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 thirty-five million non-redundant 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 µM. 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.

Computational Methods in Drug Discovery

“…Pierce et al (2008) used GLIDE to dock approximately 700,000 commercially available compounds and identified four compounds with K i values less than 5 mM. Chiu et al (2009) used SURFLEX to identify novel inhibitors of anthrax toxin lethal factor responsible for anthrax-related cytotoxicity. Docking study of a compound library derived from seven data bases, including DrugBank (Wishart et al, 2006), ZINC (Irwin and Shoichet, 2005), National Cancer Institute data base (Milne et al, 1994), etc., identified lead compounds that eventually led to the development of nanomolar inhibitors upon optimization.…”

Inhibitors of the Metalloproteinase Anthrax Lethal Factor

“…Amin and co-workers virtually screened 35 million compounds by “topomeric” shape-based screening, in which compounds were scored by 3D shape similarity to the Merck inhibitor L915 [51]. The 22,133 top scoring hits were then computationally docked onto the LF crystal structure, yielding about 300 compounds scoring better than L915 and predicted to have drug-like properties.…”

Small molecule inhibitors of anthrax lethal factor toxin

“…9 Although these proteins are independently nontoxic, their concerted action disrupts cell signaling events and can lead to cell death. Importantly, lethal factor combines with protective antigen to form the lethal factor toxin.…”