W0150

Designer Gene Therapy Using an Escherichia coli Purine Nucleoside Phosphorylase/Prodrug System. Ruchi Anand, Eric Bennett, Steven Ealick, Paula Allan, Abdalla Hassan, William Parker, John Secrist III, Eric Sorscher, Chemistry and Chemical Biology, Cornell Univ., 377 St. Olin Lab, Ithaca, NY 14853 USA.

Activation of prodrugs by Escherichia coli purine nucleoside phosphorylase (PNP) provides a method for selectively killing tumor cells expressing a transfected PNP gene. This gene therapy approach requires matching a prodrug with a known enzymatic activity present only in tumor cells. Otherwise, the prodrug may be activated by enzymes already present in the cell or the intestinal flora. Using crystallographic and computer modeling methods as guides, we have redesigned E. coli PNP to cleave modified prodrug substrates more efficiently than the wild-type enzyme. In particular, the M64V PNP mutant cleaves 9-(5'-methyl[talo]-β-D-ribofuranosyl)-6-methylpurine riboside 1,000-fold more efficiently than does wild-type E. coli PNP.

Computational studies using the AMBER force fied were done and MeP-dR 5' allo and talo isomers were docked to both wild type and M64V mutant PNP. The results show low-energy conformations with catalytically favorable geometries for enzyme/ligand combinations which show high activity in kinetic experiments. In particular, the 5' methyl group of the talo isomer fills the hole created by mutating M64 to Val while still maintaining acceptable geometry for catalysis. Crystallographic structures of the same enzyme-ligand combinations support the docking results. Strong ligand density is generally observed for enzyme/ligand combinations with high activity in kinetic experiments.