Biochemical Journal

Research article

Cyclodextrin, a novel therapeutic tool for suppressing amyloidogenic transthyretin misfolding in transthyretin-related amyloidosis

Hirofumi Jono, Takayuki Anno, Keiichi Motoyama, Yohei Misumi, Masayoshi Tasaki, Toshinori Oshima, Yoshimasa Mori, Mineyuki Mizuguchi, Mitsuharu Ueda, Makoto Shono, Konen Obayashi, Hidetoshi Arima, Yukio Ando

Abstract

TTR (transthyretin), a β-sheet-rich protein, is the precursor protein of familial amyloidotic polyneuropathy and senile systemic amyloidosis. Although it has been widely accepted that protein misfolding of the monomeric form of TTR is a rate-limiting step for amyloid formation, no effective therapy targeting this misfolding step is available. In the present study, we focused on CyDs (cyclodextrins), cyclic oligosaccharides composed of glucose units, and reported the inhibitory effect of CyDs on TTR amyloid formation. Of various branched β-CyDs, GUG-β-CyD [6-O-α-(4-O-α-D-glucuronyl)-D-glucosyl-β-CyD] showed potent inhibition of TTR amyloid formation. Far-UV CD spectra analysis showed that GUG-β-CyD reduced the conformational change of TTR in the process of amyloid formation. In addition, tryptophan fluorescence and 1H-NMR spectroscopy analyses indicated that GUG-β-CyD stabilized the TTR conformation via interaction with the hydrophobic amino acids of TTR, especially tryptophan. Moreover, GUG-β-CyD exerted its inhibitory effect by reducing TTR deposition in transgenic rats possessing a human variant TTR gene in vivo. Collectively, these results indicate that GUG-β-CyD may inhibit TTR misfolding by stabilizing its conformation, which, in turn, suppresses TTR amyloid formation.

  • amyloidosis
  • cyclodextrin (CyD)
  • familial amyloidotic polyneuropathy (FAP)
  • protein misfolding
  • transthyretin (TTR)

INTRODUCTION

Diverse human diseases, including various neurodegenerative disorders and amyloidoses, are thought to result from the misfolding and aggregation of precursor proteins [13]. Natively folded proteins generally undergo a β-sheet conformational transition under various pathological conditions, and further polymerize into amyloid fibrils. Accumulation of amyloid fibrils, which are produced via self-assembly of a naturally folded protein into an insoluble cross-β-sheet structure, is considered to be the hallmark of amyloid-related diseases such as Alzheimer's disease, prion disease and TTR (transthyretin)-related amyloidosis [1].

TTR, a β-sheet-rich plasma protein, is a transport protein for RBP (retinol-binding protein) and T4 (thyroxin), and serves as a rapid turnover protein in plasma [4]. TTR exists in its native form as a tetramer; each subunit monomer exhibits an extensive β-sheet structure, which is closely related to its amyloidogenicity [5]. In TTR-related amyloidosis, mutated TTRs and WT TTR (wild-type TTR) play crucial roles in the pathogenesis of FAP (familial amyloidotic polyneuropathy) and SSA (senile systemic amyloidosis) respectively [69]. FAP, which is caused by a point mutation or a deletion in the TTR gene, is an autosomal dominant form of fatal heredity amyloidosis characterized by systemic accumulation of amyloid fibrils in the peripheral nerves and other organs [6,7]. To date, more than 100 different points of mutation or a deletion in the TTR gene have been reported, and most of these mutations are amyloidogenic [1012]. Of the different types of ATTR (amyloidogenic TTR)-related amyloidosis, ATTR V30M is the most common and this form of FAP is found worldwide [6]. In addition to mutated TTRs, it is well documented that WT TTR can also cause amyloid fibrils to form and accumulate in extracellular spaces in patients with FAP and SSA [8,9].

It has been proposed that tetrameric TTR is not itself amyloidogenic, but dissociation of the tetramer into a non-native monomer with low conformational stability can lead to amyloid fibril formation [13]. Previous work has also shown that further structural changes within the monomer caused by protein misfolding are the crucial step to form TTR amyloid fibril aggregation [14,15]. Although it has been widely accepted that misfolding of monomeric form of TTR is critical for amyloid formation, no effective therapy targeting this step is available as of this moment.

