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

TL;DR

Engineered yeast cytosine deaminase improves cancer treatment by activating a prodrug within macrophages, enhancing drug delivery and tumor targeting.

Contribution

A rationally engineered yeast cytosine deaminase with improved stability and catalytic activity for macrophage-mediated prodrug therapy.

Findings

yCD-Met100His exhibits significantly enhanced activity and thermal stability.

Cry3Aa-yCD-M100H-loaded macrophages effectively penetrate tumor spheroids and convert 5-FC to 5-FU.

Engineered yCD demonstrates potential as a tumor-specific enzyme/prodrug activator.

Abstract

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.

An engineered yCD with enhanced oxidation resistance and intracellular stability encapsulated in macrophages showed excellent spheroid penetration and cancer cell killing, demonstrating its use as tumor-specific enzyme/prodrug activator.

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