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 (<12.8 g/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-Å 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.

Inhibition of Antibiotic-Resistant Staphylococcus aureus by the Broad-Spectrum Dihydrofolate Reductase Inhibitor RAB1

“…All contacts between the phthalazine moiety and the protein are hydrophobic. The enantiomeric preference for the propyl group in this conformation is the same as previously observed, with the S-enantiomer appearing to be the only occupant (4). While the propyl tail is not in direct contact with protein atoms, its path follows that of residue Lys 29.…”

“…For B. anthracis, the half-maximal inhibitory concentration (IC 50 ) of purified DHFR enzyme with TMP is 77 M, indicating a natural resistance, while with RAB1 it is Ϸ60 nM (3). Selectivity for the binding site in B. anthracis DHFR is controlled by the large hydrophobic phthalazine moiety, which is embedded within and causes extension of the binding site (4). Concomitant with the current work, the broad-spectrum activities of related RAB1-like molecules have been demonstrated by Basilea Pharmaceutica International AG.…”

“…Synthesis of RAB1 and purification of its component enantiomers have been previously described (4,5).…”

Modified 2,4-diaminopyrimidine-based dihydrofolate reductase inhibitors as potential drug scaffolds against Bacillus anthracis

“…2 It is also well known that these engineered strains have innate resistance to current commercial drugs. 3-6 Thus, there is a compelling and imminent need to develop new therapeutic agents to treat these resistant bacteria.…”

4,5-Dimethylbenzene-1,2-dimethanol

“…The rigid planarity of the ring system allows close positioning of reactive centers such that additional fused rings can be readily appended. We recently needed multi-gram quantities of the title compound as a building block to prepare 6,7-dimethylphthalazine for use in the synthesis of dihydrophthalazine-based antibiotics [1][2][3][4][5][6]. Though the compound is commercially available, it is quite expensive, and the supplier was unable to meet our requirements.…”