Abstract
Two double (F31A/F34A, I60A/L67G) and one quadruple (F31A/F34A/I60A/L67G) mutant murine dihydrofolate reductases were constructed and evaluated for their ability to impart antifolate resistance. Both I60A/L67G and F31A/F34A/I60A/L67G were found to be unstable and devoid of catalytic activity. The K(i) values for F31A/F34A, methotrexate (MTX), bis-MTX, and PT-523 were found to be 10100-, 4410-, and 617-fold higher than the wild-type enzyme, respectively, but only 13.5-fold higher for trimetrexate (TMTX). These findings suggest that F31A/F34A could be used for gene therapy to render normal cells resistant to MTX but sensitive to TMTX.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Antineoplastic Agents / chemical synthesis*
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Antineoplastic Agents / chemistry
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Drug Resistance, Neoplasm
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Folic Acid Antagonists / chemical synthesis*
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Folic Acid Antagonists / chemistry
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Kinetics
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Methotrexate / chemistry*
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Mutation
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Tetrahydrofolate Dehydrogenase / chemistry*
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Tetrahydrofolate Dehydrogenase / genetics*
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Trimetrexate / chemistry*
Substances
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Antineoplastic Agents
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Folic Acid Antagonists
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Recombinant Proteins
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Tetrahydrofolate Dehydrogenase
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Trimetrexate
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Methotrexate