NMR SOLUTION STRUCTURE OF THE ANTITUMOR COMPOUND PT523 AND NADPH IN THE TERNARY COMPLEX WITH HUMAN DIHYDROFOLATE-REDUCTASE

The antitumor compound PT523 4-deoxypteroyl)-N-delta-hemiphthaloyl-L-o rnithine] was found to have an inhibition constant (K-i) of 0.35 +/- 0 .10 pM against human dihydrofolate reductase (hDHFR), 15-fold lower th an that of the classical antifolate drug methotrexate (MTX). The struc ture of PT523 bound to hDHFR and hDHFR-NADPH was investigated using mu ltinuclear NMR techniques. NMR data indicate that the binary complex h as two distinct conformations in solution which are in slow exchange a nd that the addition of NADPH stabilizes the ternary complex in a sing le bound state. Comparison of resonance assignments in the PT523 and M TX ternary complexes revealed that substantial protein chemical shift differences are limited to small regions of hDHFR tertiary structure. A restrained molecular dynamics and energy minimization protocol was p erformed for the hDHFR-PT523-NADPH complex, using 185 NOE restraints ( 33 intermolecular) to define the ligand-binding region. The positions of the pteridine and pABA rings of PT523 and the nicotinamide and ribo se rings of NADPH are well defined in the solution structures (RMSD = 0.59 Angstrom) and are consistent with previously determined structure s of DHFR complexes. The N-delta-hemiphthaloyl-L-omithine group of PT5 23 is less well defined, and the calculated model structures suggest t he hemiphthaloyl ring may adopt more than one conformation in solution . Contacts between the hemiphthaloyl ring and hDHFR, which are not pos sible in the hDHFR-MTX-NADPH complex, may explain the greater inhibiti on potency of PT523.