Published by Portland Press on behalf of the Biochemical Society
Biochemical Journal vol:448 issue:1 pages:73-82
The proprotein convertase (PC) 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. Those nanobodies bind only to furin but not to other PCs. Upon overexpression in cell lines, they can inhibit the cleavage of two different furin substrates, TGFβ and GPC3. Purified nanobodies can inhibit the cleavage of diphtheria toxin into its enzymatically active A fragment, but do 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 are non-competitive inhibitors with an inhibitory constant in the micromolar range as demonstrated by Dixon plot. Furthermore, anti-furin nanobodies can protect HEK293T 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.