COMPARISON OF TERNARY COMPLEXES OF PNEUMOCYSTIS-CARINII AND WILD-TYPEHUMAN DIHYDROFOLATE-REDUCTASE WITH COENZYME NADPH AND A NOVEL CLASSICAL ANTITUMOR FURO[2,3-D]PYRIMIDINE ANTIFOLATE

The novel furopyrimidine ,3-d]pyrimidin-5-yl)methyl]methylamino}benzoy l)-L- glutamate (MTXO), a classical antifolate with antitumor activity comparable to that of methotrexate (MTX), has been studied as inhibit or-cofactor ternary crystal complexes with wild-type Pneumocystis cari nii (pc) and recombinant human wild-type dihydrofolate reductase (hDHF R). These structural data provide the first direct comparison of the b inding interactions of the same antifolate inhibitor in the active sit e for pc and human DHFR. The human ternary DHFR complex crystallizes i n the rhombohedral space group R3 and is isomorphous to the ternary co mplex reported for a gamma-tetrazole methotrexate analogue, MTXT. The pcDHFR complex crystallizes in the monoclinic space group P2(1) and is isomorphous to that reported for a trimethoprim (TMP) complex. Interp retation of difference Fourier electron-density maps for these ternary complexes revealed that MTXO binds with its 2,4-diaminofuropyrimidine ring interacting with Glu32 in pc and Glu30 in human DHFR, as observe d for MTXT. The presence of the 6-5 furopyrimidine ring instead of the 6-6 pteridine ring results in a different bridge conformation compare d with that of MTXT. The bridge torsion angles for MTXO, i.e. C(4a)-C( 5)-C(8)-N(9) and C(5)-C(8)-N(9)-C(1'), are -156.5/51.9 degrees and -16 2.6/51.8 degrees, respectively for h and pc, compared with -146.8/57.4 degrees for MTXT. In each case, the p-aminobenzoylglutamate conformat ion is similar to that observed for MTXT. In the pcDHFR complex, the a ctive-site region is conserved and the additional 20 residues gin the sequence compared with the human enzyme are located in external loop r egions. There is a significant change in the nicotinamide ribose confo rmation of the cofactor which places the nicotinamide O atom close to the 4NH(2) group of MTXO (2.7 Angstrom), a shift not observed in hDHFR structures. As a consequence of this, there is a loss of a hydrogen b ond between the nicotinamide carbonyl group and the backbone of Ala12 in pcDHFR. In the human ternary complexes, the cofactor NADPH is bound with a more extended conformation, and the nicotinamide O atom makes a 3.5 Angstrom contact with the 4NH(2) group of MTXO. Although the nov el classical antifolate MTXO is not highly active against pcDHFR, ther e are correlations between its binding interactions consistent with it s lower potency as an inhibitor of h and pcDHFR compared with MTX.