Crystal structure of Bacillus anthracis dihydrofolate reductase with the dihydrophthalazine-based trimethoprim derivative RAB1 provides a structural explanation of potency and selectivity

Abstract

Bacillus anthracis possesses an innate resistance to the antibiotic trimethoprim due to poor binding to dihydrofolate reductase (DHFR); currently, there are no commercial antibacterials that target this enzyme in B. anthracis. We have previously reported a series of dihydrophthalazine-based trimethoprim derivatives that are inhibitors for this target. In the present work, we have synthesized one compound (RAB1) displaying favorable 50% inhibitory concentration (54 nM) and MIC (< or =12.8 microg/ml) values. RAB1 was cocrystallized with the B. anthracis DHFR in the space group P2(1)2(1)2(1), and X-ray diffraction data were collected to a 2.3-A resolution. Binding of RAB1 causes a conformational change of the side chain of Arg58 and Met37 to accommodate the dihydrophthalazine moiety. Unlike the natural substrate or trimethoprim, the dihydrophthalazine group provides a large hydrophobic anchor that embeds within the DHFR active site and accounts for its selective inhibitory activity against B. anthracis.

FIG. 1.

Chemical synthesis of RAB1.

FIG. 2.

The well-defined density for RAB1 highlights a fit to the binding site that is complementary in shape and composition. (A) Electron density from refinement prior to the addition of RAB1, contoured at 1σ. (B) RAB1 structure and plot of intermolecular contacts: magenta, 2,4-diaminopyrimidine group; blue, dimethoxybenzyl ring; green, dihydrophthalazine moiety. The acryloyl linker is uncolored; the enantiomeric carbon is denoted by an asterisk. Residues depicted in blue are involved in hydrogen bonds to RAB1, denoted by green dashed lines. Red hatched semicircles indicate hydrophobic interactions. (C) B. anthracis DHFR structure with the surface indicated in gray and RAB1 surface indicated in red. Both the N and C termini are obscured at the back of the molecule in this view.

FIG. 3.

Key interactions modulate the conformation between B. anthracis DHFR and RAB1. (A) The side chain of Arg53 would sterically interfere with binding of the opposite enantiomer; only the S-enantiomer of RAB1 can be accommodated. (B) The side chain of Leu55 conforms to the observed bend in the dihydropyridazine ring of RAB1's dihydrophthalazine moiety. (C) The side chains of Arg58 and Met37 undergo a conformational change (indicated from gray to yellow) to accommodate the dihydrophthalazine moiety of RAB1.

FIG. 4.

Superposition of ligands with RAB1 (gray) reveals minor shifts in Phe96 and otherwise conserved binding site architecture reflected by the similarity of ligand conformation. (A) TMP (yellow); (B) MTX (cyan); (C) C17 (magenta).

FIG. 5.

RAB1 cannot fit in the human DHFR binding site due to steric clashes, and no other inhibitors with structural data possess the same determinants. (A) Human DHFR (PDB ID code 1OHJ [18]) with RAB1 (van der Waals surface shown) superpositioned into the binding site. Human residues Phe34, Asn64, and Arg70 are shown as van der Waals spheres. (B and C) Two-dimensional structures of Iclaprim (B) and AR-709 (C). (D to F) Superposition of RAB1 (gray) with 5-[ω-carboxy(alkyloxy)] TMP derivatives (cyan, PDB ID code 2FZH) (21) (D); PT523, also a TMP derivative (magenta, PDB ID code 1OHJ) (18) (E); and pyrimethamine (yellow, PDB ID code 2BLB) (37) (F). A fortuitous buffer molecule (MES) is in a position roughly equivalent with the acryloyl linker-dihydrophthalazine moiety of RAB1. This indicates unfilled binding site volume in the presence of pyrimethamine, allowing the buffer molecule room to bind, that in turn is filled by RAB1.