The virulent spore-forming bacterium Bacillus anthracis secretes anthrax toxin composed of protective antigen (PA), lethal factor (LF) and edema factor (EF). LF is a Zn-dependent metalloprotease that inactivates key signaling molecules, such as mitogen-activated protein kinase kinases (MAPKK), to ultimately cause cell death. We report here the identification of small molecule (nonpeptidic) inhibitors of LF. Using a two-stage screening assay, we determined the LF inhibitory properties of 19 compounds. Here, we describe six inhibitors on the basis of a pharmacophoric relationship determined using X-ray crystallographic data, molecular docking studies and three-dimensional (3D) database mining from the US National Cancer Institute (NCI) chemical repository. Three of these compounds have K(i) values in the 0.5-5 microM range and show competitive inhibition. These molecular scaffolds may be used to develop therapeutically viable inhibitors of LF.
An efficient research strategy integrating empirically guided, structure-based modeling and chemoinformatics was used to discover potent small molecule inhibitors of the botulinum neurotoxin serotype A light chain. First, a modeled binding mode for inhibitor 2-mercapto-3-phenylpropionyl-RATKML (K i ؍ 330 nM) was generated, and required the use of a molecular dynamic conformer of the enzyme displaying the reorientation of surface loops bordering the substrate binding cleft. These flexible loops are conformationally variable in x-ray crystal structures, and the model predicted that they were pivotal for providing complementary binding surfaces and solvent shielding for the pseudo-peptide. The docked conformation of 2-mercapto-3-phenylpropionyl-RATKML was then used to refine our pharmacophore for botulinum serotype A light chain inhibition. Data base search queries derived from the pharmacophore were employed to mine small molecule (non-peptidic) inhibitors from the National Cancer Institute's Open Repository. Four of the inhibitors possess K i values ranging from 3.0 to 10.0 M.Of these, NSC 240898 is a promising lead for therapeutic development, as it readily enters neurons, exhibits no neuronal toxicity, and elicits dose-dependent protection of synaptosomalassociated protein (of 25 kDa) in a primary culture of embryonic chicken neurons. Isothermal titration calorimetry showed that the interaction between NSC 240898 and the botulinum A light chain is largely entropy-driven, and occurs with a 1:1 stoichiometry and a dissociation constant of 4.6 M.
Botulinum neurotoxins (BoNTs)3 are the most potent of the biological toxins (1), and are listed as category A (highest priority) bioterror agents by the Centers for Disease Control and Prevention. They may be delivered by aerosol route (1, 2), and consequently represent a serious threat to both military personnel and civilians (3, 4). Moreover, BoNTs are now established biotherapeutics for a range of physical ailments and cosmetic treatments (2, 5-8), making their misuse and/or adverse side effects (9) more likely. Neither the currently available BoNT antitoxin nor antibodies can counter these toxins once they are inside neurons; currently, critical care mechanical ventilation is the only life-saving treatment option. However, the effects of internalized BoNTs can last for months (10), and mechanical ventilation would be impractical if even a limited number of individuals were clandestinely/accidentally intoxicated. Thus, there is an urgent need to identify and develop small molecule (non-peptidic) inhibitors (SMNPIs) that can serve as both prophylactics and post-exposure therapeutics.BoNTs are composed of a heavy chain and a light chain (LC) that are connected by a disulfide bridge (11). The heavy chain binds to neurons and transports the LC into the cytosol (12). The LC is a zinc metalloprotease. Each of the seven BoNT serotypes (A-G) cleaves a component of the SNARE (soluble NSFethylmaleimide-sensitive factor attachment protein receptor) proteins (13), which mediate ...
Type A botulinum neurotoxin catalyzed the hydrolysis of synthetic peptides based on the sequence of the 25-kD synaptosomal protein SNAP-25. In each peptide, the toxin cleaved at a single glutaminyl-arginine bond corresponding to residues 197 and 198 of SNAP-25, confirming earlier reports on the enzymatic specificity of the toxin in synaptosomal preparations. Metal chelators inhibited catalysis, consistent with a metalloprotease activity. In contrast to tetanus toxin and other botulinum toxin serotypes, type A toxin hydrolyzed relatively short, 17- to 20-residue peptides. In the substrates, SNAP-25 residue 202 and one or more of residues 187-191 were required for efficient hydrolysis, but residues 167-186 and 203-206 were not. The highest rates of hydrolysis were found when the C-terminal residues of the peptides were amidated.
The 2.7 A structure of the tetanus neurotoxin receptor binding fragment Hc reveals a jelly-roll domain and a beta-trefoil domain. Hc retains the unique transport properties of the holotoxin and is capable of eliciting a protective immunological response against the full length holotoxin.
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