CyDs (cyclodextrins), cyclic oligosaccharides composed of six to eight glucose units, are widely used as prospective drug carriers in the pharmaceutical field [16,17]. There are three common types of natural CyDs depending on how many glucose units are present: α-CyD (six), β-CyD (seven) and γ-CyD (eight). Since CyDs contain a central hydrophobic cavity, and this cavity can serve as an inclusion site for hydrophobic molecules, CyDs are mainly used as multifunctional drug carriers by enhancing the bioavailability of lipophilic drugs, improving efficacy of drugs and reducing side effects [18,19]. In addition, it has been proposed that branched β-CyDs, in which one of the primary hydroxy groups of β-CyD is replaced by mono- or di-saccharides, also have the potential to improve the bioavailability of protein drug formulation. Previous studies revealed that branched β-CyDs interacted with hydrophobic amino acids on the protein surface and increased its stability, which, in turn, suppressed protein misfolding and aggregation [20,21]. Because it is well documented that multiple hydrophobic regions of TTR are exposed in the process of TTR amyloid formation [22], these lines of evidence suggest that branched β-CyDs may have the potential to suppress TTR amyloid formation by inhibiting the misfolding of the monomeric form of TTR.

The aim of the present study was to elucidate the inhibitory effect of CyDs on TTR amyloid formation both in vitro and in vivo. To investigate the detailed mechanism of how CyDs inhibit TTR amyloid formation, we employed multiple biochemical approaches. In addition, to obtain further evidence about the inhibitory effect of CyDs under physiological conditions, we evaluated the effect of CyDs on TTR deposition in transgenic rats possessing a gene encoding human ATTR V30M, an existing useful FAP animal model.

EXPERIMENTAL

Materials

Both WT TTR and ATTR V30M were purified from serum samples obtained from healthy volunteers and homozygotic FAP ATTR V30M patients as described previously [23]. The research followed the guidelines of the Kumamoto University ethical committee. Both recombinant WT TTR and ATTR V30M were prepared as described previously [24]. HP-β-CyD (hydroxypropyl-β-CyD) was supplied by Nihon Shokuhin Kako. Various branched β-CyDs {G1-β-CyD (6-O-α-glucosyl-β-CyD), G2-β-CyD (6-O-α-maltosyl-β-CyD), GUG-β-CyD [6-O-α-(4-O-α-D-glucuronyl)-D-glucosyl-β-CyD], Gal-β-CyD (galactosylated-β-CyD) and Man-β-CyD (mannosylated-β-CyD)} were obtained from the Bio Research Corporation of Yokohama. All chemicals used in the studies were of analytical grade.

Amyloid fibril formation induced by WT TTR and ATTR V30M

To evaluate the effect of various CyDs on TTR amyloid fibril formation, TTRs were diluted in 20 mM sodium acetate and 100 mM NaCl at pH 3.5 in an Eppendorf tube to obtain a final TTR concentration of 20 μM with or without various CyDs. The resulting stationary solutions were incubated at 37 °C in the dark [25].

ThT (thioflavin T)-based fluorimetric assay

To assess the number of amyloid fibrils induced by TTRs in vitro, we measured the intensity of fluorescence of ThT with a Hitachi F-2000 spectrofluorimeter (Hitachi High Technologies), as described previously [26,27]. All assays used a λex of 442 nm and a λem of 489 nm. The reaction mixture contained 5 μM ThT and 50 mM glycine/NaOH buffer, pH 9.5; 5 μl of 20 μM TTR solution was added to 1 ml of this reaction mixture. Spectra were recorded at 25 °C within minutes of adding the TTR sample to the reaction mixture [28]. All assays were carried out in triplicate and repeated three times independently.

Electron microscopic observations

Using an electron microscope (H-7500; Hitachi High Technologies), we examined the morphological features of fibrils induced by TTR as described previously [29]. A 3 μl aliquot of incubated sample was placed on a carbon/collodion-coated grid and allowed to adhere for 1 min, after which it was drained by use of a strip of filter paper. The sample was then stained with a drop of 0.2% uranyl acetate for 30 s. After the excess stain was drained, the grid was air-dried and viewed under an electron microscope at an accelerating voltage of 100 kV and a magnification of ×10 000–80 000.

