Protein engineering: The development and characterization of GCV-specific HSV -1 TK mutant enzymes

One way to improve the overall efficacy of the HSV-1 TK/GCV suicide gene therapy in vivo is to engineer a novel GCV-kinase from the existing multi-substrate HSV-1 TK by eliminating endogenous substrate competition. Using site-directed mutagenesis, we developed sixteen different HSV-1 TK mutants. Enzymatic and kinetic analysis of these HSV-1 TK mutants identified one potential GCV-kinase, Q7530 TK. When expressed in human colon cancer cell lines, Q7530 TK had minimal thymidine kinase activity but metabolized GCV as well as cells expressing wild type HSV-1 TK. Although Q7530 TK expressing cells were equally sensitive to low GCV doses (0.1 μM) when compared to wild type HSV-1 TK expressing cells, the lack in pyrimidine metabolism observed in Q7530 TK resulted in a greater accumulation of GCV-TP incorporation into the DNA, which caused an earlier, more complete S-phase cell cycle arrest. Despite the early appearance of the cell cycle arrest, the Q7530 TK expressing cells die via apoptosis at a similar rate when compared to wild type HSV-1 TK expressing cells. Two other HSV-1 TK mutants, N7530 TK and N30-3 TK were also identified as deficient in pyrimidine metabolism, but were poor GCV kinases. Human colon cancer cells expressing N7530 TK or N30-3 TK were sensitive to high GCV doses (1–10 μM) when compared to cells expressing wild type HSV-1 TK. It was also observed that N7530 TK or N30-3 TK expressing cells did not completely enter cell cycle arrest in response to DNA damaged caused by GCV-TP incorporation or die via an apoptotic pathway. Using the FlexiDock™ molecular modeling program, we analyzed the substrate-binding complex of thymidine and GCV in the active sites of Q7530 TK, N7530 TK and N30-3 TK. Two structural abnormalities caused by mutations at position Ala-168 or Gln-125 was responsible for the observed pharmacological differences between the GCV-specific HSV-1 TK mutants. When Ala-168 was mutated to a tyrosine, it effectively eliminated thymidine binding in the active site of Q7530 TK and N7530 TK without affecting GCV catalysis. When Gln-125 was mutated to an asparagine, critical hydrogen bonds important for substrate stabilization were lost, which disrupted catalysis.