Yvette Gaumont

Stereospecificity at carbon 6 of formyltetrahydrofolate as a competitive inhibitor of transport and cytotoxicity of methotrexate in vitro

Abstract The unnatural diastereoisomer of l -5-formyltetrahydrofolate was 20-fold less effective as a competitive inhibitor of [ 3 H] methotrexate influx than the natural diastereoisomer during carrier-mediated membrane transport in L1210, S180 and Ehrlich cells. Values derived for K i , were 1.84 to 2.29 μM for the natural derivative and 35.2 to 53.8 μM for the unnatural derivative. Values for K i derived with a chemically synthesized mixture containing equal amounts of both natural and unnatural diastereoisomers were 2-fold greater than values obtained for the natural diastereoisomer. The unnatural diastereoisomer was 100-fold less effective and the chemically synthesized mixture was 2-fold less effective than the natural diastereoisomer in preventing inhibition by methotrexate of L1210 cell growth in culture. These results indicate that the unnatural diastereoisomer competes relatively ineffectively with the natural diastereoisomer or methotrexate for transport in these murine tumor cells.

Carrier-mediated transport of folate compounds in L1210 cells. Initial rate kinetics and extent of duality of entry routes for folic acid and diastereomers of 5-methyltetrahydrohomofolate in the presence of physiological anions.

Comparison of the kinetic parameters for influx of highly purified [3H]folic acid versus [3H]methotrexate in L1210 cells under anionic buffer conditions showed a marked discordancy. In addition, the kinetics for influx of [3H]folic acid were unchanged in variant L1210 cells defective in [3H]methotrexate transport. In these variant cells, the Vmax for methotrexate was reduced 17-fold and the Km was increased 3-fold. The results show that [3H]folic acid influx is mediated by a system which has a low affinity, but a 20-fold higher capacity, for folate compounds than the classical high-affinity system mediating [3H]methotrexate influx. Since the latter system also exhibits very low affinity for [3H]folic acid, it would not be expected to contribute significantly to the total influx of [3H]folic acid. The high-capacity system for [3H]folic acid influx is different from that believed to mediate pterin influx in L1210 cells since it was not inhibited by adenine, a potent inhibitor of pterin influx. However, exposure of cells to [3H]folic acid in a nonanionic buffer resulted in marked stimulation of initial influx, and a fraction of influx under these conditions was inhibited by methotrexate. These results suggest that anions modulate the extent of multiplicity of [3H]folic acid influx by their known effects on the high-affinity, reduced folate/methotrexate system. The diastereomers, at carbon 6, of [14C]5-methyltetrahydrohomofolate shared both transport systems. The influx Km for the natural diastereomer was one-half that of the unnatural form for both transport systems. Both diastereomers showed a much greater differential in affinity between the two transport systems than did [3H]folic acid. Our results suggest that an analog which could be effectively transported by the low-affinity/high-capacity route may be useful in the treatment of tumors resistant to methotrexate due to a defective high-affinity/low capacity influx system. We also found that incubation of L1210 cells with [3H]folic acid or the natural diastereomer [14C]5-methyltetrahydrohomofolate for 10 min resulted in the formation of a nonexchangeable fraction of radioactivity amounting to 20-40% of the total accumulation. This non-exchangeable fraction may be explained by the accumulation of metabolites other than polyglutamates. Preloading of cells with methotrexate prior to incubation with [3H]folic acid prevented the accumulation of radioactivity as a nonexchangeable fraction.

Effects of the diastereoisomers of methylenetetrahydrofolate on the reaction catalyzed by thymidylate synthetase

Abstract Diastereoisomers of methylenetetrahydrofolate were prepared and shown to have equal and opposite CD spectra. In addition to being inactive in the spectrophotometric assay for Lactobacillus casei thymidylate synthetase, the diastereoisomer having the unnatural configuration at carbon 6, does not promote removal of tritium from 5-(3H)-dUMP. It is a competitive inhibitor of the reaction with a K1 of 5 × 10−5 M .

A calorimetric study of the binding of 2'-deoxyuridine-5'-phosphate and 5-fluoro-2'-deoxyuridine-5'-phosphate to thymidylate synthetase.

