Sulphonamide Antifolates Inhibiting Thymidylate Synthase. Synthesis, Enzyme Inhibition and Cytotoxicity (original) (raw)
Related papers
Biochemistry, 2007
Thymidylate synthase (TS) is a target in the chemotherapy of colorectal cancer and some other neoplasms. It catalyses the transfer of a methyl group from methylenetrahydrofolate to dUMP to form dTMP. Based on structural considerations, we have introduced 1,3-propanediphosphonic acid (PDPA) as an allosteric inhibitor of human TS (hTS); it is proposed that PDPA acts by stabilizing an inactive conformer of loop 181-197. Kinetic studies showed that PDPA is a mixed (noncompetitive) inhibitor vs dUMP. In contrast, vs methylenetrahydrofolate at concentrations lower than 0.25 μM PDPA is an uncompetitive inhibitor, while at PDPA concentrations higher than 1 μM the inhibiton is noncompetive, as expected. At the concentrations corresponding to uncompetitive inhibition, PDPA shows positive cooperativity with an antifolate inhibitor, ZD9331, which binds to the active conformer. PDPA binding leads to the formation of hTS tetramers, but not higher oligomers. These data are consistent with a model in which hTS exists preferably as an asymmetric dimer with one subunit in the active conformation of loop 181-197 and the other in the inactive conformation. Thymidylate synthase (TS) catalyzes the reaction in which the nucleotide deoxyuridylate (dUMP) is reductively methylated by the folate co-substrate 5,10-methylenetetrahydrofolate (CH 2 H 4 folate) to form thymidylate (TMP) and dihydrofolate (1). Substrates are bound in an ordered manner, with dUMP binding at the active site prior to CH 2 H 4 PteGlu. A cysteine residue (Cys195 in hTS) at the active site attacks the 6-position of the pyrimidine base of the nucleotide, resulting in the formation of a covalent bond between TS and the nucleotide and activating the 5-position of the nucleotide for subsequent covalent-bond formation with the C-11 substituent of CH 2 H 4 folate (reviewed in 2-4). The enzyme is the sole source of de novo synthesized thymidylate and its inhibition leads to apoptosis of rapidly dividing cells such as cancer cells,
Mode of Action of Site-Directed Irreversible Folate Analogue Inhibitors of Thymidylate Synthase †
Biochemistry, 1998
5,8-Dideazafolate analogues are tight binding but not irreversible inhibitors of thymidylate synthase (TS). However, when a chloroacetyl (ClAc) group is substituted at the N 10 -position of 2-desamino-2-methyl-5,8-dideazafolate (DMDDF), the resulting compound, ClAc-DMDDF, although still a reversible inhibitor (K I ) 3.4 × 10 -3 M), gradually inactivates thyA-TS irreversibly at a rate of 0.37 min -1 . The corresponding iodoacetyl derivative alkylated the enzyme somewhat slower (k 3 ) 0.15 min -1 ) than ClAc-DMDDF but was bound more tightly (K I ) 1.4 × 10 -5 M), resulting in a second-order rate constant (k 3 /K I ) of inactivation that was 100-fold greater than that of ClAc-DMDDF. A tryptic digest of the ClAc-DMDDF-inactivated enzyme yielded a peptide on HPLC, which revealed that cysteine-146, the residue at the active site that is intimately involved in the catalytic process, had reacted with ClAc-DMDDF to form a covalent bond. This derivative was confirmed indirectly by Edman analysis and more directly by mass spectrometry. Deoxyuridine 5′-monophosphate, a substrate in the catalytic reaction, protected against inactivation. Similar to previously described Lactobacillus casei TS inhibition studies with sulfhydryl reagents Arch. Biochem. Biophys. 184,[336][337][338][339][340][341][342][343][344][345], the kinetics of inhibition suggested that complete inhibition occurs on reaction of only one of the two active site cysteines, although sequence and amino acid analysis revealed that iodoacetate and ClAc-DMDDF had reacted with both active site cysteines. These studies demonstrate that a sulfhydryl reactive compound that is directed to the folate binding site of TS may diffuse to the active site cysteine, and form a covalent bond with this residue. How this inhibition comes about is suggested in a stereoscopic view of the ligand when modeled to the known crystal structure of Escherichia coli TS.
