title: Counter Selection Substrate Library Strategy for Developing Specific Protease Substrates and Probes.
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.
authors.affiliation: 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.
authors.forename: Marcin
authors.initials: M
authors.lastname: Poreba
authors.affiliation: 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.
authors.forename: Rigmor
authors.initials: R
authors.lastname: Solberg
authors.affiliation: Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb.Wyspianskiego 27, 50-370 Wroclaw, Poland.
authors.forename: Wioletta
authors.initials: W
authors.lastname: Rut
authors.affiliation: Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, 0316 Oslo, Norway.
authors.forename: Ngoc Nguyen
authors.initials: NN
authors.lastname: Lunde
authors.affiliation: Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb.Wyspianskiego 27, 50-370 Wroclaw, Poland.
authors.forename: Paulina
authors.initials: P
authors.lastname: Kasperkiewicz
authors.affiliation: Program in Cell Death and Survival Networks, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA.
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Snipas
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, 1000 Ljubljana, Slovenia.
authors.forename: Marko
authors.initials: M
authors.lastname: Mihelic
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, 1000 Ljubljana, Slovenia.
authors.forename: Dusan
authors.initials: D
authors.lastname: Turk
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, 1000 Ljubljana, Slovenia.
authors.forename: Boris
authors.initials: B
authors.lastname: Turk
authors.affiliation: Program in Cell Death and Survival Networks, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA.
authors.forename: Guy S
authors.initials: GS
authors.lastname: Salvesen
authors.affiliation: 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.
authors.forename: Marcin
authors.initials: M
authors.lastname: Drag
mesh_terms: D002460:Cell Line; D020650:Combinatorial Chemistry Techniques; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008968:Molecular Conformation; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D013379:Substrate Specificity; D000099240:Asparaginyl Endopeptidase
chemical_list: D003546:Cysteine Endopeptidases; D004791:Enzyme Inhibitors; D015335:Molecular Probes; D000099240:Asparaginyl Endopeptidase