COMPARISON OF TERNARY CRYSTAL COMPLEXES OF F31 VARIANTS OF HUMAN DIHYDROFOLATE-REDUCTASE WITH NADPH AND A CLASSICAL ANTITUMOR FUROPYRIMIDINE

The novel furopyrimidine, minofuro[2,3-d]pyrimidin-5-yl)-methyl]methyl amino] -benzoyl]-L-glutamate (MTXO), a classical antifolate with weak antitumor activity compared with methotrexate (MTX), has been studied as inhibitor-cofactor ternary crystal complexes with recombinant Phe-3 1 to Ser (F31S) and Phe-31 to Gly (F31G) variant human dihydrofolate r eductase (hDHFR). Kinetic data show that the binding affinity of MTXO is significantly weaker for the variant hDHFR enzyme than far the wild type enzyme. Structural data for the Phe-31 variants, along with wild type hDHFR, provide the first direct comparison of the binding intera ctions of a single antifolate in a family of variant hDHFR. These tern ary hDHFR complexes crystallize in the rhombohedral space group R3, is omorphous to that reported for wild type hDHFR MTXO-NADPH ternary comp lex. MTXO binds with its 2,4-diaminofuropyrimidine ring interacting wi th Glu-30 in hDHFR. The greatest change on modification of the side ch ain at position 31 is loss of hydrophobic contacts to the inhibitor, w hich results in the significant decrease in binding affinity of MTXO f or the Phe-31 variants. The presence of the 6-5 furopyrimidine ring in stead of the 6-6 pteridine ring causes a different bridge conformation compared with MTX, and in the case of the wild type MTXO complex also results in weaker hydrophobic contacts to Phe-31 than observed for MT XT. For the design of antitumor agents related to MTXO, increasing the bridge of MTXO from two to three or four atoms should provide increas ed DHFR inhibitory potency and antitumor activity.