Novel transthyretin amyloid fibril formation inhibitors: synthesis, biological evaluation, and X-ray structural analysis

Abstract

Transthyretin (TTR) is one of thirty non-homologous proteins whose misfolding, dissociation, aggregation, and deposition is linked to human amyloid diseases. Previous studies have identified that TTR amyloidogenesis can be inhibited through stabilization of the native tetramer state by small molecule binding to the thyroid hormone sites of TTR. We have evaluated a new series of beta-aminoxypropionic acids (compounds 5-21), with a single aromatic moiety (aryl or fluorenyl) linked through a flexible oxime tether to a carboxylic acid. These compounds are structurally distinct from the native ligand thyroxine and typical halogenated biaryl NSAID-like inhibitors to avoid off-target hormonal or anti-inflammatory activity. Based on an in vitro fibril formation assay, five of these compounds showed significant inhibition of TTR amyloidogenesis, with two fluorenyl compounds displaying inhibitor efficacy comparable to the well-known TTR inhibitor diflunisal. Fluorenyl 15 is the most potent compound in this series and importantly does not show off-target anti-inflammatory activity. Crystal structures of the TTR:inhibitor complexes, in agreement with molecular docking studies, revealed that the aromatic moiety, linked to the sp(2)-hybridized oxime carbon, specifically directed the ligand in either a forward or reverse binding mode. Compared to the aryl family members, the bulkier fluorenyl analogs achieved more extensive interactions with the binding pockets of TTR and demonstrated better inhibitory activity in the fibril formation assay. Preliminary optimization efforts are described that focused on replacement of the C-terminal acid in both the aryl and fluorenyl series (compounds 22-32). The compounds presented here constitute a new class of TTR inhibitors that may hold promise in treating amyloid diseases associated with TTR misfolding.

Figure 1. (left) General structure of NSAID inhibitors of TTR amyloidosis (1–4) and schematic representation of their common pharmacophoric portions.

(Right) The two different types of spacer between the pharmacophoric portions present in synthesized compounds 5–32 of Table 1 and Table 2 with general formula A and classical NSAIDs with aryl–propionic structure, respectively.

Figure 2. The T4 hormone binding channel of TTR tetramer.

(a) Ribbon diagram of tetrameric structure of TTR with bound compound 15 (see results and discussion section). Each subunit (labeled A, B, C and D) of the tetramer is shown with its secondary structural elements and colored differently. The binding of 15 in both T4 binding pockets of TTR is shown as a stick model inside a transparent surface. The crystallographic asymmetric unit contains subunits A and B while subunits C and D were formed through crystallographic symmetry. Because of the two-fold axis along the binding channel (indicated by the arrows) a second symmetry-related binding conformation is present for the inhibitor molecule in both hormone binding sites. (b) Definition of the halogen binding pockets (HBPs) of the T4 hormone binding site of TTR based on the previously published crystal structure of T4 bound to the protein (PDB 1ROX) . T4 is shown in both of its symmetry-related binding modes (shown in magenta and green inside the molecular surface mesh) that are related by a two-fold rotation axis. The carboxyl tail of T4 is positioned at the entry port of the binding site (outer binding pocket) and the iodines occupy the HBP1, HBP2, and HBP3 pockets. The inner binding pocket, HBP3, is located between the side chains of Ser117, Thr119, Ala108, and Leu110, the central HBP2 pocket is formed by the side chains of Leu17, Ala108, Ala109 and Leu110 and the outer pocket HBP1 is located between the side chains of Lys15, Leu17, Thr106 and Val121.

Figure 3. Structures of β-aminoxypropionic acid compounds evaluated in this study.

Figure 4. Structures of β-aminoxymethylsulfonylpropionamides (compounds 22–27), (β-aminoxypropanamido) acetic acids (compounds 28–30), and (β-aminoxypropanamido) propanoic acids (compounds 31 and 32) evaluated in this study.

Figure 5. In vitro acid-mediated wt-TTR (3.6 µM) fibril formation in the presence of 7.2 µM inhibitors are shown plotted with wt-TTR control.

Fibril formation was assessed by turbidity measurments at 0, 24, 48, 72 and 96 hours time points at 400 nm, pH 4.4. For each compound, the inhibition values are reported as the mean value (less than±5standard error), from three independent determinations.

