title: Designed ankyrin repeat proteins (DARPins) as novel isoform-specific intracellular inhibitors of c-Jun N-terminal kinases.
abstract: The c-Jun N-terminal kinases (JNKs) are involved in many biological processes such as proliferation, differentiation, apoptosis, and inflammation and occur in highly similar isoforms in eukaryotic cells. Isoform-specific functions and diseases have been reported for individual JNK isoforms mainly from gene-knockout studies in mice. There is, however, a high demand for intracellular inhibitors with high selectivity to improve the understanding of isoform-specific mechanisms and for use as therapeutic tools. The commonly used JNK inhibitors are based on small molecules or peptides that often target the conserved ATP binding site or docking sites and thus show only moderate selectivity. To target novel binding epitopes, we used designed ankyrin repeat proteins (DARPins) to generate alternative intracellular JNK inhibitors that discriminate two very similar isoforms, JNK1 and JNK2. DARPins are small binding proteins that are well expressed, stable, and cysteine-free, which makes them ideal candidates for applications in the reducing intracellular environment. We performed ribosome display selections against JNK1α1 and JNK2α1 using highly diverse combinatorial libraries of DARPins. The selected binders specifically recognize either JNK1 or JNK2 or both isoforms in vitro and in mammalian cells. All analyzed DARPins show affinities in the low nanomolar range and isoform-specific inhibition of JNK activation in vitro at physiological ATP concentrations. Importantly, DARPins that selectively inhibit JNK activation in human cells were also identified. These results emphasize the great potential of DARPins as a novel class of highly specific intracellular inhibitors of distinct enzyme isoforms for use in biological studies and as possible therapeutic leads.
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Petra
authors.initials: P
authors.lastname: Parizek
authors.forename: Lutz
authors.initials: L
authors.lastname: Kummer
authors.forename: Peter
authors.initials: P
authors.lastname: Rube
authors.forename: Anke
authors.initials: A
authors.lastname: Prinz
authors.forename: Friedrich W
authors.initials: FW
authors.lastname: Herberg
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D017089:Ankyrin Repeat / Q000235:genetics*; D001665:Binding Sites; D019921:Biotinylation; D002845:Chromatography / Q000379:methods; D015195:Drug Design; D057809:HEK293 Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000737:chemistry*; D007700:Kinetics; D051379:Mice; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D020033:Protein Isoforms; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D020349:Surface Plasmon Resonance
chemical_list: D010455:Peptides; D020033:Protein Isoforms; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D048031:JNK Mitogen-Activated Protein Kinases