X-ray structure of the ternary MTX.NADPH complex of the anthrax dihydrofolate reductase: a pharmacophore for dual-site inhibitor design

Abstract

For reasons of bioterrorism and drug resistance, it is imperative to identify and develop new molecular points of intervention against anthrax. Dihydrofolate reductase (DHFR) is a highly conserved enzyme and an established target in a number of species for a variety of chemotherapeutic programs. Recently, the crystal structure of Bacillus anthracis DHFR (baDHFR) in complex with methotrexate (MTX) was determined and, based on the structure, proposals were made for drug design strategies directed against the substrate-binding site. However, little is gleaned about the binding site for NADPH, the cofactor responsible for hydride transfer in the catalytic mechanism. In the present study, X-ray crystallography at 100 K was used to determine the structure of baDHFR in complex with MTX and NADPH. Although the NADPH binding mode is nearly identical to that seen in other DHFR ternary complex structures, the adenine moiety adopts an off-plane tilt of nearly 90 degrees and this orientation is stabilized by hydrogen bonds to functionally conserved Arg residues. A comparison of the binding site, focusing on this region, between baDHFR and the human enzyme is discussed, with an aim at designing species-selective therapeutics. Indeed, the ternary model, refined to 2.3 A resolution, provides an accurate template for testing the feasibility of identifying dual-site inhibitors, compounds that target both the substrate and cofactor-binding site. With the ternary model in hand, using in silico methods, several compounds were identified which could potentially form key bonding contacts in the substrate and cofactor-binding sites. Ultimately, two structurally distinct compounds were verified that inhibit baDHFR at low microM concentrations. The apparent Kd for one of these, (2-(3-(2-(hydroxyimino)-2-(pyridine-4-yl)-6,7-dimethylquinoxalin-2-yl)-1-(pyridine-4-yl)ethanone oxime), was measured by fluorescence spectroscopy to be 5.3 microM.

Fig. 1

Chemical structures of MTX, NADPH, DHF, compounds 1357 and 373265. For clarity, only the atom names are provided for the moieties of MTX, NADPH and DHF that bind within the baDHFR active site.

Fig. 2

Crystal structure of (a) Bacillus anthracis (ba) DHFR in a ternary complex with MTX and NADPH. DHFR is shown as a cartoon representation. 2F_o−F_c electron density for MTX (magenta) and NADPH (dark green) is shown contoured at 1.2σ. (b) and (c) Schematic representation of the (d) MTX and (e) NADPH binding sites in baDHFR, as generated by LIGPLOT (Wallace et al., 1995).

Fig. 3

Characterization of the MTX and NADPH binding sites. (a) Superposition of ba (green), ec (magenta) and hs (cyan) DHFRs. The structures are displayed as Cα traces. Labeled numbers correspond to the four major contiguous insertion regions in the hsDHFR sequence, not found in the baDHFR and ecDHFR sequences (Bennett et al., 2007). Two views are shown, related by a 90° turn about the y-axis. The hsDHFR 61–64 insertion, seen in the left-hand panel, protrudes into the MTX p-ABA binding site of prokaryotic DHFRs including baDHFR and can be exploited in species-selective drug design, as described in (Bennett et al., 2007). (b) The nicotinamide and pteridine rings of NADPH and MTX, respectively, partially overlap in the active site. The closest interligand atoms that contact each other occur between the nicotinamide O7 and the pteridine N3 atoms: the contact distances are 3.5Å, 3.8Å and 3.7Å in baDHFR, ecDHFR and hsDHFR, respectively. (c) The adenine ring of NADPH in baDHFR is severely tilted (~85°) relative to the position of this same moiety in ecDHFR and hsDHFR. In ba and ecDHFR, functionally conserved Arg residues form stabilizing hydrogen bonds to the NADPH adenine; however, the Arg residue in hsDHFR is only close enough to the adenine to form van der Waals interactions.

Fig. 4

In silico design of a pharmacophore against the MTX and NADPH binding sites of baDHFR and in vitro inhibition assays. (a) The pharmacophore was designed to have 5 interaction features as constraints. Three of them are hydrophobic, which all are aromatic rings: the diaminopyrimidine ring of the MTX pteridine ring, the p-ABA ring of MTX and the nicotinamide ring of NADPH. (b) and (c) Curves used for determining IC₅₀ values against baDHFR for (b) compound 1357 and (c) compound 373265. (d) Docking of compound 1357 in the baDHFR active site. (e) Docking of compound 373265 in the baDHFR active site. To demonstrate that both compounds dock into both the substrate and cofactor binding sites, MTX and NADPH are shown from the baDHFR crystal structure. In (d) and (e), carbons are colored green for compounds 1357 and 373265, magenta for MTX, and cyan for NADPH. Residues from baDHFR that form hydrogen bonds with the docked compound are shown in red. For clarity, moieties of the NADPH distant from the active site are not shown.

Fig. 5

Tryptophan fluorescence quenching of baDHFR due to the binding of compound 373265. (a) Tryptophan fluorescence spectra of compound only (black squares) and baDHFR (0.09 mg/ml (5 μM) in 15 mM Bis-Tris (pH 5.5) containing 500 mM NaCl) at various μM concentrations of 373265 (0 μM, red squares; 4, pink; 10, blue; and 40, green). (b) Variation of the extent of fluorescence quenching (F₀−F/F₀, where F₀ and F are the fluorescence intensities at 342 nm in the absence and presence of compound 373265) of 0.09 mg/ml (5μM) of DHFR as a function of 373265 concentration.