Identification of novel non-hydroxamate anthrax toxin lethal factor inhibitors by topomeric searching, docking and scoring, and in vitro screening

Abstract

Anthrax is an infectious disease caused by Bacillus anthracis, a Gram-positive, rod-shaped, anaerobic bacterium. The lethal factor (LF) enzyme is secreted by B. anthracis as part of a tripartite exotoxin and is chiefly responsible for anthrax-related cytotoxicity. As LF can remain in the system long after antibiotics have eradicated B. anthracis from the body, the preferred therapeutic modality would be the administration of antibiotics together with an effective LF inhibitor. Although LF has garnered a great deal of attention as an attractive target for rational drug design, relatively few published inhibitors have demonstrated activity in cell-based assays and, to date, no LF inhibitor is available as a therapeutic or preventive agent. Here we present a novel in silico high-throughput virtual screening protocol that successfully identified 5 non-hydroxamic acid small molecules as new, preliminary LF inhibitor scaffolds with low micromolar inhibition against that target, resulting in a 12.8% experimental hit rate. This protocol screened approximately 35 million nonredundant compounds for potential activity against LF and comprised topomeric searching, docking and scoring, and drug-like filtering. Among these 5 hit compounds, none of which has previously been identified as a LF inhibitor, three exhibited experimental IC(50) values less than 100 microM. These three preliminary hits may potentially serve as scaffolds for lead optimization as well as templates for probe compounds to be used in mechanistic studies. Notably, our docking simulations predicted that these novel hits are likely to engage in critical ligand-receptor interactions with nearby residues in at least two of the three (S1', S1-S2, and S2') subsites in the LF substrate binding area. Further experimental characterization of these compounds is in process. We found that micromolar-level LF inhibition can be attained by compounds with non-hydroxamate zinc-binding groups that exhibit monodentate zinc chelation as long as key hydrophobic interactions with at least two LF subsites are retained.

Figure 1

Anthrax toxin lethal factor domains II–IV (residues 297–809) (1YQY.pdb14), colored by residuum order (N-terminus=blue, C-terminus=red), with catalytic Zn²⁺ (yellow sphere).

Figure 2

Substrate cleavage site of anthrax toxin lethal factor (1YQY.pdb14), with electrostatic potential mapping (red = positive, purple = negative); catalytic Zn²⁺ (pink sphere); Zn-chelating residues His686, His690, and Glu735; and illustrating three key subsite regions: S1’, S1–S2, and S2’.

Figure 3

Large-scale virtual screening protocol for small molecules against the anthrax toxin lethal factor.

Figure 4

Potent sulfonamide hydroxamate LF inhibitor 40, used as the topomeric searching template.

Figure 5

Ligand-receptor interaction diagram of GM6001/M364205 docked into the LF active site (1YQY.pdb14) (MOE 2007.09, Chemical Computing Group, Inc.) Green spheres = “greasy” residues; spheres with red outline = acidic residues; spheres with blue outline = basic residues; spheres with black outline = polar residues; blue background spheres = receptor exposure to solvent; blue spheres on ligand atoms = ligand exposure to solvent. Green dotted lines = sidechain donors/acceptors; blue dotted lines = backbone donors/acceptors; purple dotted line = metal contact; grey dotted line = proximity contour.

Figure 6

Three-dimensional renderings of the top three in vitro hits (hydrogens undisplayed) docked into the LF active site (1YQY.pdb14), with catalytic Zn²⁺ (grey sphere): (a) 5426202; (b) 5421384; and (c) 5428736 (SYBYL 8.1, Tripos, Inc).

Figure 6

Three-dimensional renderings of the top three in vitro hits (hydrogens undisplayed) docked into the LF active site (1YQY.pdb14), with catalytic Zn²⁺ (grey sphere): (a) 5426202; (b) 5421384; and (c) 5428736 (SYBYL 8.1, Tripos, Inc).

Figure 6

Three-dimensional renderings of the top three in vitro hits (hydrogens undisplayed) docked into the LF active site (1YQY.pdb14), with catalytic Zn²⁺ (grey sphere): (a) 5426202; (b) 5421384; and (c) 5428736 (SYBYL 8.1, Tripos, Inc).

Figure 7

Ligand-receptor interaction diagram of the top in vitro hit (5426202) docked into the LF active site (1YQY.pdb14) (MOE 2007.09, Chemical Computing Group, Inc.) Green spheres = “greasy” residues; spheres with red outline = acidic residues; spheres with blue outline = basic residues; spheres with black outline = polar residues; blue background spheres = receptor exposure to solvent; blue spheres on ligand atoms = ligand exposure to solvent. Green dotted lines = sidechain donors/acceptors; blue dotted lines = backbone donors/acceptors; purple dotted lines = metal contact; grey dotted line = proximity contour.