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

Abstract

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. D64, 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 mM K2HPO4 pH 6.9, 30% ammonium sulfate for hDHFR; 15 mM Tris pH 8.3, 75 mM 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.

The Structural Basis for Nonsteroidal Anti-Inflammatory Drug Inhibition of Human Dihydrofolate Reductase

“…Information derived from the observed NMR ILOE interactions and the quaternary hsDHFR·NADP + ·DAP·R-naproxen crystal structure enabled us to position additional NSAIDs in the pABG subsite of DHFR (Figure and Table S1). Although it is remarkable that this site can accommodate the range of structures characterizing these NSAIDs, a review of the relevant literature indicates that antifolate drugs exhibit limited variability in the pteridine-binding pharmacophore but a similarly enormous variability of the pABG-site pharmacophore. − …”

Manipulating the substrate specificity of murine dihydrofolate reductase enzyme using an expanded set of amino acids

“…Therefore, the delivery of human DHFR variants efficiently catalyzing the conversion reaction of FOL or the human DHFR genes into megaloblastic anemia patients is a potential therapeutic strategy. Second, the crystal structures of DHFRs bound to substrates are readily available facilitating the identification of key residues for the substrate binding/computational active site design [43][44][45][46]. Third, both DHF and THF are much more expensive than FOL, restricting their therapeutic applications.…”

Controlling enzyme inhibition using an expanded set of genetically encoded amino acids

“…Therefore, inhibiting DHFR activities is a very active research area (Cody et al, 2005(Cody et al, , 2006(Cody et al, , 2011bVolpato and Pelletier, 2009). Second, the crystal structures of DHFRs bound to their inhibitors and substrates are readily available facilitating the identification of key residues for the inhibitor and substrate binding (Cody et al, 2005(Cody et al, , 2006(Cody et al, , 2011a. Third, the expression and purification of DHFRs in E. coli are well established (Kwon and Tirrell, 2007;Kwon et al, 2006).…”