Abstract
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 and a known enzymatic activity present only in tumor cells. The specificity of the method relies on avoiding prodrug cleavage by enzymes already present in the host cells or the intestinal flora. Using crystallographic and computer modeling methods as guides, we have redesigned E. coli PNP to cleave new prodrug substrates more efficiently than does the wild-type enzyme. In particular, the M64V PNP mutant cleaves 9-(6-deoxy-alpha-L-talofuranosyl)-6-methylpurine with a kcat/Km over 100 times greater than for native E. coli PNP. In a xenograft tumor experiment, this compound caused regression of tumors expressing the M64V PNP gene.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Binding Sites
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Cell Line, Tumor
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Computer Simulation
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Computer-Aided Design
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Crystallography, X-Ray
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Escherichia coli / enzymology*
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Escherichia coli / genetics
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Female
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Genetic Therapy / methods*
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Humans
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Kinetics
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Mice
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Models, Molecular
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Molecular Structure
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Mutation / genetics
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Neoplasms / drug therapy
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Neoplasms / genetics
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Neoplasms / pathology
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Organ Specificity
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Prodrugs / adverse effects
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Prodrugs / metabolism*
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Prodrugs / therapeutic use*
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Protein Conformation
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Protein Engineering*
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Purine-Nucleoside Phosphorylase / chemistry*
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Purine-Nucleoside Phosphorylase / genetics
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Purine-Nucleoside Phosphorylase / metabolism*
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Substrate Specificity
Substances
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Prodrugs
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Purine-Nucleoside Phosphorylase
Associated data
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PDB/1OTX
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PDB/1OTY
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PDB/1OU4
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PDB/1OUM
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PDB/1OV6
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PDB/1OVG