NMR solution structure of the antitumor compound PT523 and NADPH in the ternary complex with human dihydrofolate reductase.
The antitumor compound PT523 [N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L- ornithine] was found to have an inhibition constant (K(i)) of 0.35 +/- 0.10 pM against human dihydrofolate reductase (hDHFR), 15-fold lower than that of the classical antifolate drug methotrexate (MTX). The structure of PT523 bound to hDHFR and hDHFR-NADPH was investigated using multinuclear NMR techniques. NMR data indicate that the binary complex has two distinct conformations in solution which are in slow exchange and that the addition of NADPH stabilizes the ternary complex in a single bound state. Comparison of resonance assignments in the PT523 and MTX 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 performed 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 ribose rings of NADPH are well defined in the solution structures (RMSD = 0.59 A) and are consistent with previously determined structures of DHFR complexes. The N(delta)-hemiphthaloyl-L-ornithine group of PT523 is less well defined, and the calculated model structures suggest the hemiphthaloyl ring may adopt more than one conformation in solution. Contacts between the hemiphthaloyl ring and hDHFR, which are not possible in the hDHFR-MTX-NADPH complex, may explain the greater inhibition potency of PT523.
Johnson
J M
JM
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Meiering
E M
EM
Wright
J E
JE
Pardo
J
J
Rosowsky
A
A
Wagner
G
G
D000970:Antineoplastic Agents / Q000737:chemistry*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D005493:Folic Acid Antagonists / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008727:Methotrexate / Q000737:chemistry; D009249:NADP / Q000737:chemistry*; D009952:Ornithine / Q000031:analogs & derivatives* / Q000737:chemistry; D011487:Protein Conformation; D011622:Pterins / Q000737:chemistry*; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*
D000970:Antineoplastic Agents; D005493:Folic Acid Antagonists; D008024:Ligands; D011622:Pterins; C085590:N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine; D009249:NADP; D009952:Ornithine; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate