Synthesis and evaluation of inhibitors of transthyretin amyloid formation based on the non-steroidal anti-inflammatory drug, flufenamic acid

doi:10.1016/S0968-0896(99)00066-8

Bioorganic & Medicinal Chemistry

Volume 7, Issue 7, July 1999, Pages 1339-1347

https://doi.org/10.1016/S0968-0896(99)00066-8 Get rights and content

Abstract

A light scattering-based amyloid fibril formation assay was employed to evaluate potential inhibitors of transthyretin (TTR) amyloid fibril formation in vitro. Twenty nine aromatic small molecules, some with homology to flufenamic acid (a known non-steroidal anti-inflammatory drug) were tested to identify important structural features for inhibitor efficacy. The results of these experiments and earlier data suggest that likely inhibitors will have aromatic-based structures with at least two aromatic rings. The ring or fused ring system occupying the outermost TTR binding pocket needs to be substituted with an acidic functional group (e.g. a carboxylic acid) to interact with complimentary charges in the TTR binding site. The promising TTR amyloid fibril inhibitors ranked in order of efficacy are: 2>4≈7>3>9>6>21 (see Fig. 5

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Introduction

One of the common features of human amyloid disease is the deposition of insoluble high molecular weight cross-β-sheet fibrils derived from the self-assembly of one of 20 human proteins.1, 2, 3, 4, 5, 6, 7In the case of transthyretin (TTR), the process of amyloid fibril formation appears to be the causative agent in senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP).7, 8Wild-type TTR can be transformed into amyloid by partial denaturation (e.g. at reduced pH), affording a monomeric amyloidogenic intermediate with an alternatively folded structure that self-assembles into amyloid fibrils.1, 2, 3, 9, 10, 11, 12, 13, 14, 15Transthyretin is a stable tetramer under physiological conditions; however, a low pH environment such as that encountered in an endosome or lysosome (or an analogous non-acidic denaturing environment) dissociates TTR into monomeric subunits as well as facilitating a conformational change within the TTR monomer making it amyloidogenic.[16]The >50 mutations associated with FAP make the kinetics and thermodynamics of this process more facile.12, 13, 14, 17

Transthyretin normally binds to the thyroid hormone thyroxine (1, T₄) with negative cooperativity and high affinity (K_A1 10⁸; K_A2 10⁶), serving as the primary carrier of T₄ in the cerebral spinal fluid (CSF). However in plasma, TTR serves as the backup carrier for T₄, thyroid binding globulin being the primary carrier (K_A 6×10⁹). Our strategy for inhibiting transthyretin amyloid fibril formation is to identify a ligand that will bind to TTR in human plasma using the largely unoccupied (≈90% unoccupied) T₄ binding site. Ligand binding stabilizes transthyretin and as a result also increases the activation barrier associated with the tetramer to monomeric amyloidogenic intermediate transition, i.e. the enabling event in amyloid fibril formation.[18]Previous results from our laboratory demonstrate that thyroxine is capable of stabilizing the tetrameric form of transthyretin, preventing amyloid fibril formation at a pH below 5.5 where TTR normally self-associates into amyloid fibrils.[15]Thyroid hormone also appears to stabilize TTR in the CSF in vivo, preventing fibril formation. Numerous pathological evaluations suggest that TTR amyloid fibrils are generally not observed in the brain.[15]This is the case even when a particularly unstable and pathogenic FAP variant such as L55P is expressed in the CSF, consistent with the role of T₄ as a TTR stabilizing agent in the CSF.

The stagnant fibril formation assay developed by our laboratory11, 15was used to discover the non-steroidal anti-inflammatory drug flufenamic acid (Flu, 2), which is an excellent TTR fibril inhibitor.[19]Flufenamic acid binds with high affinity and negative cooperativity (pH 7.6) to wild-type (K_D1=30±14 nM, K_D2=255±97 nM), V30M (K_D1=41±10 nM, K_D2=320±125 nM) and L55P TTR (K_D1=74±16 nM, K_D2=682±137 nM), completely inhibiting amyloid fibril formation at a concentration of 10.8 μM, 3× the physiological concentration of TTR (3.6 μM), under conditions where TTR amyloid fibril formation is maximal (pH 4.4).[19]A cocrystal structure of TTR with flufenamic acid was determined to 2.0 Å resolution in collaboration with the Sacchettini laboratory.[19]This structural information provides the basis for a rational drug design effort centered on identifying the important pharmacophoric substructures of flufenamic acid, such that subsequent parallel syntheses could be utilized to make an optimized ligand.

The compounds synthesized and reported in this paper were designed to test the important structural features of flufenamic acid that render it a good inhibitor (Fig. 1). Each of the five molecular fragments of Flu (fragments A–E) were evaluated in the structure–activity analysis described herein to differing extents. Such information should eventually lead to a generalizable pharmacophore hypothesis and help identify other novel ligands as represented by the generic structure on the right side of Fig. 1. In this paper we report fibril inhibition data for several NSAIDs including flufenamic acid analogues utilizing the proven stagnant amyloid fibril formation assay.11, 15, 18The data suggests that the carboxylic acid functionality plays an important role in the binding of amyloid fibril inhibitors to TTR. In addition, there are also clear preferences for the aromatic platform that is capable of making van der Waals interactions with the TTR binding site.

Section snippets

Results

The structures of compounds either purchased or prepared for this study are shown in Fig. 2 and Fig. 3. The NSAIDs diclofenamic acid (3), niflumic acid (4), indomethacin (5), sulindac (6), diflunisal (7), and tolmetin (8) (Fig. 2), as well as N-phenylanthranilic acid (9), meta-trifluoromethylaniline (10), and para-aminobenzoic acid (12) (Fig. 3) were all purchased and used without further purification. N-Acetyl-meta-trifluoromethylaniline (11) was prepared by acylation of 10 with acetic

Discussion

A collaboration between the Sacchettini laboratory and our own produced a 2 Å resolution crystal structure of the Flu₂·TTR complex.[19]Owing to the symmetry associated with the T₄ binding site, Flu is able to bind in two symmetry equivalent modes in each of two symmetry equivalent binding sites shown in Fig. 6. In addition, Flu is able to bind in two conformations where the CF₃ group is either in a cis or trans relationship to the intramolecularly hydrogen bonded NH and COOH functional groups (

Conclusion

We have utilized the stagnant fibril formation assay to evaluate potential inhibitors of TTR amyloid fibril formation in vitro. Twenty nine aromatic small molecules, some with homology to flufenamic acid, were tested to identify important structural features for inhibitor efficacy. The results of these experiments and earlier screens suggest that likely inhibitors will have aromatic-based structures with at least two aromatic rings (one of the two rings can be a bi- or tricyclic aromatic ring

General aspects

All glassware were oven-dried and cooled in a dessicator containing CaSO₄. THF was distilled from Na/benzophenone. Anhydrous methanol was obtained by distilling HPLC grade methanol from magnesium methoxide which was stored over activated 4 Å molecular sieves. Thin-layer chromatography was performed on Kodak plastic backed silica gel plates 250 μ that were visualized by UV irradiation or I₂ staining or both. Chromatographic purification on silica gel (Merck, grade 60, 240–400 mesh, 60 Å) was

Acknowledgements

We thank the National Institutes of Health (NIH R01 DK46335), the Lita Annenberg Hazen Family, the Skaggs Family, and the NIH-NRSA to P.B. (F32 GM18451-01) for partial funding of this work.

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