Biochemical Pharmacology, 1991
Nl°-propargyl-5,8-dideazafolic acid (ICI 198583) is a more watersoluble analogue of the quinazoline-based thymidylate synthase (TS) inhibitor, Nl°-propargyl-5,8-dideazafolic acid (CB3717). A 3-fold loss in TS inhibitory activity (murine and human TS, K~ = 10 nM) was accompanied by a 40-fold increase in growth inhibitory potency against L1210 and W1L2 cells in vitro 0Cs0 = 0.085 and 0.05 tzM, respectively) when compared with CB3717. In L1210 cells a concentrative uptake mechanism was demonstrated for [3H]ICI 198583 (Kt = 2.9/~M). The L1210:1565 cell line, with an impaired ability to transport reduced folates or methotrexate (MTX), was resistant (100fold relative to the wild-type L1210 line) to ICI 198583 (but not CB3717) and did not take up [SH]ICI 198583 significantly. The measurement of folylpolyglutamate synthetase (FlaGS) substrate activity demonstrated a g m of 40/aM for ICI 198583 and a Vmax/Km (relative to folic acid) of 3.5. The formation of intracellular polyglutamate derivatives was demonstrated in both L1210 (mouse) and W1L2 (human) cells grown in vitro after exposure to 1/xM [3H]ICI 198583. In L1210 cells, by 4 hr,-50% of the intracellular 3H(~l laM) was found as polyglutamate forms of ICI 198583, principally as tri-and tetraglutamates. After 24 hr the ICI 198583 polyglutamate pool had expanded, the tetraglutamate metabolite predominanted and there was significant formation of the pentaglutamate. Upon resuspension of L1210 cells in drug free medium, ICI 198583 was largely lost from the cells but the polyglutamates were preferentially retained, after 24 hr-70% remained. Synthetic ICI 198583 polyglutamates were shown to be up to 100-fold more potent as inhibitors of isolated TS than the parent compound. Following in vivo administration (500mg/kg i.v.) ICI 198583 was cleared rapidly from the plasma of mice (Tz/2# = 16 min, clearance = 42 mL/min/kg). Despite this clearance there was prolonged, dose-dependent inhibition of TS in L1210 : NCI cells in vivo. Thus, following 500 mg/kg i.v. the flux through TS was inhibited by >80% for at least 24 hr. Administration of five doses at 5 mg/kg daily of ICI 198583 to L1210 : ICR tumour-bearing mice resulted in >60% of the mice being cured, a 10-fold improvement in potency over CB3717. The maximum tolerated dose (MTD) for ICI 198583 using this schedule was >500 mg/kg/day compared with 200 mg/kg/day of CB3717. ICI 198583 is therefore a potent inhibitor of TS in vitro and in vivo with a marked improvement in therapeutic index over CB3717 in mice.
Human thymidylate synthetase—III
Biochemical Pharmacology, 1979
The structure-activity relationship of human thymidylate synthetase (EC 2.1.1.45) was studied with two groups of folate analogs: (I) methotrexate (MTX) analogs modified at the glutamate residue and N": and (2) tetrahydrofolate (H,PteGlu) analogs modified at N' and N". With respect to MTX analogs, it was found that: (I) substitution of the glutamate side chain by z-aminoadipic acid. z-aminopimclic acid or /3aminoglutaric acid slightly affects its Ki: (2) a free z-carboxyl group on the amino acid side chain of MTX. or any free carboxyl group in that vicinity plays an important role in the inhibitory potency of MTX analogs to the enzyme; (3)esterilication or amidation ofthe z-carboxyl group of MTX decreases the inhibitory potency; and (4) free aspartyl or glutamyl conjugation through a peptide linkage to the ;~~boxyl group of the glutamate side chain decreases its K, to the enzyme by 5-and 8-fold respectively. Tetrahydrofolate analogs formed by inserting an ethylene, iminyl or a carbonyl bridge between the nitrogen at N" and N'" or by substitution at the NJ position were found to be poor inhibitors under our assay conditions.
