Bacterial cytosine deaminase mutants created by molecular engineering show improved 5-fluorocytosine-mediated cell killing in vitro and in vivo

Abstract

Cytosine deaminase is used in combination with 5-fluorocytosine as an enzyme-prodrug combination for targeted genetic cancer treatment. This approach is limited by inefficient gene delivery and poor prodrug conversion activities. Previously, we reported individual point mutations within the substrate binding pocket of bacterial cytosine deaminase (bCD) that result in marginal improvements in the ability to sensitize cells to 5-fluorocytosine (5FC). Here, we describe an expanded random mutagenesis and selection experiment that yielded enzyme variants, which provide significant improvement in prodrug sensitization. Three of these mutants were evaluated using enzyme kinetic analyses and then assayed in three cancer cell lines for 5FC sensitization, bystander effects, and formation of 5-fluorouracil metabolites. All variants displayed 18- to 19-fold shifts in substrate preference toward 5FC, a significant reduction in IC(50) values and improved bystander effect compared with wild-type bCD. In a xenograft tumor model, the best enzyme mutant was shown to prevent tumor growth at much lower doses of 5FC than is observed when tumor cells express wild-type bCD. Crystallographic analyses of this construct show the basis for improved activity toward 5FC, and also how two different mutagenesis strategies yield closely related but mutually exclusive mutations that each result in a significant alteration of enzyme specificity.

Figure 1

Cartoon diagram of wild-type bCD monomer. The 22 residues that were subjected to randomization and selection for 5FC sensitization are in blue. The location and identity of mutations (V152A, F316C and D317G; 1525) that gave rise to highest specificity and activity toward 5FC are labeled.

Figure 2

5FC sensitivity assays of cells stably transfected with wild-type or mutant bCDs. Pools of stably transfected (A) C6, DU145 and HCT116 cells containing pCDNA, pCDNA:bCD, pCDNA:1246, pCDNA:1525 and pCDNA:1779 were evaluated for 5FC sensitivity. Pools of stably transfected HCT116 cells containing pCDNA, pCDNA:bCD, (B) pCDNA:D314G, pCDNA:D314A and pCDNA:D314S; or (C) pCDNA:1525, pCDNA:D314S and pCDNA:1525/D314S were evaluated for 5FC sensitivity. After 6 days of 5FC treatment, cell survival was determined using Alamar Blue according to the manufacturer’s instructions. Each data point (mean ± SEM; n=3; performed with 24 replicates) is expressed as a percentage of the value for control wells with no 5FC treatment. Student’s t-test analysis determined that the differences in IC₅₀ values between mutant bCD-and wild-type bCD-transfectants are statistically significant (P≤0.05).

Figure 3

Bystander analysis of stable transfectants exposed to 5FC. Pools of (A) C6, (B) DU145, (C) HCT116 cells containing pCDNA were mixed with cells harboring either pCDNA:bCD, pCDNA:1246, pCDNA:1525 or pCDNA:1779 at different ratios and were subjected to 4mM (C6) or 10mM (DU145 and HCT116) 5FC for a period of 6 days and cell survival was determined using Alamar Blue. Each data point (mean ± SEM; n=3; performed with 24 replicates) is expressed as a percentage of the value for control wells with vector-transfected cells. Student’s t-test analysis determined that the differences between percentage of mutant bCD-and wild-type bCD-transfectants needed to achieve 50% tumor cell killing are statistically significant (P≤0.05). (D) HPLC analysis of 5FU levels in transfected cells. Stably transfected HCT116 cells were incubated with 2.5mM 5FC for up to seven days and metabolite levels calculated relative to a known concentration of an internal standard. Student’s t-test was done to compare 1525 versus wild-type bCD transfected cell lysates. Asterisks denote p≤0.005 (days 3–4) and P≤0.0003 (days 5–7).

Figure 4

Tumor growth during and after 5FC treatment in a xenograft model. Pools of HCT116 cells transfected with pCDNA, pCDNA:bCD or pCDNA:1525 were used to seed tumors in nude mice (n=5). When tumor size reached 3–4mm (day 0), (A) 5FC (375mg/kg) or (B) PBS was intraperitoneally administered twice a day for 8 days. Tumor growth was measured every other day for the duration of the experiment. Tumor volume was calculated using the formula 4/3π ((width ×length ×height)/2), plotted and analyzed for statistical significance using Student’s t-test. Asterisks denote statistical significance (p≤0.05) in tumor sizes between mice harboring 1525-expressing tumor cells and those that received either empty vector- or wild-type bCD-expressing tumor cells in the presence of 375mg/kg 5FC.

Figure 5

(A)Electron density for bound substrate analogue 2-hydroxypyrimidine (or dihydroxypyrimidine, DHP) (left) and 5-fluoro-DHP (right) bound in the active site of bCD. As noted in the text, the density for the bound compound indicates disorder, particularly near the C1 carbon. However, the appearance of additional difference density corresponding to the 5-fluoro substituent (arrow) unambiguously confirms the orientation of the inhibitor. (B) Comparison of the active sites of wild-type and mutant bCD enzymes. Superposition of the active sites of wild-type bCD in complex with DHP (PDB accession code-1K70), D314A mutant in complex with 5F-DHP (PDB accession code-1RA5) versus 1525 soaked with 5F-DHP. The motion of residue D314 (arrow), into the cavity vacated by removal of a side chain in the D317G mutation, mimics the structural effect of the previously described D314A and D314G mutations.