Measurements of CD spectra

CD spectra were obtained by a Jasco J-720 spectropolarimeter. The samples were WT TTR (20 μM) incubated for 7 days at 37 °C in PBS, pH 3.5, with GUG-β-CyD. Far-UV spectra (200–240 nm) were recorded in a 1-mm-path-length cell. The spectra were recorded with a response time of 4 s, sensitivity of 10 mdeg and scan speed of 10 nm/min at 25 °C and converted into mean residue ellipticity in deg·cm2·dmol−1. CD spectra were accumulated three times for data collection. Each data point was an average of three accumulations. The experiments were repeated three times independently.

Tryptophan fluorescence intensity

The fluorescence intensity of tryptophan was measured by a Hitachi F-4500 spectrofluorimeter at 25 °C. All assays used excitation at 295 nm and emission at 340 nm. Excitation and emission slits were set at 5 nm. The fluorescence intensity was recorded at a protein concentration of 10 μM, in 67 mM sodium phosphate, pH 7.4. All experiments were repeated three times independently.

NMR spectroscopy

The 500 MHz proton NMR spectra were recorded on a JNM-500 spectrometer at 80 °C. WT TTR (2.0 mg) was dissolved in 0.6 ml of 2H2O, which was 0.1 and 0.05 M with respect to sodium chloride and sodium phosphate respectively, and adjusted to register a pH-meter reading of 3.5.

Animals

Tg (transgenic) rats possessing a human ATTR V30M gene (ATTR V30M Tg rats) were generated as described previously [30]. Genomic DNA was extracted from tail samples of transgenic rats by the QIAamp DNA Mini Kit (Qiagen). To screen for transgenic rats with human ATTR V30M cDNA, E2-S (5′-GGCACCGGTGAATCCAAGTGT-3′) and E4-AS (5′-TTCCTTGGGATTGGTGACGAC-3′) were used as the forward and reverse primers respectively. Animals were maintained in a specific pathogen-free environment at the Center for Animal Resources and Development, Kumamoto University, Kuamoto, Japan and all animal work followed the guidelines of the Kumamoto University ethical committee.

GUG-β-CyD inoculation in vivo

Male ATTR V30M Tg rats (9 months old) were used in the present study. ATTR V30M Tg rats were injected intravenously with saline containing GUG-β-CyD (200 mg/kg of body weight n=9) or saline alone (n=9) and received the same treatment twice a week for 3 months. After 3 months, ATTR V30M Tg rats were analysed by immunohistochemistry for human ATTR V30M. Analysis of blood biochemistry was performed by the central clinical laboratory at Kumamoto University, Kuamoto, Japan.

Immunohistochemical staining

Paraffin-embedded 4-μm-thick sections were prepared and deparaffinized in xylene and rehydrated in graded alcohols. Deparaffinized sections were pretreated with 20 min of heating in an autoclave apparatus. Slides were then treated with periodic acid for 10 min at room temperature (25 °C), after which they were incubated in 5% (v/v) normal serum for 1 h at room temperature in a moist chamber. The primary antibodies were rabbit polyclonal anti-TTR (Dako) used at a 1:50 dilution. The secondary antibody was a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin antibody (Dako) diluted 1:100 in PBS-T plus 5% BSA. Reactivity was visualized with the DAB (diaminobenzidine) Liquid System (Dako). Sections were counterstained with haematoxylin.

Digital quantification of TTR deposition

The sections were examined under light microscopy and the entire field of the colon was digitized using an Olympus DP71 camera and DP-BSW-V3.1 software. Briefly, individual images of the section were captured, and then merged to produce an image of the colon section. Semiquantitative analysis of immunohistochemical images was performed using public domain ImageJ software (National Institutes of Health), which performed automated particle analysis in a measured area, that is, the area occupied by pixels corresponding to the immunohistochemical substrate colour was counted [31]. TTR staining in the colon area was assessed by the colour selection tool of the program and the volume of TTR deposition per total area was determined. Each slide used in semiquantitative immunohistochemistry was analysed in 30 different selected areas by two investigators independently.

