Scientific RepoRts | 7: 182 | DOI:10.1038/s41598-017-00338-x
Inhibition of the mechano-
of transthyretin: role of ligand
anity, binding cooperativity and
occupancy of the inner channel
, P. Patrizia Mangione
, Sara Raimondi
, Soa Giorgetti
, Giulia Faravelli
, Alessandra Corazza
, Julian D. Gillmore
, Philip N. Hawkins
, Mark B.
, Graham W. Taylor
& Vittorio Bellotti
Dissociation of the native transthyretin (TTR) tetramer is widely accepted as the critical step in TTR
amyloid brillogenesis. 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 identied
a new mechano-enzymatic pathway of TTR brillogenesis in vitro, catalysed by selective proteolytic
cleavage, which produces a high yield of genuine amyloid brils. This pathway is eciently inhibited
only by ligands that occupy both binding sites in TTR. Tolcapone, which is bound with similar high
anity in both TTR binding sites without the usual negative cooperativity, is therefore of interest. Here
we show that TTR brillogenesis 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
bril 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 reects the critical additional interactions of its linker within the TTR central channel. Our
ndings have major implications for therapeutic approaches in TTR amyloidosis.
e seminal observation that the native non-covalent TTR homotetramer dissociates at low pH into dimers and
monomers that self-assemble into amyloid brils
is the basis for the current, widely accepted model for TTR
amyloid formation. However low pH treatment of both wild type and amyloidogenic TTR variants produces
mostly heterogeneous amorphous aggregates with a very low yield of authentic amyloid brils showing pathog-
nomonic green birefringence in polarized light aer Congo red staining, characteristic brillar electron micro-
scopic appearance and the cross-β X-ray bre diraction signature. Ex vivo TTR amyloid deposits, especially
in the heart, usually contain a substantial proportion of the C-terminal TTR fragment generated by proteolytic
cleavage at Lys48-r49
. Following this observation we have identied and characterized a novel mechanism of
TTR amyloid brillogenesis mediated by selective tryptic cleavage at residue 48. e highly amyloidogenic TTR
C-terminal residue 49–127 polypeptide is released, catalysing amyloid bril formation in vitro, and the whole
process is strongly enhanced by biomechanical forces
. Abundant authentic amyloid brils are produced with
pathognomonic features indistinguishable from natural ex vivo brils.
Compounds able to stabilize TTR, intended for use as drugs to treat and prevent systemic ATTR amyloidosis,
have hitherto been identied exclusively by their capacity to inhibit TTR dissociation and aggregation induced
by low pH in vitro. However, we have found that inhibition of the mechano-enzymatic pathway of TTR amyloid
brillogenesis at physiological pH, ionic strength and temperature requires occupation of both thyroxine binding
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.
Department of Medical and Biological Sciences (DSMB), University of Udine, Piazzale
Kolbe 4, 33100, Udine, Italy.
National Amyloidosis Centre, University College London, London, NW3 2PF, UK.
Correspondence and requests for materials should be addressed to V.B. (email: firstname.lastname@example.org)
Received: 15 November 2016
Accepted: 21 February 2017
Published: xx xx xxxx