Synthetic and crystallographic studies of a new inhibitor series targeting Bacillus anthracis dihydrofolate reductase

Abstract

Bacillus anthracis, the causative agent of anthrax, poses a significant biodefense danger. Serious limitations in approved therapeutics and the generation of resistance have produced a compelling need for new therapeutic agents against this organism. Bacillus anthracis is known to be insensitive to the clinically used antifolate, trimethoprim, because of a lack of potency against the dihydrofolate reductase enzyme. Herein, we describe a novel lead series of B. anthracis dihydrofolate reductase inhibitors characterized by an extended trimethoprim-like scaffold. The best lead compound adds only 22 Da to the molecular weight and is 82-fold more potent than trimethoprim. An X-ray crystal structure of this lead compound bound to B. anthracis dihydrofolate reductase in the presence of NADPH was determined to 2.25 A resolution. The structure reveals several features that can be exploited for further development of this lead series.

Figure 1

Homology model of BaDHFR bound to compound.

Figure 2

Homology model of BaDHFR bound to compound 4.

Figure 3

Electron density for the active site of the ternary complex of BaDHFR including compound 17. Density was contoured at 2σ.

Figure 4

(A) overall structure of BaDHFR bound to NADPH and 17 and a comparison with (B) overall binary complex with MTX (PDB 2QK815) and (C) residues and ligands at the active site.

Figure 5

Detailed interactions of the ligand with the active site residues. (A) Stereoview of the active site with NADPH in magenta and compound 17 in blue. Water molecules are shown as “x”, and orange dashed lines indicate hydrogen bonds. (B) Two-dimensional depiction of protein-ligand interactions. Hydrogen bonds are noted with dashed lines and distance measurements.

Figure 6

Structural alignment of BaDHFR and hDHFR: (left) structure-based sequence alignment, where residues in the active site are shown in red; (right) superposition of BaDHFR (green) and hDHFR (purple) with active site residue substitutions labeled.

Figure 7

Surface depiction of binding pocket surrounding compound 17. The surface is colored with a lipophilicity gradient. Compound 17 and NADPH are shown as gray sticks with atom colors. Figure was generated by Sybyl 8.0.

Scheme 1

(a) Ph₃PCH₂OMeCl, NaO^tBu, THF, 0° C; (b) 10% HCl, THF, reflux; (c) CBr₄, PPh₃, CH₂Cl₂, 0° C; (d) Mg, THF; (e) CuI, Pd(PPh₃)₂Cl₂, Et₃N, DMF, 60° C.

Scheme 2