The pyrimidine salvage pathway enzyme cytosine deaminase (CD; EC 126.96.36.199) is responsible for converting the nucleobase cytosine to uracil and ammonia. This activity is found primarily in microbes1
, including Saccharomyces cerevisiae
and Escherichia coli
, and has arisen at least twice, using completely separate protein folds2,3
. In addition to cytosine, cytosine deaminase catalyzes the conversion of 5-fluorocytosine (5FC) to the potent chemotherapeutic drug, 5-fluorouracil (5FU). Thus, the combination of CD enzyme activity and 5FC as its substrate forms the basis of a potential anti-tumor gene therapy, where CD plays the role of a 'suicide gene'4
In suicide gene therapy applications, the gene for cytosine deaminase is introduced into cancer cells, followed by systemic administrations of the prodrug 5FC. Following deamination by CD, 5FU is converted by cellular enzymes to 5FdUMP, an irreversible inhibitor of thymidylate synthase (TS). Inhibition of TS blocks dTTP production and prevents DNA synthesis5–7
. The CD/5FC system has been used in numerous animal models8–10
and is currently being evaluated in clinical trials for solid tumors11–15
. One limitation to this approach is the poor transfection efficiency of current vector delivery systems. Consequently, high 5FC doses must be administered to achieve therapeutic value and are associated with unwanted side effects suggested to be a result of the generation of 5FU by intestinal bacteria16
Key to suicide gene therapy is the phenomenon known as the bystander effect, in which non-transfected neighboring cells are killed through the transfer of antimetabolites from CD expressing cells in close proximity. A strong bystander effect has been associated with CD and 5FC because 5FU is a small, uncharged molecule capable of non-facilitated diffusion through cellular membranes17–21
. Unlike other suicide gene therapy systems such as herpes simplex virus thymidine kinase (HSVTK) and ganciclovir (GCV) that rely on transfer of metabolites through gap junctions, the CD/5FC bystander effect is not dependent upon cell-to-cell contact17
Another advantage of the CD/5FC combination is that 5FU has radiosensitizing properties22–23
. Since it is unlikely that treatment with gene therapy would be the only course of action in patients, radiosensitizing effects can augment treatment regimens. Several groups have reported in vivo
results with CD to have a significant bystander effect22
at clinically relevant 5FC doses and radiation regimens22,25–26
Two completely separate forms of CD have evolved in nature, and both are being studied in anti-tumor gene therapy investigations. Yeast CD (yCD) belongs to the amidohydrolase protein fold family (CATH topology class 3.40.140) and shares homology with bacterial and eukaryotic cytidine deaminases3
. The enzyme is assembled into a homodimer comprised of 17.5 kDa subunits that contain a catalytic zinc ion. In contrast, bacterial CD (bCD) belongs to the alpha-beta ‘TIM’ barrel fold family (CATH topology class 2.30.40) and most closely resembles human adenosine deaminase2
. In E. coli
, the enzyme is assembled into a hexamer of 60 kDa subunits that contain a catalytic iron.
Although product release from the yeast cytosine deaminase is rate-limiting27
, yCD has been observed to display superior kinetic properties towards 5FC (corresponding to a 22-fold lower Km
for the prodrug) and slightly improved efficacy for treating tumors in mice than bCD28
. However, wild-type yCD is relatively thermolabile as compared to bCD28–29
, a property that may limit its performance in therapeutic applications.
Using computational protein engineering, we previously created a series of mutant yCD variants that display elevated unfolding temperatures in denaturation experiments and increased half-lives of catalytic activity at elevated temperatures30
. An enzyme mutant with two substitutions (A23L/I1140L or 'yCD-double') and a subsequent, final redesigned mutant (A23L/V108I/I140L or 'yCD-triple') display wild-type catalytic efficiencies, and 5- and 30-fold increases in the half-lives of those catalytic activities. Furthermore, yCD-double and yCD-triple display apparent melting temperatures 6°C and 10°C greater, respectively, than wild-type yCD. The latter construct also displayed improved complementation of cytosine deaminase activity at elevated temperatures in bacteria.
In the present study, we have randomly mutagenized residues that were selected based on sequence conservation with similar enzymes, and have used positive and negative genetic complementation strategies to identity mutations that confer increased 5FC sensitivity (i.e., that lead to toxicity at the lowest possible concentrations of 5FC). The resulting mutations were analyzed alone and in combination with those previously engineered in the protein core for their effect on (1) prodrug sensitivity in mammalian cells and in a mouse xenograft tumor model, (2) substrate specificity, (3) kinetic efficiency, and (4) thermal unfolding of the enzyme and the half-life of catalytic activity. The crystal structure of the mutant from the screen that induced the most significant increase in 5FC sensitivity was determined. The computationally engineered yCD-triple and the genetically selected yCD-D92E variant each separately provide enhanced 5FC-sensitivity to cells and therefore may serve as improved candidates for future suicide gene therapy studies. In contrast, the combination of these mutations abrogates their individual contribution to improved performance in PGT assays, possibly due to the accumulation of individual small reductions in catalytic efficiencies that reach a critical threshold for sensitization to 5- FC.