Novel inhibitors of anthrax edema factor

Abstract

Several pathogenic bacteria produce adenylyl cyclase toxins, such as the edema factor (EF) of Bacillus anthracis. These disturb cellular metabolism by catalyzing production of excessive amounts of the regulatory molecule cAMP. Here, a structure-based method, where a 3D-pharmacophore that fit the active site of EF was constructed from fragments, was used to identify non-nucleotide inhibitors of EF. A library of small molecule fragments was docked to the EF-active site in existing crystal structures and those with the highest HINT scores were assembled into a 3D-pharmacophore. About 10,000 compounds, from over 2.7 million compounds in the ZINC database, had a similar molecular framework. These were ranked according to their docking scores, using methodology that was shown to achieve maximum accuracy (i.e., how well the docked position matched the experimentally determined site for ATP analogues in crystal structures of the complex). Finally, 19 diverse compounds with the best AutoDock binding/docking scores were assayed in a cell based assay for their ability to reduce cAMP secretion induced by EF. Four of the test compounds, from different structural groups, inhibited in the low micromolar range. One of these has a core structure common to phosphatase inhibitors previously identified by high-throughput assays of a diversity library. Thus, the fragment based pharmacophore identified a small number of diverse compounds for assay, and greatly enhanced the selection process of advanced lead compounds for combinatorial design.

Effects of Introducing a Single Charged Residue into the Phenylalanine Clamp of Multimeric Anthrax Protective Antigen

“…The exact location of the electrostatic barrier within the pore is not known, but mutating Phe 427 to Ala caused the pore to become less selective for K ϩ over Cl Ϫ and lowered the barrier to translocation of LF N bearing an SO 3 Ϫ group (36). These findings, together with the fact that two acidic residues, Asp 425 and Asp 426 , are immediately adjacent to Phe 427 , suggest that the main barrier lies at the level of the Phe clamp.…”

“…Anthrax toxin consists of three nontoxic proteins that co-assemble to produce a series of free or cell-bound toxic complexes (1). Two of the proteins, Lethal Factor (LF) 3 and Edema Factor (EF), are enzymes that modify cytosolic targets (2,3). The third, Protective Antigen (PA), is a receptor-binding and pore-forming protein that transports LF and EF to the cytosol (4).…”

Use of allostery to identify inhibitors of calmodulin-induced activation of Bacillus anthracis edema factor

“…TUAdiCl compares favorably with several compounds previously reported to inhibit EF-CaM association in vitro or in vivo with IC 50 values in the 10-100 μM range (25,27,28) and represents a new chemical family.…”

“…It is able to enter into eukaryotic cells where it is activated upon binding to endogenous calmodulin (CaM) to produce supraphysiological levels of cAMP that alter the cell physiology. By targeting immune cells, EF contributes to the virulence of B. anthracis and is therefore considered as a target for anti-anthrax drugs (25)(26)(27)(28).…”

Virtual Screening of Penicillin-derived Inhibitors for the Metallo-β-lactamase from Bacillus cereus

“…[14][15][16] AutoDock is a molecular modeling program which has been previously used for designing inhibitors. [17][18][19][20] It predicts the docking of flexible small molecules, or drug candidates, to receptors of enzymes and macromolecules. This program also provides a free-energy force field to evaluate docking conformations and binding energies during the docking simulations.…”