Probing the Folate-binding Site of Human Thymidylate Synthase by Site-directed Mutagenesis

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 IC 50 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 IC 50 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 IC 50 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.Thymidylate synthase (TS), 1 which catalyzes the conversion of dUMP to dTMP, is an attractive target for drug design (1-5). TS inhibitors, which occupy either the substrate or cofactorbinding site, have been designed based on the structure and properties of the enzyme. Fluoropyrimidines, such as 5-fluorouracil (5-FU) and fluorodeoxyuridine (FdUrd), are metabolized to 5-fluoro-2-deoxyuridine monophosphate (FdUMP) and compete subsequently with the substrate, dUMP, for its binding site and have been used in the clinic for over 40 years to treat breast and gastrointestinal cancers. However, fluoropyrimidines, due to their incorporation into DNA and RNA, are not pure TS inhibitors. Also, they are susceptible to metabolic degradation in vivo. In contrast, the cofactor CH 2 H 4 folate is a relatively large molecule and has a variety of binding sites that may be altered in drug design. In recent years folate analogues have been designed as highly specific and stable TS inhibitors (6). The inhibitor CB3717 was the first folate analogue inhibitor of TS tested in the clinic and although anti-tumor activity was demonstrated, its further development was abandoned due to renal and hepatic toxicity (7,8). The information provided by the crystal structures of TS from bacterial and mammalian sources (9 -18) has led to the design and synthesis of novel analogues of CH 2 H 4 folate, e.g. raltitrexed (Tomudex ® , ZD1694), BW1843U89, Thymitaq (AG337), and AG331 (19 -27). These new and promising agents have entered clinical trials...

Resistance to antifolates

“…For instance, Ile108Ala produced marked resistance to ZD1694 and AG337, with lesser resistance to GW1843U89. A Phe225Trp mutation produced 17-fold resistance to the latter compound and about half the level of resistance to fluorouracil, without any increase in the IC 50 , or the kinetics of enzyme inhibition by ZD1694 and AG337 (Tong et al, 1998b). As observed for DHFR, mutations in TS that arise during selection can be accompanied by amplification of the enzyme, probably required for sufficient catalytic activity for the natural substrate to sustain cell replication (Tong et al, 1998a).…”

Protection of hematopoietic stem cells from pemetrexed toxicity by retroviral gene transfer with a mutant dihydrofolate reductase-mutant thymidylate synthase fusion gene

“…5 Our laboratory has previously generated a variety of drug resistance genes, and combinations thereof, conferring resistance to antimetabolites such as MTX, trimetrexate, 5FU, raltitrexed, thymitaq, and cytosine arabinoside. 14,[20][21][22][23][24] In this study, we show that the transfer of a DHFR F/S-TS G52S fusion gene into hematopoietic progenitor cells results in resistance to a promising new antifolate, pemetrexed.…”

Distribution of Mutations in Human Thymidylate Synthase Yielding Resistance to 5-Fluorodeoxyuridine

“…Refs. [5][6][7][8]. Here, we used a different strategy, based on the hypothesis that amino acid replacements yielding 5-FdUR resistance are located throughout the TS primary sequence.…”

“…Introduction of a drug-resistant TS into bone marrow stem cells could protect patients from the severe side effects of TS inhibitors. Based on this premise, mutations have been identified that render human TS resistant to 5-fluoropyrimidines and anti-folate analogs (5)(6)(7)(8)(9)(10). Nearly all of these mutations map within the catalytic site.…”

“…Earlier work involving mutagenesis of human HT1080 cells with ethylmethane sulfonate, followed by selection with the anti-folate Thymitaq (AG337), yielded three Arg 50 loop mutants (D49G, G52S, and K47E) that were resistant to both the anti-folate and 5-FdUR (7). Two other mutants that are resistant to both anti-folates and 5-FdUR, I108A and F225W, were obtained by site-directed mutagenesis of highly conserved residues important in folate binding (8).…”