Comparative thermodynamic study of the interaction of some antifolates with dihydrofolate reductase

The thermodynamic parameters of the binding of antifolate drugs to bovine liver dihydrofolate reductase (EC 1.5.1.3., 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase) have been measured with a flow microcalorimetric method. These parameters are greatly influenced by the structure of the inhibitor and/or by the presence of NADPH and above all by temperature. For all the compounds studied, binding at 37 degrees C is driven by favourable enthalpy variations, whereas entropy variations are unfavourable. At 10 degrees C, reactions are both enthalpically and entropically driven. These effects can be explained by a partial thermal denaturation of dihydrofolate reductase at 37 degrees C, which is restructured by NADPH and/or the antifolate. The refolding induced by the antifolate trimetrexate may explain its high association constant in the binary system (without NADPH), and the weaker cooperative effect of NADPH in the ternary system, as compared to methotrexate. In contrast, the poor affinity of trimethoprim for mammalian dihydrofolate reductase in binary and ternary systems at 37 degrees C is the result of a weaker stabilizing effect of this compound as regards temperature increase. Heat capacity variation linked to the complex formation reaction showed that this conformational transition is more pronounced between 25 and 37 degrees C than between 10 and 25 degrees C. Thus, the ability of the inhibitors to give to dihydrofolate reductase a more stable thermal behaviour at 37 degrees C is determinant in their binding.