In situ assembly of enzyme inhibitors using extended tethering

Nat Biotechnol. 2003 Mar;21(3):308-14. doi: 10.1038/nbt786. Epub 2003 Feb 3.

Abstract

Cysteine aspartyl protease-3 (caspase-3) is a mediator of apoptosis and a therapeutic target for a wide range of diseases. Using a dynamic combinatorial technology, 'extended tethering', we identified unique nonpeptidic inhibitors for this enzyme. Extended tethering allowed the identification of ligands that bind to discrete regions of caspase-3 and also helped direct the assembly of these ligands into small-molecule inhibitors. We first designed a small-molecule 'extender' that irreversibly alkylates the cysteine residue of caspase-3 and also contains a thiol group. The modified protein was then screened against a library of disulfide-containing small-molecule fragments. Mass-spectrometry was used to identify ligands that bind noncovalently to the protein and that also form a disulfide linkage with the extender. Linking the selected fragments with binding elements from the extenders generates reversible, tight-binding molecules that are druglike and distinct from known inhibitors. One molecule derived from this approach inhibited apoptosis in cells.

Publication types

  • Evaluation Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Apoptosis / drug effects
  • Caspase 3
  • Caspase Inhibitors*
  • Caspases / chemistry*
  • Caspases / metabolism
  • Combinatorial Chemistry Techniques / methods*
  • Enzyme Inhibitors / chemical synthesis*
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / classification
  • Enzyme Inhibitors / pharmacology*
  • Humans
  • Jurkat Cells / drug effects*
  • Jurkat Cells / metabolism
  • Mass Spectrometry / methods
  • Models, Molecular
  • Peptide Library

Substances

  • Caspase Inhibitors
  • Enzyme Inhibitors
  • Peptide Library
  • CASP3 protein, human
  • Caspase 3
  • Caspases

Associated data

  • PDB/1NME
  • PDB/1NMQ
  • PDB/1NMS