Isolation and Characterization of Thymitaq (AG337) and 5-Fluoro-2-deoxyuridylate-resistant Mutants of Human Thymidylate Synthase from Ethyl Methanesulfonate-exposed Human Sarcoma HT1080 Cells

Abstract

Thymidylate synthase plays an essential role in the synthesis of DNA. Recently, several new and specific thymidylate synthase inhibitors that occupy the folate binding site, including Tomudex®, BW1843U89, and Thymitaq, have demonstrated therapeutic activity in patients with advanced cancer. In order to find drugresistant forms of human thymidylate synthase for gene therapy applications, human sarcoma HT1080 cells were exposed to ethyl methanesulfonate and Thymitaq selection. Thymitaq-resistant clonal derived sublines were established, and analysis indicated that both gene amplification and point mutations contributed to drug resistance. Eight mutant cDNAs that were identified from Thymitaq-resistant sublines were generated by site-directed mutagenesis and transfected into thymidylate synthase-negative cells. Only K47E, D49G, or G52S mutants retain enzyme activity. Moreover, cytotoxicity studies demonstrated that D49G and G52S transfected cells, besides displaying resistance to Thymitaq with IC 50 values 40-and 12-fold greater than wild-type enzyme transfected cells, respectively, also lead to fluorodeoxyuridine resistance (26-and 97-fold in IC 50 values, respectively) but not to Tomudex or BW1843U89. Characterization of the purified altered enzymes obtained from expression in Escherichia coli is consistent with the cell growth inhibition results. We postulate that the D49G or G52S mutation leads to the structural perturbation of the highly conserved Arg 50 loop, decreasing the binding of thymidylate synthase to the inhibitors, Thymitaq and fluorodeoxyuridylate.Thymidylate synthase (TS, 1 EC 2.1.1.45) catalyzes the de novo biosynthesis of thymidylate, which is necessary for DNA synthesis and repair (1). The mechanism of TS activity involves the reductive methylation of the substrate, 2Ј-deoxyuridine 5Ј-monophosphate (dUMP) by transfer of a methylene group from the cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate (CH 2 H 4 folate), to generate 2Ј-deoxythymidine 5Ј-monophosphate (dTMP) and 7,8-dihydrofolate. Human TS has been sequenced (2), purified (3, 4), and crystallized (5). As an attractive target for anti-cancer drug design, since the 1950s, many TS analogues of both the substrate, dUMP, and the cofactor, CH 2 H 4 folate, have been synthesized and tested as potential anti-cancer therapeutics. Until recently, 5-fluorouracil and fluorodeoxyuridine (FdUrd) were the sole TS-targeted drugs approved for clinical application. In vivo, 5-fluorouracil and FdUrd are metabolized to 5-fluoro-2-deoxyuridylate (FdUMP), a compound that subsequently occupies the pyrimidine binding site forming a ternary complex with TS and the folate cofactor, resulting in inhibition of enzyme function. The recent determination of the three-dimensional structure of human TS has allowed the design of highly specific inhibitors, leading to the emergence of novel folate analogues, such as Tomudex (ZD1694), BW1843U89, and Thymitaq (AG337) (Fig. 1) (6). These promising compounds have entered clinical trials in recent years (7).Previous studies h...

Resistance to antifolates

“…Changes induced by the first two mutations were highly selective. Cells were crossresistant to fluorouracil but not to ZD1694 or GW1843U89, consistent with differences in the binding sites for these antifolates (Tong et al, 1998a). Similarly, site-directed mutagenesis of highly conserved residues confirmed the selective effects of mutations of this enzyme.…”

“…Several mutations in a highly conserved region (Lys47Glu, Asp49Gly, Gly52Ser) were identified in human sarcoma HT1080 cells selected for resistance to AG337, a lipid-soluble inhibitor that does not form polyglutamate derivatives and is transported by passive diffusion (Webber et al, 1996;Tong et al, 1998a). Changes induced by the first two mutations were highly selective.…”

“…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).…”

“…As observed with DHFR, this is frequently associated with gene amplification. For example, resistance to ZD1694 in human MCF-7 breast cancer, lymphoblastoid, and ovarian carcinoma cell lines, or to ICI-198584 was associated with TS amplification (O'Connor et al, 1992;Freemantle et al, 1995;Jackman et al, 1995;Drake et al, 1996;Tong et al, 1998a;Kitchens et al, 1999a). The relationship between the increase in expression and the level of resistance can vary.…”

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

“…K m values for the TS substrate dUMP were determined as described previously. 15 Using the above assay for TS activity, the initial velocity measurements of wtTS (200 nM), TS G52S (100 nM) or DHFR-F/S-TS G52S (80 nM) were measured at a saturating concentration of cofactor, 6-R-CH 2 H 4 folate (400 mM), while varying the concentration of dUMP over the range of 5-500 mM. Similarly, K m values for TS cofactor were determined at a saturating concentration of dUMP (500 mM), while varying the concentration of 6-R-CH 2 H 4 folate over the range of 2-500 mM.…”