Abstract The thermodynamic parameters, Δ H ′, Δ G ′, and Δ S ′, and the stoichiometry for the binding of the substrate 2′-deoxyuridine-5′-phosphate (dUMP) and the inhibitor 5-fluoro-2′-deoxyuridine-5′-phosphate (FdUMP) to Lactobacillus casei thymidylate synthetase (TSase) have been investigated using both direct calorimetric methods and gel filtration methods. The data obtained show that two ligand binding sites are available but that the binding of the second mole of dUMP is extremely weak. Binding of the first mole of dUMP can best be illustrated by dUMP + TSase + H + ⇌(dUMP-TSase-H + ). [1] The enthalpy, Δ H 1 ′, for reaction [1] was measured directly on a flow modification of a Beckman Model 190B microcalorimeter. Experiments in two different buffers ( I = 0.10 m) show that Δ H 1 ′ = −28 kJ mol −1 and that 0.87 mol of protons enters into the reaction. Analysis of thermal titrations for reaction [1] indicates a free energy change of Δ G 1 ′ = −30 kJ mol −1 ( K 1 = 1.7 × 10 5 m −1 ). From these parameters, Δ S 1 ′ was calculated to be +5 J mol −1 degree −1 , showing that the reaction is almost totally driven by enthalpy changes. Gel filtration experiments show that at very high substrate concentrations, binding to a second site can be observed. Gel filtration experiments performed at low ionic strength ( I = 0.05 m) reveal a stronger binding, with Δ G 1 ′ = −35 kJ mol −1 ( K 1 = 1.2 × 10 6 m −1 ), suggesting that the forces driving the interaction are, in part, electrostatic. Addition of 2-mercaptoethanol (0.10 m ) had the effect of slightly increasing the dUMP binding constant. Binding of FdUMP to TSase is best illustrated by 2FdUMP + TSase + n H H + ⇌FdUMP 2 − TSase − (H + ) n H . [2] The enthalpy for this reaction, Δ H 2 , was also measured calorimetrically and found to be −30 kJ mol −1 with n H = 1.24 at pH 7.4 Assuming two FdUMP binding sites per dimer as established by Galivan et al. [ Biochemistry 15 , 356–362 (1976)] our calorimetric results indicate different binding energies for each site. Based on the binding data, a thermodynamic model is presented which serves to rationalize much of the confusing physical and chemical data characterizing thymidylate synthetase.

Further studies of stereospecificity at carbon 6 for membrane transport of tetrahydrofolates: Diastereoisomers of 5-methyltetrahydrofolates as competitive inhibitors of transport of methotrexate in L1210 cells

The unnatural d diastereoisomer at carbon 6 of 5-methyltetrahydrofolate was only slightly less effective than the natural 1 diastereoisomer as a competitive inhibitor of the carrier-mediated membrane transport of [3H]methotrexate into L1210 murine leukemia cells. The apparent Ki for a mixture containing equal amounts of both natural and unnatural diastereoisomers was not significantly different from that found for the unnatural form. These results show that the reduced folate carrier system in these cells has a strong affinity for the unnatural stereoisomer, a finding in contrast to that obtained with the corresponding diastereoisomer of 5-formyltetrahydrofolate.

Synthesis and evaluation of 8,10-dideazatetrahydrofolic acid and derivatives

Syntheses of 8,10-dideazatetrahydrofolic acid and its 5-N-methyl and 5-N-formyl derivatives are reported. Hydrolysis of 2,4-diamino-4-deoxy-8,10-dideazapteroic acid in hot alkali afforded 8, 10-dideazapteroic acid. Coupling with diethyl L-glutamate followed by saponification gave 8,10-dideazafolic acid. Hydrogenation in acidic media gave the tetrahydro compound, while hydrogenation in the presence of formaldehyde yielded the 5-N-CH 3 analog. The 5-N-CH 3 compound was more potent than 5,10-DDTHF as an inhibitor of growth for L1210 cells in culture. In contrast to 5,10-DDTHF, the locus of action was apparently unrelated to inhibition of GAR formyltransferase. Unfortunatly, 5-CH 3 -8,10-DDTHF was not active in vivo against an L1210 challenge in mice

Tetrahydrohomofolate polyglutamates as inhibitors of thymidylate synthase and glycinamide ribonucleotide formyltransferase in Lactobacillus casei

In order to determine the mechanism for the effects of homofolates on growth of Lactobacillus casei, polyglutamated derivatives of homofolate (HPteGlu), dihydrohomofolate and tetrahydrohomofolate (H4HPteGlu) were synthesized and tested as inhibitors of folate-requiring enzymes. The following L. casei enzymes were examined: thymidylate synthase (TS), glycinamide ribonucleotide formyltransferase (GARFT), aminoimidazolecarboxamide ribonucleotide formyltransferase, serine hydroxymethyltransferase and dihydrofolate reductase. Polyglutamates of (6R,S)-H4HPteGlu are potent inhibitors of TS and GARFT. For example, the IC50 values of (6R,S)-H4HPteGlu6 are 0.7 microM for TS and 0.3 microM for GARFT. By contrast, the value for HPteGlu6 is greater than 10 microM for both TS and GARFT. Inhibition of TS and GARFT by (6R,S)-H4HPteGlu derivatives increases with polyglutamate chain length. For TS, the Glu5 and Glu6 derivatives of (6R,S)-H4HPteGlu are 20 and 30 times more potent than the monoglutamate, respectively. For GARFT, the Glu2-6 derivatives are 2-3 times more potent than Glu1. Inhibition of TS and GARFT by (6R,S)-H4HPteGlu polyglutamates is almost entirely due to the unnatural (6R) diastereomer at C-6. Homofolate derivatives are only weak inhibitors of aminoimidazolecarboxamide ribonucleotide formyltransferase, serine hydroxymethyltransferase, and dihydrofolate reductase. We conclude that both TS and GARFT are potential targets of (6R)-H4HPteGlu polyglutamates.

Nα-Ipteroyltetra (γ-Glutamyl)]-Lysine as a Ligand for the Purification of Thymidylate Synthetase by Affinity Chromatography

Abstract Nα[Pteroyltetra (γ-glutamyl)]-lysine Sepharose was synthesized and shown to be a stable, high capacity affinity matrix capable of bringing about the purification of Lactobacillus casei thymidylate synthetase to maximum specific activity from crude extracts in high yield. Under conditions optimal for binding of thymidylate synthetase, dihydrofolate reductase was not bound.