Inhibition of Metalloprotease Botulinum Serotype A from a Pseudo- Peptide Binding Mode to a Small Molecule that is Active in Primary Neurons

摘要

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. Database 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 synaptosomal-associated 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 a1:1 inhibitor:enzyme ratio and a dissociation constant of 4.6 M.