title: Inhibition of the mechano-enzymatic amyloidogenesis of transthyretin: role of ligand affinity, binding cooperativity and occupancy of the inner channel.
abstract: Dissociation of the native transthyretin (TTR) tetramer is widely accepted as the critical step in TTR amyloid fibrillogenesis. It is modelled by exposure of the protein to non-physiological low pH in vitro and is inhibited by small molecule compounds, such as the drug tafamidis. We have recently identified a new mechano-enzymatic pathway of TTR fibrillogenesis in vitro, catalysed by selective proteolytic cleavage, which produces a high yield of genuine amyloid fibrils. This pathway is efficiently inhibited only by ligands that occupy both binding sites in TTR. Tolcapone, which is bound with similar high affinity in both TTR binding sites without the usual negative cooperativity, is therefore of interest. Here we show that TTR fibrillogenesis by the mechano-enzymatic pathway is indeed more potently inhibited by tolcapone than by tafamidis but neither, even in large molar excess, completely prevents amyloid fibril formation. In contrast, mds84, the prototype of our previously reported bivalent ligand TTR 'superstabiliser' family, is notably more potent than the monovalent ligands and we show here that this apparently reflects the critical additional interactions of its linker within the TTR central channel. Our findings have major implications for therapeutic approaches in TTR amyloidosis.
authors.affiliation: Wolfson Drug Discovery Unit, Centre for Amyloidosis and Acute Phase Proteins, University College London, London, NW3 2PF, UK.
authors.forename: Guglielmo
authors.initials: G
authors.lastname: Verona
authors.affiliation: Wolfson Drug Discovery Unit, Centre for Amyloidosis and Acute Phase Proteins, University College London, London, NW3 2PF, UK.|Department of Molecular Medicine, Institute of Biochemistry, University of Pavia, Via Taramelli 3b, Pavia, 27100, Italy.
authors.forename: P Patrizia
authors.initials: PP
authors.lastname: Mangione
authors.affiliation: Department of Molecular Medicine, Institute of Biochemistry, University of Pavia, Via Taramelli 3b, Pavia, 27100, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Raimondi
authors.affiliation: Department of Molecular Medicine, Institute of Biochemistry, University of Pavia, Via Taramelli 3b, Pavia, 27100, Italy.
authors.forename: Sofia
authors.initials: S
authors.lastname: Giorgetti
authors.affiliation: Department of Molecular Medicine, Institute of Biochemistry, University of Pavia, Via Taramelli 3b, Pavia, 27100, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Faravelli
authors.affiliation: Wolfson Drug Discovery Unit, Centre for Amyloidosis and Acute Phase Proteins, University College London, London, NW3 2PF, UK.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Porcari
authors.affiliation: Wolfson Drug Discovery Unit, Centre for Amyloidosis and Acute Phase Proteins, University College London, London, NW3 2PF, UK.|Department of Medical and Biological Sciences (DSMB), University of Udine, Piazzale Kolbe 4, 33100, Udine, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Corazza
authors.affiliation: National Amyloidosis Centre, University College London, London, NW3 2PF, UK.
authors.forename: Julian D
authors.initials: JD
authors.lastname: Gillmore
authors.affiliation: National Amyloidosis Centre, University College London, London, NW3 2PF, UK.
authors.forename: Philip N
authors.initials: PN
authors.lastname: Hawkins
authors.affiliation: Wolfson Drug Discovery Unit, Centre for Amyloidosis and Acute Phase Proteins, University College London, London, NW3 2PF, UK.|National Amyloidosis Centre, University College London, London, NW3 2PF, UK.
authors.forename: Mark B
authors.initials: MB
authors.lastname: Pepys
authors.affiliation: Wolfson Drug Discovery Unit, Centre for Amyloidosis and Acute Phase Proteins, University College London, London, NW3 2PF, UK.
authors.forename: Graham W
authors.identifier: 0000-0002-9672-8463
authors.initials: GW
authors.lastname: Taylor
authors.affiliation: Wolfson Drug Discovery Unit, Centre for Amyloidosis and Acute Phase Proteins, University College London, London, NW3 2PF, UK. v.bellotti@ucl.ac.uk.|Department of Molecular Medicine, Institute of Biochemistry, University of Pavia, Via Taramelli 3b, Pavia, 27100, Italy. v.bellotti@ucl.ac.uk.
authors.forename: Vittorio
authors.identifier: 0000-0002-0467-864X
authors.initials: V
authors.lastname: Bellotti
mesh_terms: D000682:Amyloid / Q000378:metabolism*; D001577:Benzophenones / Q000494:pharmacology*; D001583:Benzoxazoles / Q000494:pharmacology*; D001665:Binding Sites / Q000187:drug effects; D054361:Fenamates / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D009596:Nitrophenols / Q000494:pharmacology*; D011228:Prealbumin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization; D059748:Proteolysis; D000077867:Tolcapone
chemical_list: C555138:2,2'-(4,4'-(heptane-1,7-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene))bis(azanediyl)dibenzoic acid; D000682:Amyloid; D001577:Benzophenones; D001583:Benzoxazoles; D054361:Fenamates; D009596:Nitrophenols; D011228:Prealbumin; C547076:tafamidis; D000077867:Tolcapone