Figure 6. Crystal structures of inhibitors 11 and 13 bound to TTR.

(a) and (b) Electron density of 11 and 13 bound with both hormone binding pockets of wt-TTR. Electron density of Shake&wARP omit maps are contoured at the 1 σ level. The blob feature in XtalView was applied to limit the electron density display to within 1.5 Å of the inhibitor and the final figure was rendered with Raster3D . The inhibitor molecules were omitted from the model before the map calculation. (c) The binding interactions of 11 with the hormone binding pocket of TTR, the better ordered BD binding pocket is shown here. Like most of the TTR bound ligands, 11 also binds in two symmetry-related binding modes (shown in magenta and green). The key interacting residues are labeled, primed and unprimed residues refer to two neighboring symmetry related monomers comprising the T4 site. Compound 11 binds in the forward binding mode by orienting its carboxylate substituent to the outer binding pocket residue Lys15. The aryl moiety of 11 is anchored by its trifluro group to HBP3 and HBP3' of the inner most binding pocket. Like compound 11, compound 13 also binds in the forward binding mode with similar interactions (not shown here).

Figure 7. Crystal structures of inhibitors 15 and 16 bound to TTR.

(a) and (b) Electron density of 15 and 16 bound with both hormone binding pockets of wt-TTR. Electron density of Shake&wARP omit maps were contoured at the 1 σ level. The blob feature in XtalView has been applied to limit the electron density display to within 1.5 Å of the inhibitor and the final figure was rendered with Raster3D . The inhibitor molecules were omitted from the model before the map calculation. (c) The binding interactions of 15 with the hormone binding pocket of TTR, the better ordered BD binding pocket is shown here. Inhibitor 15 also binds in two symmetry-related binding modes (shown in magenta and green). The key interacting residues are labeled, primed and unprimed residues refer to two neighboring symmetry related monomers comprising the T4 site. Compound 15 binds in the reverse binding mode by orienting its carboxylate substituent to the inner binding pocket and its fluroneyl ring to the outer binding pocket. The fluroneyl ring is optimally sandwiched between the side chain atoms of the outer binding pocket, while the corboxylate group makes a network of direct and water mediated hydrogen bonds. Leu110 and Leu110' stack on top of the linker region but are not shown for clarity. Compound 16 also binds in similar fashion (not shown here).

Figure 8. Docking of β-aminoxypropionic acids to TTR.

(a) and (b) Schematic representation of compounds 11 (left) and 15 (right) in one of the TTR hormone binding sites shown with the best docking results of compounds 11 and 15, evidencing the HBP3 surface; both possible binding modes are shown for 15 (red and black). (c)–(e) Gold docking results. (c): front view of compounds 16 (magenta) and 17 (green), depicting the HBP3 surface and Lys15 of A and C monomers, (d): side view of compound 20, (e): front view of compound 24, (f): front view of compound 26.

Figure 9. Comparison of the TTR∶11 and TTR∶15 structures with the TTR∶OE1, TTR∶DDBF, and TTR∶PHENOX structures.

(a) Overlay of the binding pocket of previously published TTR∶OE1 (shown in orange red [39]) on the TTR∶11 crystal structure. Residues Ser117 and Ser117' point to the inner binding pocket while Lys15, Lys15', Glu54, and Glu54' denote the outer binding pocket of one of the T4 sites of TTR. The two binding conformations of 11 are shown in light and dark green stick format. Compared to OE1, 11 binds more deeply in the inner binding pocket and positions its carboxyl group closer to the outer binding pocket residue Lys15. (b) Overlay of the binding pocket of TTR∶DDBF and TTR∶PHENOX on the TTR∶15 crystal structure. Residues Ser117 and Ser117' point to the inner binding pocket while Lys15, Lys15', Glu54, and Glu54' denote the outer binding pocket of one of the T4 sites of TTR. The two binding conformations of 15 are shown in light and dark green stick format, PHENOX is shown in red and orange sticks. Both binding conformations of DDBF are shown in thin black sticks. The DDBF binds mainly to HBP1 near Lys15 while PHENOX extended its interaction from Glu54 to HBP2. The new compound 15 effectively utilizes all the HBPs located between Lys15 and Ser117. Suitable substitution in the fluorenyl ring of 15 should cover the entire binding pocket starting from Glu54 to Ser117.