PROBING THE FOLATE-BINDING SITE OF HUMAN THYMIDYLATE SYNTHASE BY SITE-DIRECTED MUTAGENESIS - GENERATION OF MUTANTS THAT CONFER RESISTANCE TO RALTITREXED, THYMITAQ, AND BW1843U89

Human thymidylate synthase (TS) contains three highly conserved residu es Ile-108, Leu-221, and Phe-225 that have been suggested to be import ant for cofactor and antifolate binding. To elucidate the role of thes e residues and generate drug-resistant human TS mutants, 14 variants w ith multiple substitutions of these three hydrophobic residues were cr eated by site-directed mutagenesis and transfected into mouse TS-negat ive 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 i ncrease in IC50 values), but no resistance to raltitrexed and Thymitaq , It also confers 8-fold resistance to fluorodeoxyuridine. Both the ki netic characterization of the altered enzymes and formation of antifol ate-resistant colonies in mouse bone marrow cells that express mutant TS are in accord with the IC50 values for cytotoxicity noted above. Th e human TS mutants (I108A and F225W), by virtue of their desirable pro perties, including good catalytic function and resistance to antifolat e TS inhibitors, confirm the importance of amino acid residues Ile-108 and Phe-225 in the binding of folate and its analogues, These novel m utants may be useful for gene transfer experiments to protect hematopo ietic progenitor cells from the toxic effects of these drugs.