Development of a Highly Potent Transthyretin Amyloidogenesis Inhibitor: Design, Synthesis, and Evaluation

Abstract

Transthyretin amyloidosis (ATTR) is a group of fatal diseases described by the misfolding and amyloid deposition of transthyretin (TTR). Discovering small molecules that bind and stabilize the TTR tetramer, preventing its dissociation and subsequent aggregation, is a therapeutic strategy for these pathologies. Departing from the crystal structure of TTR in complex with tolcapone, a potent binder in clinical trials for ATTR, we combined rational design and molecular dynamics (MD) simulations to generate a series of novel halogenated kinetic stabilizers. Among them, M-23 displays one of the highest affinities for TTR described so far. The TTR/M-23 crystal structure confirmed the formation of unprecedented protein−ligand contacts, as predicted by MD simulations, leading to an enhanced tetramer stability both in vitro and in whole serum. We demonstrate that MD-assisted design of TTR ligands constitutes a new avenue for discovering molecules that, like M-23, hold the potential to become highly potent drugs to treat ATTR.

3-O-methyltolcapone and Its Lipophilic Analogues are Potent Inhibitors of Transthyretin Amyloidogenesis with High Permeability and Low Toxicity

“…As expected for specific binding, integration of the titration peaks produced a sigmoidal enthalpy curve for each interaction (Figure 5 Previous ITC studies used different models for interpolating ITC data. The calorimetric data of the binary complexes of tafamidis with TTR were interpolated using a sequential model [12,18], revealing a strong negative cooperativity between the two binding sites. The result showed that the first binding site had an extremely low K d ' of 9.9 nM and a ΔH = -6.0 kcal mol -1 , while the second binding site resulted in a loss of affinity with a very high K d " of 260 nM and a ΔH = -6.5 kcal mol -1 .…”

“…The effect of adding an extra methyl to 3-OMT in the non-phenolic ring worsen the affinity in derivative 1 even further and restores in derivative 2 the high affinity observed for tolcapone, accompanied by higher permeability and selectivity than 3-OMT, hence of tolcapone (Table 4). The enhanced ability to penetrate the blood-brain barrier represents a major key point in developing a treatment for CNS TTR amyloidosis, such as familial leptomeningeal amyloidosis [59,60], as demonstrated by the studies attempting to increase the penetration both in the brain and in the periphery of tafamidis [61] and tolcapone itself [18].…”

“…While recently reported new tolcapone analogues conserve its 3-4-dihydroxy-5-nitrophenyl ring but present modifications in the non-phenolic aromatic ring to achieve the highest affinities [18],…”

“…TTR stabilisers establish interactions by bridging the two TTR monomers that form each T4 binding site present in the oligomeric protein, thereby stabilising its tetrameric native state and inhibiting amyloidogenesis [6,12–18].…”

Binding of a Pyrene-Based Fluorescent Amyloid Ligand to Transthyretin: A Combined Crystallographic and Molecular Dynamics Study

“…A first clinical trial of CRISPR/Cas9 has recently been performed with successful results in the reduction of TTR production in humans . Currently, work is ongoing to better diagnose patients early, to follow up and monitor the various available treatments, and to find improved small-molecule ligands as kinetic stabilizers. − …”