Inhibitory Activities of Propolis and Its Promising Component, Caffeic Acid Phenethyl Ester, against Amyloidogenesis of Human Transthyretin Takeshi Yokoyama, Yuto Kosaka, and Mineyuki Mizuguchi* Faculty of Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0914, Japan S Supporting Information *

ABSTRACT: Transthyretin (TTR) is a homotetrameric serum protein associated with amyloidoses such as familial amyloid polyneuropathy and senile systemic amyloidosis. The amyloid fibril formation of TTR can be inhibited through stabilization of the TTR tetramer by the binding of small molecules. In this study, we examined the inhibitory potency of caffeic acid phenethyl ester (CAPE) and its derivatives. Thioflavin T assay showed that CAPE suppressed the amyloid fibril formation of TTR. Comparative analysis of the inhibitory potencies revealed that phenethyl ferulate was the most potent among the CAPE derivatives. The binding of phenethyl ferulate and the selected compounds to TTR were confirmed by the 8-anilino-1-naphthalenesulfonic acid displacement and X-ray crystallography. It was also demonstrated that Bio 30, which is a CAPE-rich commercially available New Zealand propolis, inhibited TTR amyloidogenesis and stabilized the TTR tetramer. These results suggested that a propolis may be efficient for preventing TTR amyloidosis.

INTRODUCTION Transthyretin (TTR) is a homotetrameric protein found in the human blood plasma, where it transports thyroxine (T4) and retinol by binding to retinol-binding protein. TTR variants are related to hereditary amyloidoses such as familial amyloid polyneuropathy and familial cardiomyopathy.1,2 To date, over 100 disease-related point mutations of TTR have been identified, and V30M mutation is the most frequent among them.3−5 Amyloidogenic mutants, including V30M, present a destabilized quaternary and/or tertiary structure and tend to dissociate into monomers, aggregate into amyloid fibrils, and deposit in organs such as the peripheral nerves, heart, and eyes.1,2,6−8 Since the amyloid fibrils of TTR are formed through the misfolding of monomers, stabilization of the TTR tetramer is an efficient strategy for TTR amyloidosis.9−11 TTR is a βsheet-rich protein composed of four subunits termed A, B, C, and D, each of which is composed of 127 amino acid residues. TTR has two funnel-shaped T4-binding sites which are created between subunits A and C (AC site) or B and D (BD site). It has been shown that T4 molecules bind to TTR with negative cooperativity and the association constants for the first and the second T4 molecule differ by a factor of about 100.12,13 Several small molecules are known to bind to the T4-binding site of TTR, stabilize the TTR structure, and inhibit the amyloid fibril formation of TTR.14−16 In the search for a new inhibitor, some natural products such as genistein, resveratrol, and (−)-epigallocatechin 3-gallate have recently been shown to inhibit the TTR amyloid fibril formation.17−19 © XXXX American Chemical Society

Propolis is a sticky resinous substance that honeybees collect from plants to reinforce the hive and prevent parasites from entering. It has been adopted as a form of folk medicine since ancient times.20,21 In the modern era, propolis has been shown to exhibit a broad spectrum of biological activities, such as antioxidant, anti-inflammatory, immunomodulatory, and anticancer properties, and these activities are considered to be derived from the various compounds in the ethanol extract of propolis.22−24 The chemical composition of propolis is variable, depending on the vegetation at the area from which it was collected.25,26 Caffeic acid phenethyl ester (1, CAPE) (Table 1) is one of the most active of the compounds isolated from New Zealand,27 Uruguayan,28 and Netherlands propolis.29 Propolis dietary supplements are commercially available and considered to be very safe, and therefore, if propolis or its constituents inhibit the amyloid fibril formation of TTR, they could be adopted as reasonable auxiliary materials. Additionally, the structural similarity between CAPE and the known inhibitors such as diflunisal and resveratrol is a driving force to test CAPE and its derivatives (Supporting Information Figure 1). In this study, we set out to determine the inhibitory activities of CAPE and its analogues against the amyloid fibril formation of TTR. In addition, we tested several lignans, including nordihydroguaiaretic acid (14, NDGA) to gain further insight. Their binding to TTR was verified using an ANS (8-anilino-1naphthalenesulfonic acid) displacement experiment and X-ray Received: July 3, 2014

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Table 1. Inhibitory Activities of Caffeic Acid Phenethyl Ester and Caffeic Acid Derivatives

compd no.

compd name




EC50 (μM)

Control 5.6 ± 0.39

Diflunisal 1 2 3 4 5

phenethyl phenethyl phenethyl phenethyl phenethyl

caffeate ferulate 4-methylcaffeate dimethylcaffeate cinnamate

6 7 8 9 10 11 12 13

caffeic acid methyl caffeate ethyl caffeate 1,1-dimethylallyl caffeate n-octyl caffeate benzyl caffeate chlorogenic acid rosmarinic acid

Caffeic Acid Phenethyl Ester Derivatives −OH −OH −(CH2)2Ph −OCH3 −OH −(CH2)2Ph −OH −OCH3 −(CH2)2Ph −OCH3 −OCH3 −(CH2)2Ph −H −H −(CH2)2Ph Caffeic Acid Derivatives −OH −OH −H −OH −OH −CH3 −OH −OH −CH2CH3 −OH −OH −CH2CHCCH3CH3 −OH −OH −(CH2)7CH3 −OH −OH −CH2Ph −OH −OH quinic acid −OH −OH caffeic acid