Statistics

All data are expressed as means±S.D. Statistical comparison of control with treated groups was carried out by Student's t test. The accepted level of significance was P<0.05.

RESULTS

Effect of branched β-CyDs on TTR amyloid formation

Because recent studies revealed that branched β-CyDs interacted with hydrophobic amino acids on the protein surface and increased its stability, which, in turn, suppressed the protein misfolding and aggregation [20,21], we first screened the effect of possible branched β-CyDs on TTR amyloid formation by ThT-based fluorimetric assay. Of various branched β-CyDs, GUG-β-CyD significantly showed an inhibitory effect on TTR amyloid formation, compared with the other branched β-CyDs (Figure 1).

Figure 1 Effect of branched β-CyDs on TTR amyloid formation

Samples of 20 μM WT TTR were incubated with or without various branched β-CyDs (HP-β-CyD, G1-β-CyD, G2-β-CyD, GUG-β-CyD, Gal-β-CyD and Man-β-CyD; 40 mM for each β-CyD) at 37 °C for 14 days in sodium acetate buffer, pH 3.5. WT TTR amyloid formation was assessed by ThT-based fluorimetric assays. Each bar represents the mean±S.D. (n=3). *P<0.05 compared with WT TTR alone.

Effect of GUG-β-CyD on TTR amyloid formation

To determine the inhibitory effect of GUG-β-CyD on TTR amyloid formation further, we next evaluated the effect of GUG-β-CyD on amyloid formation of both WT TTR and ATTR V30M by ThT-based fluorimetric assays. As shown in Figures 2(A) and 2(B), GUG-β-CyD significantly suppressed the amyloid formation of both WT TTR and ATTR V30M in a dose-dependent manner. The effect of GUG-β-CyD was sustained and increased in a time-dependent manner, and the significant inhibition of both WT TTR and ATTR V30M amyloid formation was observed even at 14 days after GUG-β-CyD administration (Figures 2A and 2B). There was no effect of the addition of GUG-β-CyD on ThT binding to TTR amyloid fibrils (see Supplementary Figure S1 at http://www.BiochemJ.org/bj/437/bj4370035add.htm). To confirm the inhibitory effect of GUG-β-CyD morphologically, electron microscopic analysis was performed to detect TTR amyloid fibrils directly. Consistent with the results described above, TTR amyloid formation was significantly suppressed in the presence of GUG-β-CyD (Figure 2C).

Figure 2 Effect of GUG-β-CyD on TTR amyloid formation

Samples of 20 μM WT TTR (A) and V30M ATTR (B) were incubated with or without GUG-β-CyD at 37 °C in sodium acetate buffer, pH 3.5. TTR amyloid formation was assessed by ThT-based fluorimetric assays in a dose-dependent (left-hand panel) and a time-dependent (right-hand panel) manner, as indicated. Each value represents the mean±S.D. (n=5). *P<0.01 compared with WT TTR alone; **P<0.01 compared with ATTR V30M alone. (C) Electron micrograph of WT TTR incubated with or without GUG-β-CyD at 37 °C for 14 days in sodium acetate buffer, pH 3.5. Scale bars=100 nm.

Effect of GUG-β-CyD on the conformational change of TTR

To investigate the detailed mechanism of how GUG-β-CyD inhibited TTR amyloid formation, the conformational change of TTR by GUG-β-CyD was next examined by CD spectroscopic analysis. As shown in Figure 3, the CD spectrum of TTR (5.0 μM) showed negative bands at 215 nm in PBS, pH 3.5. Under the appropriate value of the high voltage (HT; photomultiplier voltage), the positive and negative CD bands of TTR (1.25 μM) at 195 nm and 215 nm respectively in the TTR-alone system clearly showed a β-sheet structure (a predominant feature of monomers or dimers) of TTR (see Supplementary Figure S2 at http://www.BiochemJ.org/bj/437/bj4370035add.htm). In the presence of GUG-β-CyD, the negative band in the CD spectrum of TTR was significantly increased in a dose-dependent manner (Figure 3), suggesting that the inhibitory effect was caused by the reduction of conformational changes of TTR in the process of amyloid formation. In contrast, GUG-β-CyD showed no significant effect on the stabilization of tetrameric TTR (see Supplementary Figure S3 at http://www.BiochemJ.org/bj/437/bj4370035add.htm).

