Tolerance of 5-fluorodeoxyuridine resistant human thymidylate synthases to alterations in active site residues.
Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in cancer therapy. In the cell, 5-FU is metabolized to 5-fluorodeoxyuridylate (5-FdUMP), a tight binding covalent inhibitor of thymidylate synthase (TS). In order to create 5-FdUMP resistant enzymes to protect chemosensitive normal cells and further understand mechanisms of 5-FdUMP resistance, we have randomized four residues within the active site of TS. Our previous studies identified alterations in residues which produce active TS with enhanced resistance to 5-fluorouridine (5-FdUR). By remutagenizing a subset of the 13 previously targeted residues (A197, L198, C199 and V204), an unbiased random library can be created allowing for extensive testing of all possible amino acid substitutions at each of the sites. Using genetic complementation and selection in Escherichia coli, we identified the spectrum of substitutions that yield active TS as well as those that resulted in 5-FdUR resistant mutants of TS. The 5-FdUR resistant TS were found to share several structural features including hydrophobic substitutions at residue 197, retention of the wild-type leucine 198, the alteration C199L (present in 64% of the drug-resistant library), and polar alterations of valine 204. The catalytic activity of mutants with these features was approximately equal to that of the wild-type TS.
Landis
D M
DM
The Joseph Gottstein Memorial Cancer Research Laboratory, Departments of Pathology and Biochemistry, University of Washington School of Medicine, Box 357705, Seattle, WA 98195-7705, USA.
Gerlach
J L
JL
Adman
E T
ET
Loeb
L A
LA
D019943:Amino Acid Substitution; D000964:Antimetabolites, Antineoplastic / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D005467:Floxuridine / Q000378:metabolism / Q000494:pharmacology*; D015723:Gene Library; D005816:Genetic Complementation Test; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D016008:Statistical Distributions; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D000964:Antimetabolites, Antineoplastic; D005467:Floxuridine; D013940:Thymidylate Synthase