Structural studies on bioactive compounds. 30. Crystal structure and molecular modeling studies on the Pneumocystis carinii dihydrofolate reductase cofactor complex with TAB, a highly selective antifolate

The crystal structure of the ternary complex of NADPH, the potent antifolat e [2,4-diamino-5-{3-[3-(2-acetyloxyethyl)-3-benzyltriazen-1-yl]-4-chlorophe nyl}-6-ethylpyrimidine] (TAB, 1) and Pneumocystis carinii dihydrofolate red uctase (pcDHFR), refined to 2.1 Angstrom resolution, reveals that TAB binds similar to the antifolates trimethoprim and methotrexate. These data also reveal multiple conformations for the binding geometry of TAB with two pref erred orientations of the acetyloxy and benzyl groups that results from a 1 80 degrees rotation about the N2-N3 triazenyl bond. The methyl of the acety loxy and benzyl ring of TAB probes large hydrophobic regions of the p-amino benzoyl folate binding pocket of the active site, in particular the region near Phe69, which is unique to the pcDHFR sequence. These results confirm p rior molecular modeling investigations of the binding of TAB to pcDHFR that identified four low-energy binding geometries, two involving rotations abo ut the terminal N(2)-N(3) triazenyl linkage and two involving atropisomeris m about the pivotal pyrimethamine-phenyl bend. The primary differences in t he molecular dynamics (MD) models and those observed in this crystal comple x result from small conformational changes in active-sits residues on energ y minimization. However, two MD models place the acetyloxy and benzyl ring groups in a region of the active site between the cofactor-binding region a nd the p-aminobenzoyl folate pocket; an orientation never observed in any D HFR crystal structure to date. These conformers interact with solvent near the enzyme surface and are probably not observed due to the loss of specifi c hydrogen bonds with the enzyme. The high species pcDHFR selectivity of TA B could be the result of ligand flexibility that enables multiple binding o rientations at the enzyme active site. Further modification of the acetylox y region of TAB could increase its potency and selectivity for pcDHFR.