Figure 3 Effect of GUG-β-CyD on the conformational change of TTR

Samples of 20 μM WT TTR incubated with or without GUG-β-CyD at 37 °C for 7 days in sodium acetate buffer, pH 3.5, were applied to a Jasco J-720 spectropolarimeter, as indicated. Far-UV spectra (200–240 nm) were recorded in a 1-mm-path-length cell. TTR alone (continuous line); TTR with 10 mM (– –), 20 mM (—) and 40 mM (dotted line) GUG-β-CyD.

Effect of GUG-β-CyD on intrinsic tryptophan fluorescence of TTR

Because previous studies revealed that branched β-CyDs inhibited protein aggregation by stabilization of a molten globule intermediate upon the refolding process via the interaction with a hydrophobic side chain, such as tryptophan [20,21], the effect of GUG-β-CyD on intrinsic tryptophan fluorescence of TTR was next evaluated. The fluorescence intensity of tryptophan (340 nm) of TTR was quenched by the addition of GUG-β-CyD in a dose-dependent manner (Figure 4A). Consistent with the result shown in Figure 1, the quenching ability for fluorescence of tryptophan was decreased in the order GUG-β-CyD>G2-β-CyD>HP-β-CyD (Figure 4B).

Figure 4 Effect of GUG-β-CyD on tryptophan fluorescence intensity of TTR

The tryptophan fluorescence intensity of WT TTR was monitored by a Hitachi F-4500 spectrofluorimeter at 25 °C. (A) Samples of 20 μM WT TTR were incubated with or without GUG-β-CyD. TTR alone (continuous line); TTR with 10 mM (——), 20 mM (––) and 40 mM (—) GUG-β-CyD. (B) Samples of 20 μM WT TTR were incubated with or without various branched β-CyDs. TTR alone (continuous line), HP-β-CyD (——), G2-β-CyD (––) and GUG-β-CyD (—); 40 mM for each β-CyD.

Effect of GUG-β-CyD on 1H-NMR spectrum of TTR

To gain insight into the mechanism of the interaction mode of the TTR/GUG-β-CyD system, we performed 1H-NMR spectroscopic analysis in which TTR was dissolved in 2H2O, pH 3.5, at 80 °C (Figure 5). Reid and Saunders [32] reported that four amino acids (Trp79, Phe44, His88 and Tyr105) of TTR gave NMR signals around 7.4–7.8 p.p.m. As shown in Figure 5, although no significant chemical shift was observed in 1H-NMR spectra in the presence of GUG-β-CyD, the NMR signals around 7.4–7.8 p.p.m. were apparently sharpened as the concentration of GUG-β-CyD was increased.

Figure 5 Effect of GUG-β-CyD on 1H-NMR Spectrum of TTR

(A) 1H-NMR spectra were recorded on a JNM-500 spectrometer at 80 °C. WT TTR (2.0 mg) was dissolved in 0.6 ml of 2H2O, which was 0.1 and 0.05 M with respect to sodium chloride and sodium phosphate respectively, and adjusted to register a pH-meter reading of 3.5. (B) Enlarged 1H-NMR spectra of TTR in the range 7.65–7.55 p.p.m.

Effect of GUG-β-CyD on TTR deposition in ATTR V30M Tg rats

To confirm the inhibitory effect of GUG-β-CyD further in vivo, we evaluated the effect of GUG-β-CyD on TTR deposition in transgenic rats possessing a gene encoding human ATTR V30M. Besides no side effect on blood biochemical findings such as aspartate aminotransferase, alanine aminotransferase, creatinine and blood urea nitrogen being observed in GUG-β-CyD-treated rats (see Supplementary Figure S4 at http://www.BiochemJ.org/bj/437/bj4370035add.htm), serum levels of human ATTR V30M showed no significant change (Figure 6A). Because our previous report showed that nonfibrillar deposits of human ATTR V30M were detected in the gastrointestinal tracts of ATTR V30M Tg rats, considered as an index of TTR deposition [30], we next sought to determine whether GUG-β-CyD inoculation suppressed TTR deposition in the gastrointestinal tract. In accordance with our results from the present study, TTR deposition in the colon was significantly reduced by GUG-β-CyD treatment (Figures 6B and 6C).

