Inhibition of metalloprotease botulinum serotype A from a pseudo-peptide binding mode to a small molecule that is active in primary neurons

J Biol Chem. 2007 Feb 16;282(7):5004-5014. doi: 10.1074/jbc.M608166200. Epub 2006 Nov 8.

Abstract

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 microM. 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 a 1:1 stoichiometry and a dissociation constant of 4.6 microM.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Botulinum Toxins, Type A / chemistry*
  • Botulinum Toxins, Type A / metabolism
  • Botulism / drug therapy
  • Botulism / enzymology
  • Cells, Cultured
  • Chick Embryo
  • Metalloproteases / chemistry*
  • Metalloproteases / metabolism
  • Models, Molecular*
  • Neurons / chemistry*
  • Neurons / enzymology
  • Protease Inhibitors / chemistry*
  • Protease Inhibitors / metabolism
  • Protease Inhibitors / therapeutic use

Substances

  • Protease Inhibitors
  • Metalloproteases
  • Botulinum Toxins, Type A

Associated data

  • PDB/2ISE
  • PDB/2ISG
  • PDB/2ISH