Promotion of purine nucleotide binding to thymidylate synthase by a potent folate analogue inhibitor, 1843U89.

Weichsel, A.; Montfort, W.R.; Cieśla, Jarosław M.; Maley, Frank

doi:10.1073/pnas.92.8.3493

Cited by 19 publications

(12 citation statements)

References 27 publications

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“…In the presence of dUMP, the enzyme was stabilized to the extent that the T 1/ 2 was now 49°C and on the addition of FH 2 the T 1/ 2 was improved further to 53°C. However, FH 2 alone had no affect (Table 3), which is not surprising in that the structural stability of the enzyme appears to be improved by the stacking energy resulting from the van der Waals interaction of folate and folate analogues with dUMP and its derivatives at the active site of TS (21). It is probable that if a tighter binding folate analogue had been employed an even greater stabilizing response would have been observed, as was found in the case of dUMP and GW1843, where the unfolding temperature for E. coli TS was increased from 45 to 72°C (19).…”

Section: Expression and Isolation Of E Coli And B Subtilis Tsmentioning

confidence: 80%

“…Basically, these results demonstrate that although B. subtilis is about 7°C more stable than E. coli TS, the combination of dUMP plus FH 2 increases both their respective heat stabilities by 9 -10°C. The most probable explanation for this increase in stability is that the conformational change induced by the substrate or substrate analogues is associated with the extra free energy of stabilization imparted to the protein as a consequence of the stacking effect (21). a Temperature at which 50% of the original TS activity was lost.…”

Section: Expression and Isolation Of E Coli And B Subtilis Tsmentioning

confidence: 99%

See 1 more Smart Citation

High-Level Expression of Escherichia coli and Bacillus subtilis Thymidylate Synthases

Changchien

Garibian

Frasca

et al. 2000

Protein Expression and Purification

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Section: Expression and Isolation Of E Coli And B Subtilis Tsmentioning

confidence: 80%

Section: Expression and Isolation Of E Coli And B Subtilis Tsmentioning

confidence: 99%

High-Level Expression of Escherichia coli and Bacillus subtilis Thymidylate Synthases

Changchien

Garibian

Frasca

et al. 2000

Protein Expression and Purification

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“…This effect is even more dramatically shown in the effect that BW1843U89 has on the binding of dUMP and even dGMP (GMP) where the stacking energy between the benzoquinazoline ring of BW1843U89 and TS, and also indicated nucleotides greatly enhances their binding (42). Any mutation that impairs the binding of folate analogues would naturally impair the binding of dUMP or FdUMP and would thus explain the resistance of the indicated cell lines containing I108A and F225W to both BW1843U89 and FdUMP.…”

Section: Drug-resistant Mutants Of Human Thymidylate Synthasementioning

confidence: 99%

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

Tong¹,

Liu-Chen²,

Ercikan-Abali³

et al. 1998

Journal of Biological Chemistry

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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...

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“…We used near-UV circular dichroism (240-400 nm) as a screening method (Danenberg, Langenbach & Heidelberger, 1974), so that changes in the difference spectra would be indicative of productive binding as in our previous work with the binding of purine and pyrimidine nucleotides to the TS active site (Galivan, Maley & Maley, 1975;Weichsel, Montfort, Ciesla & Maley, 1995) (Fig. 3).…”

Section: Ligand Selectionmentioning

confidence: 99%

“…Ellipticities were measured from 240 to 400 nm at room temperature using a Jasco J-720 (Jasco Inc., Easton, MD) spectropolarimeter, essentially as previously described (Galivan, Maley & Maley, 1975;Weichsel, Montfort, Ciesla & Maley, 1995). Difference spectra were obtained by subtracting the ellipticities of TS (WT or mutant) to the TS-nucleotide-folate solution complex using a program provided by Jasco.…”

Section: Near Uv Circular Dichroism (Cd)mentioning

confidence: 99%

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: Calorimetric and crystallographic analysis of the K48Q mutant

Arvizu‐Flores

Sugich-Miranda

Arreola

et al. 2008

The International Journal of Biochemistry & Cell Biology

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Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH 2 THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen-bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k cat of the K48Q mutant was 430 fold lower than wild-type TS in activity, while the the K m for the (R)-stereoisomer of CH 2 THF was 300 µM, about 30 fold larger than K m from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideaza folate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutamt, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH 2 THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group.

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Promotion of purine nucleotide binding to thymidylate synthase by a potent folate analogue inhibitor, 1843U89.

Cited by 19 publications

References 27 publications

High-Level Expression of Escherichia coli and Bacillus subtilis Thymidylate Synthases

High-Level Expression of Escherichia coli and Bacillus subtilis Thymidylate Synthases

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

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: Calorimetric and crystallographic analysis of the K48Q mutant

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