Figure 6 Effect of GUG-β-CyD on TTR deposition in ATTR V30M Tg rats

(A) Serum levels of human ATTR V30M in Tg rats. n.s., not significant. (B) Immunoreactivity with polyclonal anti-human TTR antibody in the colon of ATTR V30M Tg rats. The panels shown are representative images of 30 fields in four colonal sections collected from nine ATTR V30M Tg rats. Scale bars=50 μm. (C) Semi-quantitative analysis of TTR deposition in the colon of ATTR V30M Tg rats. Bars represent the relative quantity of TTR deposition reported as the mean±S.D. (n=9). *P<0.05 compared with control.

DISCUSSION

In the present study, we demonstrated that GUG-β-CyD caused significant inhibition of TTR amyloid formation. The results presented in Figure 2 clearly indicate that GUG-β-CyD suppressed the amyloid formation of both WT TTR and ATTR V30M not only in a dose-dependent, but also in a time-dependent, manner. From CD spectroscopic analysis, GUG-β-CyD significantly decreased the negative Cotton effect derived from β-sheet structures of TTR (Figure 3). The slight change of the minimal wavelengths was probably due to the conformational change of TTR through the interaction of GUG-β-CyD. The strong negative band at 200 nm in the spectrum of the TTR might be caused by the soluble aggregation of TTR in sodium acetate buffer, pH 3.5. Additionally, there was no significant effect of GUG-β-CyD on the spectrum of native TTR (see Supplementary Figure S5 at http://www.BiochemJ.org/bj/437/bj4370035add.htm). Because it has been proposed that disease-causative proteins generally undergo a β-sheet conformational transition to form aggregation intermediates and further polymerize into amyloid fibril aggregation [1], these results suggest that the inhibitory effect of GUG-β-CyD was caused by the reduction of β-sheet transitions and conformational change of TTR in the process of amyloid formation.

Since previous studies revealed that branched β-CyDs interacted with hydrophobic amino acids on the protein surface and increased its stability [20,21], the hydrophobic cavity of GUG-β-CyD may directly include the hydrophobic amino acids of TTR, resulting in the prevention of TTR amyloid formation. As expected, the fluorescence intensity of tryptophan (340 nm) of TTR was quenched by the addition of GUG-β-CyD (Figure 4). In a monomeric form of TTR, two hydrophobic tryptophan residues (at positions 41 and 79) are located at the TTR protein surface and are presumably accessible to GUG-β-CyD (see Supplementary Figure S6 at http://www.BiochemJ.org/bj/437/bj4370035add.htm). In general, the fluorescence intensity of tryptophan increases when this moiety is buried in the hydrophobic environment of the native form. Therefore these results suggest that GUG-β-CyD may cause the conformational change of TTR from the native form through the interaction with tryptophan residues of TTR. Moreover, the NMR signals of TTR around 7.4–7.8 p.p.m. derived from aromatic amino acids were apparently sharpened by the addition of GUG-β-CyD (Figure 5), suggesting that the flexibility of the aromatic amino acids including tryptophan was increased by GUG-β-CyD, probably due to a conformational change of TTR.

