Yeast Cytosine Deaminase Mutants with Increased Thermostability Impart Sensitivity to 5-Fluorocytosine

Abstract

SUMMARYProdrug gene therapy (PGT) is a treatment strategy in which tumor cells are transfected with a 'suicide' gene that encodes a metabolic enzyme capable of converting a nontoxic prodrug into a potent cytotoxin. One of the most promising PGT enzymes is cytosine deaminase (CD), a microbial salvage enzyme that converts cytosine to uracil. CD also converts 5-fluorocytosine (5FC) to 5-fluorouracil (5FU), an inhibitor of DNA synthesis and RNA function. Over 150 studies of cytosine deaminasemediated PGT applications have been reported since 2000, all using wild-type enzymes. However, various forms of cytosine deaminase are limited by inefficient turnover of 5FC and/or limited thermostability.In a previous study we stabilized and extended the half-life of yeast cytosine deaminase (yCD) by repacking of its hydrophobic core at several positions distant from the active site. Here we report that random mutagenesis of residues selected based on alignment with similar enzymes, followed by selection for enhanced sensitization to 5FC, also produces an enzyme variant (yCD-D92E) with elevated T m values and increased activity half-life. The new mutation is located at the enzyme's dimer interface, indicating that independent mutational pathways can lead to an increase in the temperature that induces protein unfolding and aggregation in thermal denaturation experiments measured by circular dichroism spectroscopy, and an increase in the half-life of enzyme activity at physiological temperature, as well as more subtle effect on enzyme kinetics. Each independently derived set of mutations significantly improves the enzyme's performance in PGT assays both in cell culture and in animal models.

Retroviral Replicating Vectors Deliver Cytosine Deaminase Leading to Targeted 5-Fluorouracil-Mediated Cytotoxicity in Multiple Human Cancer Types

“…Parenthetically, the Kd for 5-FC of the yCD protein used in this study has been measured as 18 mg/ ml (140 mM). 34 Consequently, all the 5-FC concentrations used here are well below this Kd. In agreement with the Kd, we have shown that yields of 5-FU continue to increase with increasing 5-FC up to 5.0 mg/ml (38.7 mM) in assays performed using infected U-87 cell extracts (Supplementary Fig.…”

“…CD-based cancer gene therapy has proven to be effective in chemotherapyresistant cancer cell lines, 31,32 in combination with chemotherapy, 4 and to enhance the effect of radiotherapy. 33 To further improve the wild-type CD, we developed a codon-optimized, 7 heat-stabilized 34 yeast CD (yCD) gene, which resulted in an approximate 3-fold greater enzyme-specific activity compared with the wild-type protein when incorporated into Toca 511.…”

Gene-Directed Enzyme Prodrug Cancer Therapy

“…One of the limitations of the yCD/5FC system is the thermolabile nature of yCD at 37 C. To overcome this problem, two enzyme mutants (yCDdouble and yCDtriple) with increased half-lives and melting temperatures were identified that significantly increased the sensitivity of cancer cells to 5FC treatment compared with wild-type yCD [50,51]. Leveille et al showed the benefits of optimizing each step in a multicomponent therapy for efficient clinical outcome [52].…”

Enhancement of Anti-tumor Activity of Newcastle Disease Virus by the Synergistic Effect of Cytosine Deaminase

“…The CD/5-FC system demonstrates stronger bystander effect and the gap-junctional intercellular communications than other suicide gene system, which results in strong inhibition of tumor growth (Khatri et al, 2006;Stolworty et al, 2008;Dachs et al, 2009). However, there is no efficient method to deliver the CD gene into the tumor target cells, which hampers the application of the suicide gene therapy.…”