Structural analysis of human dihydrofolate reductase as a binary complex with the potent and selective inhibitor 2,4‐diamino‐6‐{2′‐ O ‐(3‐carboxypropyl)oxydibenz[ b , f ]‐azepin‐5‐yl}methylpteridine reveals an unusual binding mode

In order to understand the structure–activity profile observed for a series of substituted dibenz[ b , f ]azepine antifolates, the crystal structure of the binary complex of human dihydrofolate reductase (hDHFR) with the potent and selective inhibitor 2,4‐diamino‐6‐{2′‐ O ‐(3‐carboxypropyl)oxydibenz[ b , f ]‐azepin‐5‐yl}methylpteridine (PT684) was determined to 1.8 Å resolution. These data revealed that the carboxylate side chain of PT684 occupies two alternate positions, neither of which interacts with the conserved Arg70 in the active‐site pocket, which in turn hydrogen bonds to water. These observations are in contrast to those reported for the ternary complex of mouse DHFR (mDHFR) with NADPH [Cody et al. (2008), Acta Cryst. D 64 , 977–984], in which the 3‐carboxypropyl side chain of PT684 was hydrolyzed to its hydroxyl derivative, PT684a. The crystallization conditions differed for the human and mouse DHFR crystals (100 m M K 2 HPO 4 pH 6.9, 30% ammonium sulfate for hDHFR; 15 m M Tris pH 8.3, 75 m M sodium cacodylate, PEG 4K for mDHFR). Additionally, the side chains of Phe31 and Gln35 in the hDHFR complex have a single conformation, whereas in the mDHFR complex they occupied two alternative conformations. These data show that the hDHFR complex has a decreased active‐site volume compared with the mDHFR complex, as reflected in a relative shift of helix C (residues 59–64) of 1.2 Å, and a shift of 1.5 Å compared with the ternary complex of Pneumocystis carinii DHFR (pcDHFR) with the parent dibenz[ b , f ]azepine PT653. These data suggest that the greater inhibitory potency of PT684 against pcDHFR is consistent with the larger active‐site volume of pcDHFR and the predicted interactions of the carboxylate side chain with Arg75.