It is well documented that the conversion of TTR from the native folded form into self-assembled amyloid fibrils is thought to involve multi-step processes [33]. Tetrameric TTR first dissociates into monomers. This step is essential, but not sufficient, for amyloid fibril formation, because further structural change by misfolding within the monomeric form of TTR is necessary to form aggregation intermediates, and thereafter to induce self-assembly polymerization [14]. However, no effective therapy targeting this misfolding step has been designed. For the last decade, multiple studies have focused on stabilizing the tetrameric TTR structure, as a potential therapeutic strategy [34]. Previous studies have shown that various NSAIDs (non-steroidal anti-inflammatory drugs), such as flufenamic acid and diflunisal or structurally related species, have potential for stabilizing the tetrameric TTR [3537]. However, since pharmacological effects of NSAIDs also lead to problematic side effects such as gastrointestinal ulcers and bleeding and an increase in the risk of cardiovascular events [3840], an alternative therapeutic strategy is urgently needed. In the present study, our multiple biochemical approaches revealed that GUG-β-CyD exerted its inhibitory effect on TTR amyloid formation by stabilizing TTR conformation via the interaction with the hydrophobic amino acids of TTR (Figures 3–5). In addition to the results described above, it is notable that GUG-β-CyD showed no significant effect on the stabilization of tetrameric TTR (Supplementary Figure S3). The soluble monomeric form of TTR was increased by GUG-β-CyD treatment (results not shown), suggesting that GUG-β-CyD may have potential to affect the misfolding within the monomer. Meanwhile, because the cavity of GUG-β-CyD preferentially interacts with amino acid residues responsible for the aggregation through the inclusion complex [20], the inhibitory effect of GUG-β-CyD could also be attributed to impairment of polymerization of TTR through steric hindrance and suppression of the intermolecular hydrophobic contacts between aromatic side chains across the monomer–monomer interfaces of TTR by complexation of TTR with GUG-β-CyD. Taken together, our results may raise the possibility of a novel therapeutic strategy for TTR-related amyloidosis.

Several studies suggested the inhibitory effects of CyDs on aggregation of proteins such as human serum albumin, α-chymotrypsin, recombinant growth hormones and β-amyloid peptide [4145]. For this inhibitory effect on protein aggregation, the internal cavity of CyDs, which is hydrophobic, is a key structural feature of the molecule, providing the ability to complex, and can contain a variety of guest molecules such as fatty acids and esters [18,19,46]. The guest must satisfy the size criterion of fitting at least partially into the CyD cavity to form an inclusion complex, and the cavity diameters of α-CyD, β-CyD and γ-CyD are 4.7–5.3 Å, 6.0–6.5 Å and 7.5–8.3 Å respectively (1 Å=0.1 nm). Since the effective diameter of the aromatic ring of amino acids such as phenylalanine and tryptophan, adding hydrogen, is approximately 5 Å [47], β-CyD should make a good steric fit to the tryptophan. Since GUG-β-CyD has almost the same cavity size as β-CyD, GUG-β-CyD probably forms an inclusion complex with tryptophan of TTR. GUG-β-CyD, a novel branched β-CyD derivative with a carboxylic acid moiety, was synthesized by microbial oxidation [47]. There are many advantages of GUG-β-CyD, such as higher solubility in water (>2000 mg/ml in water at 25 °C), excellent solubilizing ability for poorly water-soluble drugs, and lower haemolytic activity and cytotoxicity, compared with alkylated CyDs [48,49]. GUG-β-CyD can also be obtained in a high state of purity, which may have a significant advantage over commercially available CyDs such as HP-β-CyD and SBE-β-CyD (sulfobutyl ether-β-CyD). In addition, previous studies have revealed that GUG-β-CyD can effectively inhibit the aggregation of proteins such as bFGF (basic fibroblast growth factor) and lysozyme [20,21]. Interestingly, although the chemical structural difference between GUG-β-CyD and G2-β-CyD is only whether they have a carboxylic acid in their molecule or not [48], the inhibitory effect of GUG-β-CyD was superior to that of G2-β-CyD (Figure 1). Khajehpour et al. [50] reported that visual inspection of several protein–CyD complexes in the protein structural database shows tryptophan side chains interacting with the outside of the CyD ring. Therefore the inhibitory effect on TTR amyloid formation might be induced by not only hydrophobic interaction with the CyD cavity, but also the interaction with the carboxylic acid moiety of GUG-β-CyD. Taken together, these lines of evidence suggest that GUG-β-CyD has a great potential to be a parenteral therapeutic product as a novel inhibitor of TTR amyloid formation.

