Inhibition of Human Transthyretin Aggregation by Non-Steroidal Anti-Inflammatory Compounds: A Structural and Thermodynamic Analysis

Ricardo Sant'Anna,Igor Polikarpov,Carolina Braga,Debora Foguel,Salvador Ventura,Luis Lima

doi:10.3390/ijms14035284

Abstract

Transthyretin (TTR) is a homotetrameric protein that circulates in plasma and cerebral spinal fluid (CSF) whose aggregation into amyloid fibrils has been associated with at least two different amyloid diseases: senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP). In SSA aggregates are composed of WT-TTR, while in FAP more than 100 already-described variants have been found in deposits. Until now, TTR-related diseases have been untreatable, although a new drug called Tafamidis has been approved only in Europe to specifically treat V30M patients. Thus, new strategies are still necessary to treat FAP caused by other variants of TTR. TTR has two channels in the dimer interface that bind to the hormone thyroxin and that have been used to accommodate anti-amyloidogenic compounds. These compounds stabilize the tetramers, rendering TTR less amyloidogenic. Here, we investigated the effects of three non-steroidal anti-inflammatory compounds—sulindac (SUL), indomethacin (IND) and lumiracoxib (LUM)—as tetramer stabilizers and aggregation inhibitors. WT-TTR and the very aggressive TTR variant L55P were used as models. These compounds were able to stabilize TTR against high hydrostatic pressure (HHP), increasing the ΔGf by several kcal. They were also effective in inhibiting WT-TTR and L55P acid- or HHP-induced aggregation; in particular, LUM and IND were very effective, inhibiting almost 100% of the aggregation of both proteins under certain conditions. The species formed when aggregation was performed in the presence of these compounds were much less toxic to cells in culture. The crystal structures of WT-TTR bound to the three compounds were solved at high resolution, allowing the identification of the relevant protein:drug interactions. We discuss here the ligand-binding features of LUM, IND and SUL to TTR, emphasizing the critical interactions that render the protein more stable and less amyloidogenic.

Highlights

Transthyretin (TTR) is a homotetrameric, β-sheet–rich protein of 56 kDa produced in the liver or in cerebral spinal fluid (CSF)
We are referring to the affinities as apparent, because TTR has two hormone binding sites (HBS) in each tetramer that ligands generally bind with negative cooperativity [9,38,44,45]
The affinity constants of diclofenac, a well-known TTR stabilizer and aggregation inhibitor, for wild-type TTR (WT-TTR) were kd1 0.060 μM and kd2 1.2 μM, which were in the same range as LUM

Summary

Introduction

Transthyretin (TTR) is a homotetrameric, β-sheet–rich protein of 56 kDa produced in the liver or in cerebral spinal fluid (CSF). TTR has two channels at the dimer–dimer interface, where the hormone thyroxine and other ligands can bind. These channels are called hormone binding sites (HBS), only a low percentage of the TTR that circulates in the plasma or in CSF has its channels occupied by thyroxine [1,2,3,4]. TTR is associated with two amyloidoses: senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP) [14,15]. SSA is caused by the massive deposition of aggregates composed of wild-type TTR (WT-TTR) mainly in the heart [16,17], while FAP is caused by the deposition of aggregates at the peripheral nerves and tissues that are composed of more than 100 autosomal TTR variants [18,19]. The L55P substitution is one of the most aggressive variants and is associated with early onset of the disease with cardiac and neurologic pathologies [20,21,22]

Methods

Results

Conclusion