Counter Selection Substrate Library Strategy for Developing Specific Protease Substrates and Probes

Cell Chem Biol. 2016 Aug 18;23(8):1023-35. doi: 10.1016/j.chembiol.2016.05.020. Epub 2016 Jul 28.


Legumain (AEP) is a lysosomal cysteine protease that was first characterized in leguminous seeds and later discovered in higher eukaryotes. AEP upregulation is linked to a number of diseases including inflammation, arteriosclerosis, and tumorigenesis. Thus this protease is an excellent molecular target for the development of new chemical markers. We deployed a hybrid combinatorial substrate library (HyCoSuL) approach to obtain P1-Asp fluorogenic substrates and biotin-labeled inhibitors that targeted legumain. Since this approach led to probes that were also recognized by caspases, we introduced a Counter Selection Substrate Library (CoSeSuL) approach that biases the peptidic scaffold against caspases, thus delivering highly selective legumain probes. The selectivity of these tools was validated using M38L and HEK293 cells. We also propose that the CoSeSuL methodology can be considered as a general principle in the design of selective probes for other protease families where selectivity is difficult to achieve by conventional sequence-based profiling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Combinatorial Chemistry Techniques*
  • Cysteine Endopeptidases / chemistry
  • Cysteine Endopeptidases / metabolism*
  • Enzyme Inhibitors / chemical synthesis
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology*
  • Humans
  • Hydrogen-Ion Concentration
  • Molecular Conformation
  • Molecular Probes / chemical synthesis
  • Molecular Probes / chemistry
  • Molecular Probes / pharmacology*
  • Substrate Specificity


  • Enzyme Inhibitors
  • Molecular Probes
  • Cysteine Endopeptidases
  • asparaginylendopeptidase