Finally, our in vivo studies revealed that the significant inhibitory effect of GUG-β-CyD was also confirmed under physiological conditions in the ATTR V30M Tg rat [30], an existing useful animal model of FAP (Figure 6). Since no suitable animal model representing the amyloid deposition and clinical symptoms seen in FAP is currently available, TTR deposition (the pre-amyloid state of TTR) has been widely used as an index of therapeutic efficacy for evaluating various candidate agents for FAP [5153]. Our ATTR V30M Tg rats showed the prefibrillar deposits of human ATTR V30M in the gastrointestinal tract starting at 10–12 months after birth [30]. In FAP, it is well known that TTR deposition systematically occurs before the appearance of amyloid deposition and leads to progression of FAP symptoms. In our present study, because GUG-β-CyD administration was started at 9 months after birth, our results may suggest the preventive effect of GUG-β-CyD on TTR deposition. As suggested by the characterization of CyDs, no side effect on the blood biochemical findings, such as aspartate aminotransferase, alanine aminotransferase, creatinine and blood urea nitrogen, was observed by the administration of GUG-β-CyD (Supplementary Figure S4). It should be noted that in some cases, CyD derivatives are used in the pharmaceutical field at the concentration employed in the present study, because of their low toxicity [17,54]. Because serum levels of human ATTR V30M showed no significant change (Figure 6A), we can exclude the possibility that the reduction of TTR deposition was caused by the effect of GUG-β-CyD on TTR expression. In addition, this result may also imply that the stabilization of TTR conformation by GUG-β-CyD does not influence the metabolism of serum TTR. In general, it is well known that the hydrophilic CyDs are rapidly eliminated through renal excretion after intravenous administration [55,56]. In the present study, GUG-β-CyD still exhibited the significant inhibitory effect in ATTR V30M Tg rats (Figure 6C), implying the different disposition of GUG-β-CyD in blood circulation. The pharmacokinetic analysis of GUG-β-CyD after intravenous administration should be examined further.

TTR, a β-sheet-rich protein, is a precursor protein of both FAP and SSA [69]. In FAP, although liver transplantation has become a well-established therapy, it has given rise to several problems, and no essential therapy has been practically established [5759]. Moreover, although it is well documented that WT TTR can also cause amyloid fibrils in patients with SSA [8], no effective therapy for SSA is available as of this moment.

In conclusion, we have provided the first evidence that GUG-β-CyD significantly suppressed TTR amyloid formation in in vitro and in vivo model examinations. Since CyDs are safe and already widely used in many fields, especially in the pharmaceutical field, from a practical application perspective GUG-β-CyD may have the potential to become a novel curative medicine for TTR-related amyloidosis.

AUTHOR CONTRIBUTION

Hirofumi Jono designed and performed experiments, analysed data and wrote the paper. Takayuki Anno and Yohei Misumi designed and performed experiments. Keiichi Motoyama designed experiments, analysed data and wrote the paper. Masayoshi Tasaki, Toshinori Oshima and Yoshimasa Mori performed experiments. Mineyuki Mizuguchi provided materials. Mitsuharu Ueda, Makoto Shono and Konen Obayashi gave conceptual advice. Hidetoshi Arima designed experiments, analysed data and wrote the paper. Yukio Ando designed and supervised experiments, and wrote the paper.

FUNDING

This work was supported by Grants-in-Aid for Scientific Research (B) from the Ministry of Education, Science, Sports and Culture of Japan [grant numbers 17390254 and 21390270].

Acknowledgments

We thank Mrs Hiroko Katsura for tissue specimen preparation and Dr Teruya Nakamura for preparing the crystal structure image of TTR.

Abbreviations: CyD, cyclodextrin; FAP, familial amyloidotic polyneuropathy; G1-β-CyD, 6-O-α-glucosyl-β-CyD; G2-β-CyD, 6-O-α-maltosyl-β-CyD; Gal-β-CyD, galactosylated-β-CyD; GUG-β-CyD, 6-O-α-(4-O-α-D-glucuronyl)-D-glucosyl-β-CyD; HP-β-CyD, hydroxypropyl-β-CyD; Man-β-CyD, mannosylated-β-CyD; NSAID, non-steroidal anti-inflammatory drug; SSA, senile systemic amyloidosis; Tg, transgenic; ThT, thioflavin T; TTR, transthyretin; ATTR, amyloidogenic TTR; WT, TTR, wild-type TTR

References

View Abstract