title: Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin repeat proteins.
abstract: The specific intracellular inhibition of protein activity at the protein level allows the determination of protein function in the cellular context. We demonstrate here the use of designed ankyrin repeat proteins as tailor-made intracellular kinase inhibitors. The target was aminoglycoside phosphotransferase (3')-IIIa (APH), which mediates resistance to aminoglycoside antibiotics in pathogenic bacteria and shares structural homology with eukaryotic protein kinases. Combining a selection and screening approach, we isolated 198 potential APH inhibitors from highly diverse combinatorial libraries of designed ankyrin repeat proteins. A detailed analysis of several inhibitors revealed that they bind APH with high specificity and with affinities down to the subnanomolar range. In vitro, the most potent inhibitors showed complete enzyme inhibition, and in vivo, a phenotype comparable with the gene knockout was observed, fully restoring antibiotic sensitivity in resistant bacteria. These results underline the great potential of designed ankyrin repeat proteins for modulation of intracellular protein function.
authors.affiliation: Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, Zürich CH-8057, Switzerland.
authors.forename: Patrick
authors.initials: P
authors.lastname: Amstutz
authors.forename: H Kaspar
authors.initials: HK
authors.lastname: Binz
authors.forename: Petra
authors.initials: P
authors.lastname: Parizek
authors.forename: Michael T
authors.initials: MT
authors.lastname: Stumpp
authors.forename: Andreas
authors.initials: A
authors.lastname: Kohl
authors.forename: Markus G
authors.initials: MG
authors.lastname: Grütter
authors.forename: Patrik
authors.initials: P
authors.lastname: Forrer
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000583:Amikacin / Q000737:chemistry; D000617:Aminoglycosides / Q000737:chemistry; D017487:Ankyrins / Q000737:chemistry*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D019921:Biotinylation; D020134:Catalytic Domain; D002845:Chromatography; D003001:Cloning, Molecular; D024881:Drug Resistance, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D007612:Kanamycin / Q000737:chemistry; D019868:Kanamycin Kinase / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D010641:Phenotype; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000737:chemistry; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance
chemical_list: D000617:Aminoglycosides; D017487:Ankyrins; D000900:Anti-Bacterial Agents; D007612:Kanamycin; D000583:Amikacin; D019868:Kanamycin Kinase