THERMODYNAMIC STUDY OF DIHYDROFOLATE-REDUCTASE INHIBITOR SELECTIVITY

The thermodynamic parameters of the binding of some folate analogues ( methotrexate, trimetrexate and trimethoprim) to dihydrofolate reductas es from different species have been measured with a flow microcalorime tric method at 37 degrees C. In the absence of NADPH, the three inhibi tors exhibited a higher affinity for E. coil DHFR than for vertebrate DHFRs. This selectivity in favor of bacterial DHFR is entropy driven a nd is correlated with a weaker conformational change for bacterial DHF R than for vertebrate DHFRs, and with additional hydrophobic contacts, provided by this enzyme to the ligands. In presence of NADPH, as repo rted in the literature, trimetoprim shows a high selectivity in favor of bacterial DHFR, contrarily to methotrexate and trimetrexate, whose affinities are elevated and highly similar for mammalian and bacterial enzymes. The positive cooperative effect of NADPH, which has an entha lpic origin, fluctuates widely with inhibitor structure and with enzym e species. For trimethoprim, the cooperative effect is much more prono unced for bacterial DHFR than for vertebrate DHFRs. But the role of NA DPH is not to induce a selectivity it only increases the selectivity t hat trimethoprim already presented in absence of NADPH. Inversely, for methotrexate and trimetrexate, the cooperative effect is stronger for vertebrate enzymes than for the bacterial enzyme, and thus, NADPH can cels the selectivity the two antifolic compounds had, in the absence o f NADPH, for the bacterial enzyme.