Synthesis of 5,11-methenyltetrahydrohomofolate and its antifolate activity in vitro
Journal of Medicinal Chemistry, 1981
means of anion-exchange HPLC as described in published work from this laboratory.% Nucleosides were not retained on a Whatman Partisil-10 SAX HPLC column, whereas the 5'monophosphates of 5-FdUrd and C-B-F-2'-dUrd were retained for 7 min on a linear gradient (40 min) of NH4H2P04 (5 mM, pH 2.8. to 750 mM. DH 3.7) at a flow rate of 2 mL/min. The dUMP J. Med. Chem. 1981,24, 1086-1088 The retention times (min) for uracil (9), 5-FUra (9.4), dUrd (15), 5-PdUrd (16.6), and C-5-F-2'-dUrd (18) were such that the enzymatic conversion of nucleoside to base could be followed by means of a UV detector (254 nm). Deoxyuridine and 5-FdUrd were readily phosphorylyzed to Ura and to 5-FUra, respectively, but conversion of C-5-F-Y-dUrd to 5-FUra was not detectable. andogues were' separakd from other W-absdrbing components of the enzyme reaction mixture, primarily adenine nucleotides, so that quantitation of nucleotide formation was possible.
Theoretical Studies Suggest a New Antifolate as a More Potent Inhibitor of Thymidylate Synthase
Journal of the American Chemical Society, 2000
A set of free energy calculations were performed for selected antifolate compounds as inhibitors of thymidylate synthase (TS). These calculations reproduced the nonadditive behavior of different substitutions on selected compounds described in the experiments by Jones et al 1 . (J. Med. Chem. 1996, 39 (4), 904-917). Molecular dynamics (MD) simulations showed that the nonadditive behavior was due to the interference between the way different substituents interacting with key protein side chains. Pictorial representation of free energy change (PROFEC) and free energy calculations suggested that a compound not previously considered would bind more tightly to TS than the best binding known compounds in this series of propargyl inhibitors and, thus, could be a promising candidate in anticancer drug design.
Biochemical Pharmacology, 1981
Deoxyuridine derivatives containing styryl, 3-nitrostyryl, 4-nitrostyryl, and phenylethyl groups substituted at the 5-position of the pyrimidine ring have been evaluated for their effects on vaccinia and herpes simplex virus replication (in primary rabbit kidney cell cultures) and mouse leukemia L-1210 cell culture growth. 5-Phenylethyl-2'-deoxyuridine inhibited herpes simplex (type 1 and 2) virusinduced cytopathogenicity by 50 per cent at a dose (ID50) of 10-30/~g/ml. It was inactive against tumor cell growth. The corresponding styryl derivative showed an IDs0 of 30-70/ug/ml for herpes simplex virus, 20 t~g/ml for vaccinia virus, and 280/ug/ml for L-1210 cell growth. 5(E)-(3-Azidostyryl)-2'-deoxyuridine 5'-phosphate inhibited vaccinia replication with an IC50 of 20/*g/ml and L-1210 cell culture growth with an IO50 of 80/2g/ml. The nucleotides of these compounds were all potent reversible inhibitors of thymidylate synthetase (Lactobacillus casei) with the following Ki/Km ratios: 3-nitrostyryl, 0.035; 4nitrostyryl, 0.05; 3-azidostyryl, 0.06; styryl, 0.08; and phenylethyl, 0.31. The photodecomposition of the azidostyryl derivative, a photoaffinity labeling reagent for thymidylate synthetase, was examined at two wavelengths.