Probing the folate-binding site of human thymidylate synthase by site-directed mutagenesis: Generation of mutants that confer resistance to raltitrexed, Thymitaq, and BW1843U89 Journal Article
| Authors: | Tong, Y.; Liu-Chen, X.; Ercikan-Abali, E. A.; Zhao, S. C.; Banerjee, D.; Maley, F.; Bertino, J. R. |
| Article Title: | Probing the folate-binding site of human thymidylate synthase by site-directed mutagenesis: Generation of mutants that confer resistance to raltitrexed, Thymitaq, and BW1843U89 |
| Abstract: | Human thymidylate synthase (TS) contains three highly conserved residues Ile-108, Leu-221, and Phe-225 that have been suggested to be important for cofactor and antifolate binding. To elucidate the role of these residues and generate drug-resistant human TS mutants, 14 variants with multiple substitutions of these three hydrophobic residues were created by site- directed mutagenesis and transfected into mouse TS-negative cells for complementation assays and cytotoxicity studies, and the mutant proteins expressed and characterized. The I108A mutant confers resistance to raltitrexed and Thymitaq with respective IC50 values 54- and 80-fold greater than wild-type but less resistance to BW1843U89 (6-fold). The F225W mutant displays resistance to BW1843U89 (17-fold increase in IC50 values), but no resistance to raltitrexed and Thymitaq. It also confers 8-fold resistance to fluorodeoxyuridine. Both the kinetic characterization of the altered enzymes and formation of antifolate-resistant colonies in mouse bone marrow cells that express mutant TS are in accord with the IC50 values for cytotoxicity noted above. The human TS mutants (I108A and F225W), by virtue of their desirable properties, including good catalytic function and resistance to antifolate TS inhibitors, confirm the importance of amino acid residues Ile-108 and Phe-225 in the binding of folate and its analogues. These novel mutants may be useful for gene transfer experiments to protect hematopoietic progenitor cells from the toxic effects of these drugs. |
| Keywords: | cancer chemotherapy; controlled study; human cell; nonhuman; antineoplastic agent; mutant protein; animal cell; mouse; animals; mice; bone marrow cells; antimetabolites, antineoplastic; drug resistance; gene transfer; animalia; kinetics; recombinant proteins; stem cells; models, molecular; isoleucine; mutagenesis, site-directed; binding sites; folic acid; bone marrow cell; hematopoietic stem cell; drug cytotoxicity; catalysis; enzyme kinetics; indoles; macrophages; leucine; folic acid antagonist; thymidylate synthase; drug binding site; folic acid antagonists; quinazolines; floxuridine; site directed mutagenesis; phenylalanine; colony forming unit gm; thiophenes; raltitrexed; hydrophobicity; nolatrexed; granulocytes; humans; human; male; priority journal; article; metesind glucuronate; 2 [5 [[(1,2 dihydro 3 methyl 1 oxobenzo[f]quinazolin 9 yl)methyl]amino] 1 oxo 2 isoindolinyl]glutaric acid |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 273 |
| Issue: | 47 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 1998-11-20 |
| Start Page: | 31209 |
| End Page: | 31214 |
| Language: | English |
| DOI: | 10.1074/jbc.273.47.31209 |
| PUBMED: | 9813027 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Article -- Export Date: 12 December 2016 -- Source: Scopus |
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