active site Cys91. The engineered yCD-Met100His exhibits significantly enhanced activity and thermal stability. yCD-M100H is then genetically fused to the crystal-forming protein Cry3Aa to generate Cry3Aa-yCD-M100H fusion crystals to facilitate the enzyme’s uptake into macrophages. The resulting Cry3Aa-yCD-M100H-loaded macrophages exhibit excellent penetration into tumor spheroids and readily convert 5-FC to 5-FU leading to efficacious cancer cell killing. This study showcases a promising route for stabilizing yCD and the feasibility of enzyme-internalized macrophages to serve as tumor-specific enzyme/prodrug activators.