title: Generation and characterization of non-competitive furin-inhibiting nanobodies.
abstract: The PC (proprotein convertase) furin cleaves a large variety of proproteins and hence plays a major role in many pathologies. Therefore furin inhibition might be a good strategy for therapeutic intervention, and several furin inhibitors have been generated, although none are entirely furin-specific. To reduce potential side effects caused by cross-reactivity with other proteases, dromedary heavy-chain-derived nanobodies against catalytically active furin were developed as specific furin inhibitors. The nanobodies bound only to furin but not to other PCs. Upon overexpression in cell lines, they inhibited the cleavage of two different furin substrates, TGFβ (transforming growth factor β) and GPC3 (glypican 3). Purified nanobodies could inhibit the cleavage of diphtheria toxin into its enzymatically active A fragment, but did not inhibit cleavage of a small synthetic peptide-based substrate, suggesting a mode-of-action based on steric hindrance. The dissociation constant of purified nanobody 14 is in the nanomolar range. The nanobodies were non-competitive inhibitors with an inhibitory constant in the micromolar range as demonstrated by Dixon plot. Furthermore, anti-furin nanobodies could protect HEK (human embryonic kidney)-293T cells from diphtheria-toxin-induced cytotoxicity as efficiently as the PC inhibitor nona-D-arginine. In conclusion, these antibody-based single-domain nanobodies represent the first generation of highly specific non-competitive furin inhibitors.
authors.affiliation: Laboratory for Molecular Oncology, Department of Human Genetics, University of Leuven, Leuven, Belgium.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Zhu
authors.forename: Jeroen
authors.initials: J
authors.lastname: Declercq
authors.forename: Bart
authors.initials: B
authors.lastname: Roucourt
authors.forename: Gholamreza H
authors.initials: GH
authors.lastname: Ghassabeh
authors.forename: Sandra
authors.initials: S
authors.lastname: Meulemans
authors.forename: Jörg
authors.initials: J
authors.lastname: Kinne
authors.forename: Guido
authors.initials: G
authors.lastname: David
authors.forename: Alphons J M
authors.initials: AJ
authors.lastname: Vermorken
authors.forename: Wim J M
authors.initials: WJ
authors.lastname: Van de Ven
authors.forename: Iris
authors.initials: I
authors.lastname: Lindberg
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.forename: John W M
authors.initials: JW
authors.lastname: Creemers
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D002162:Camelus; D002384:Catalysis / Q000187:drug effects; D003374:Coumarins / Q000378:metabolism; D004167:Diphtheria Toxin / Q000378:metabolism; D004705:Endocytosis; D045683:Furin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D053673:Glypicans / Q000378:metabolism; D057809:HEK293 Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D043484:Proprotein Convertases / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D013379:Substrate Specificity; D016212:Transforming Growth Factor beta / Q000378:metabolism
chemical_list: D003374:Coumarins; D004167:Diphtheria Toxin; C105937:GPC3 protein, human; D053673:Glypicans; D009842:Oligopeptides; D010446:Peptide Fragments; D061905:Single-Domain Antibodies; D016212:Transforming Growth Factor beta; C491427:nonaarginine; D043484:Proprotein Convertases; C468731:FURIN protein, human; D045683:Furin