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

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

Author information

1
Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb.Wyspianskiego 27, 50-370 Wroclaw, Poland; Program in Cell Death and Survival Networks, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA. Electronic address: marcin.poreba@pwr.edu.pl.
2
Program in Cell Death and Survival Networks, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA; Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, 0316 Oslo, Norway. Electronic address: rigmor.solberg@farmasi.uio.no.
3
Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb.Wyspianskiego 27, 50-370 Wroclaw, Poland.
4
Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, 0316 Oslo, Norway.
5
Program in Cell Death and Survival Networks, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA.
6
Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, 1000 Ljubljana, Slovenia.
7
Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb.Wyspianskiego 27, 50-370 Wroclaw, Poland. Electronic address: marcin.drag@pwr.edu.pl.

Abstract

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.

PMID:
27478158
PMCID:
PMC5939596
DOI:
10.1016/j.chembiol.2016.05.020
[Indexed for MEDLINE]
Free PMC Article

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