Title : Structure-based discovery of kinetic stabilizers for transthyretin amyloidosis
Abstract:
Transthyretin amyloidosis (ATTR) is a group of fatal protein misfolding disorders caused
by the dissociation and aggregation of the transthyretin (TTR) tetramer. Tetramer destabilization,
driven by aging or pathogenic mutations, precedes amyloid formation, making native-state
stabilization by small chemical molecules a validated therapeutic strategy. Here, we present an
integrated, structure-based approach combining drug repurposing, rational design, molecular
dynamics (MD) simulations, and advanced biophysical methods to develop highly potent TTR
kinetic stabilizers. We first demonstrate that tolcapone, an FDA-approved drug for Parkinson’s
disease, binds selectively to TTR in plasma, stabilizes the native tetramer in vivo, and inhibits
aggregation and cytotoxicity. High-resolution crystal structures reveal superior occupancy of the
thyroxine (T4) binding pocket compared to tafamidis. Importantly, tolcapone effectively stabilizes
highly destabilized leptomeningeal TTR variants and crosses the blood–brain barrier, positioning it
as a promising disease-modifying candidate for central nervous system ATTR, a form currently
lacking effective therapies. Building on these structural insights, we applied MD-assisted design to
generate next-generation halogenated stabilizers, identifying M-23 as one of the highest-affinity
TTR ligands reported to date. Further optimization led to PITB, a selective plasma-active stabilizer
with excellent oral bioavailability, favorable pharmacokinetics, and lack of detectable toxicity,
outperforming tolcapone in inhibiting tetramer dissociation and aggregation of wild-type and
prevalent pathogenic variants. Finally, hydrogen–deuterium exchange and fast photochemical
oxidation mass spectrometry reveal mutation- and ligand-specific conformational dynamics not
captured by static crystal structures, highlighting the need for variant-aware, dynamics-informed
drug discovery strategies for ATTR. Overall, this work establishes a unified, structure- and
dynamics-informed chemical strategy for the rational discovery of small-molecule kinetic stabilizers
that are potent, selective, and clinically translatable for the treatment of the full spectrum of
transthyretin amyloidosis
