Engineering yeast cytosine deaminase for improved efficacy in cancer gene directed enzyme prodrug therapy

Abstract

Conventional treatment of cancer typically involves some combination of surgery coupled with radio- or chemotherapy. Prognosis in cases of tumor metastasis or inoperable solid-tumors remains grim as cancer cells are often refractory to the DNA damage caused by these treatments due to the loss of cell cycle regulatory proteins during oncogenesis. Higher dosages of either radiation or chemotherapeutic agents can compensate, however, lack of cancer cell specificity and low therapeutic index put strict limitations on dosages that can be administered safely.Growing understanding of oncogenesis and molecular biology has lead to the development of vectors capable of targeting and transducing tumors with high specificity. One of the most successful applications of this technology has been a form of targeted chemotherapy called gene-directed enzyme/prodrug therapy (GDEPT), in which tumor cells are transduced with a suicide gene encoding a non-endogenous enzyme that later metabolically converts a systemically administered prodrug into a potent cytotoxin locally within the tumor. These enzymes are often non-ideal due to both lack of stability and low catalytic efficiency. Protein engineering methods could be used in order to tailor the pharmacokinetic profiles of these enzymes for improved therapeutic use.Our work focused on the re-engineering the enzyme yeast cytosine deaminase for improved efficacy in GDEPT. Two distinct enzyme engineering strategies were employed: regiospecific random mutagenesis was used in order to select for mutants with increased sensitization to the prodrug 5-fluorocytosine while computational design was used to select thermostabilizing mutations. Both approaches lead to independent mutations that conferred improved pharmacokinetics in in vivo mouse tumor xenograph models. Additional study revealed that in both cases the biochemical cause of improvement appeared to be thermostabilization of the enzyme and not an improvement in catalytic efficiency.