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

Abstract

Allostery plays a key role in the regulation of the activity and function of many biomolecules. And although many ligands act through allostery, no systematic use is made of it in drug design strategies. Here we describe a procedure for identifying the regions of a protein that can be used to control its activity through allostery. This procedure is based on the construction of a plausible conformational path, which describes protein transition between known active and inactive conformations. The path is calculated by using a framework approach that steers and markedly improves the conjugate peak refinement method. The evolution of conformations along this path was used to identify a putative allosteric site that could regulate activation of Bacillus anthracis adenylyl cyclase toxin (EF) by calmodulin. Conformations of the allosteric site at different steps along the path from the inactive (free) to the active (bound to calmodulin) forms of EF were used to perform virtual screenings and propose candidate EF inhibitors. Several candidates then proved to inhibit calmodulin-induced activation in an in vitro assay. The most potent compound fully inhibited EF at a concentration of 10 μM. The compounds also inhibited the related adenylyl cyclase toxin from Bordetella pertussis (CyaA). The specific homology between the putative allosteric sites in both toxins supports that these pockets are the actual binding sites of the selected inhibitors.anthrax | transition path calculation | molecular dynamics | drug discovery | inhibition of protein-protein association

Allosteric Activation of Bordetella pertussis Adenylyl Cyclase by Calmodulin

“…The AC protein content was determined based on the absorption spectra using an extinction coefficient at 278 nm of 28,000 M Ϫ1 /cm Ϫ1 . AC Enzymatic Activity Assays-The activity of AC was measured using a sensitive colorimetric assay, which was reported recently (27). AC converts ATP into cAMP and pyrophosphate (PP i ), and the latter can be hydrolyzed further by an exogenously added pyrophosphatase into two phosphate molecules (P i ), which can be quantified using a standard colorimetric assay based on the change in the absorbance of malachite green dye in the presence of phosphomolybdate complexes.…”

Identification of a Region That Assists Membrane Insertion and Translocation of the Catalytic Domain of Bordetella pertussis CyaA Toxin

“…Molar ⑀ values of 28,880 M Ϫ1 cm Ϫ1 and 35,870 M Ϫ1 cm Ϫ1 were computed from the sequence of AC384 and AC489, respectively. Enzymatic activity was measured as described previously (32).…”

An overview of investigational toxin-directed therapies for the adjunctive management ofBacillus anthracisinfection and sepsis

“…Adefovir inhibited EF-stimulated cAMP production and its cardiovascular effects in perfused heart and aortic ring models [22,25]. Other agents shown to have the potential to inhibit EF's enzymatic activity include 4-[4-(4-nitrophenyl)-thiazolylamino]-benzene-sulfonamide [140,141], 3-{(9-oxo-9H-fluorene-1-carbonyl)-amino]-benzoic acid [142,143], and (M)Ant nucleotides [144-149] and allosteric inhibitors [150]. …”