Thermodynamic study of the interaction of methotrexate, its metabolites, and new antifolates with thymidylate synthase: influence of FdUMP.
A microcalorimetric method was used for the direct study of the interaction of methotrexate, its metabolites, and new antifolates N10-propargyl-5,8-dideazafolate (CB 3717) and 2-methyl,2-desamino N10-propargyl-5,8-dideazafolate (CB 3819), with thymidylate synthase. We show that 7-hydroxymethotrexate and dideazafolates require the prior binding of dUMP or its fluorinated derivative FdUMP to bind to thymidylate synthase, as does methotrexate. Conversely, we show that methotrexate-G2 can interact directly with the enzyme alone. On the other hand, both dUMP and FdUMP exhibited a large cooperative effect on the affinity for thymidylate synthase of the inhibitors, and surprisingly, no significant difference was shown at this level between the natural substrate dUMP and its fluorinated derivative. It was demonstrated that this cooperative effect had an enthalpic origin. In the presence of FdUMP or dUMP, all the studied compounds except 7-hydroxymethotrexate exhibited a large negative enthalpy variation when binding to thymidylate synthase (from -44 to -91 kJ/mol). CB 3717 and methotrexate-G2 are competitors for the same protein binding site. Polyglutamation of methotrexate lead to compounds with higher affinity (association constants were 6.6 x 10(3) M-1 and 2.3 x 10(6) M-1 for methotrexate and methotrexate-G2 respectively) while hydroxylation has an unfavourable effect (association constant of 7-hydroxymethotrexate inferior to 500 M-1). Evidence for the influence of polyglutamation was also provided by the relatively low affinity of dideazofolates for thymidylate synthase (association constant equal to 1.4 and 1.7 x 10(7) M-1 for CB 3717 and CB 3819, respectively), whereas these compounds are known to be strong inhibitors of the enzyme in cells in their polyglutamated forms.
Gilli
R
R
Faculté de Pharmacie de Marseille, Laboratoire de Physique Pharmaceutique, France.
Lopez
C
C
Sari
J C
JC
Briand
C
C
D002151:Calorimetry / Q000379:methods; D005468:Fluorodeoxyuridylate / Q000494:pharmacology*; D005493:Folic Acid Antagonists / Q000378:metabolism*; D008727:Methotrexate / Q000031:analogs & derivatives / Q000378:metabolism*; D013816:Thermodynamics; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D005493:Folic Acid Antagonists; D005468:Fluorodeoxyuridylate; D013940:Thymidylate Synthase; C011864:7-hydroxymethotrexate; D008727:Methotrexate