RESULTS Chemicals. The following commercially available compounds were used in this study (Table 1, Table 2): caffeic acid Table 2. Inhibitory Activities of NDGA Derivatives

compd name



EC50 (μM)

14 15 16

NDGA dihydroguaiaretic acid tetramethyl NDGA



7.3 ± 0.81 6.3 ± 0.99 ND

97 ± 21 67 ± 11 23 ± 0.82 21 ± 3.1 nonspecific inhibition 16 (competitive binding with thioflavin T) ND 8.6 ± 0.24

Information Figure 2). CAPE (1), phenethyl ferulate (2), and rosmarinic acid (13) were the most potent inhibitors among the CAPE and CA alkyl esters, and their EC50 values were below 10 μM (Table 1). While the methylation of 3-OH of CAPE increased the inhibitory activity (2, phenethyl ferulate; p < 0.05), the methylation of 4-OH of CAPE decreased it (3, phenethyl 4-methyl caffeate; p < 0.001). The methylation of both 3- and 4-OH of CAPE drastically decreased the inhibitory activity (4, phenethyl dimethyl caffeate). Phenethyl cinnamate (6, dehydroxylated of 1) did not exhibit the inhibitory activity. Caffeic acid (6, CA) was the inhibitor with the least inhibitory activity among the CA and its alkyl esters (Table 1). With the larger volume of alkyl esters, the inhibitory activities became stronger, but the activity of benzyl caffeate (11), which was the most potent among the CA derivatives, was weaker than that of CAPE. Although n-octyl caffeate (10) possessed a certain inhibitory activity, the ratio of amyloid fibril formation showed much weaker concentration dependence than that for the other compounds: 5 μM, 27%; 10 μM, 44%; 20 μM, 47%; 40 μM, 49%; 80 μM, 53%. The inhibitory activities of compound 10 may thus be due to a nonspecific interaction. Chlorogenic acid (12), the glycosylated form of CA, did not inhibit the amyloid fibril formation. We have also tested NDGA (14) and its methylated derivatives (15, 16) from lignans (Table 2). The inhibitory activities of 15 and 16 are not known, although NDGA is known as a TTR amyloid inhibitor.31 Compound 15 exhibited stronger inhibitory activity than 14 (p < 0.01), and its EC50 value was close to that of diflunisal, which is considered a candidate for clinical use.18,32,33 On the other hand, compound 16 did not inhibit the amyloid fibril formation. The comparative analysis including CAPE derivatives revealed that the methylation of 3-OH was more effective than that of 4-OH, and the methylation of both the hydroxyl groups drastically decreased the activity. L55P and I84T amyloidogenic variants have also been tested using compound 14 by thioflavin T assay. The EC50 values for the L55P and I84T mutants were 12.6 ± 0.3 and 8.5 ± 1.6 μM, respectively.

crystallographic analysis. Finally, the inhibitory activity and the stabilization effect of Bio 30, which is a commercially available propolis derived from New Zealand, were evaluated. The present results suggest that Bio 30 could be used as an auxiliary or alternative material for the amyloid disease inhibitors.

compd no.

8.6 ± 0.44 7.9 ± 0.64 11 ± 1.3 58 ± 6.3 ND

phenethyl ester (1, CAPE, phenethyl caffeate), CAPE derivatives (2−5), caffeic acid (6, CA), CA alkyl esters (7− 11), chlorogenic acid (12, glucoside CA), rosmarinic acid (13, dimer ester of CA), nordihydroguaiaretic acid (14, NDGA), dihydroguaiaretic acid (15), and tetramethyl NDGA (16). Inhibitory Activity and ANS Displacement Experiment. The inhibitory activities of compounds 1−16 and diflunisal as positive control were tested against the amyloid fibril formation of TTR. The V30M mutated TTR, the most common TTR variant in FAP patients, was used for the acidmediated aggregation experiments. The thioflavin T assays and calculation of EC50 values are the well-established method for the quantification of the amyloid fibrils.30 The amyloid fibril inhibitory activities against 10 μM V30M are listed in Table 1 and Table 2 (Supporting Information Table 1, Supporting B | J. Med. Chem. XXXX, XXX, XXX−XXX

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Figure 1. Displacement of ANS by compound 2. The TTR-ANS complex was titrated with increasing concentrations of compound 2.

Figure 2. V30M crystal structures complexed with CAPE (a) and compounds 2 (b), 8 (c), 9 (d), and 13 (e). The carbon atoms and ribbon model of TTR are colored in silver. The carbon atoms of the compounds are colored in green and cyan. Oxygen atoms are colored in red and nitrogen atoms in blue. The alphabetic characters in parentheses indicate the subunit name. The hydrogen bonds are indicated as a dashed yellow line.

In order to verify whether the tested compounds competitively bind to amyloid fibrils with thioflavin T, the fluorescence emission spectra of 20 μM thioflavin T in the presence of the preformed V30M amyloid fibrils and 0−80 μM compounds were measured (Supporting Information Figure 3 and Supporting Information Figure 4). There were no significant changes dependent on the compound concentrations, indicating that the compounds were not competitive binders at amyloid fibrils.34 It should be noted that only compound 11 showed the concentration dependence, suggesting the competitive binding or disaggregation of amyloid fibrils (Supporting Information Figure 4i). In the case of Bio 30, the fluorescence intensities were slightly decreased only at high concentration (Supporting Information Figure 4m), suggesting that most of active ingredients of Bio 30 are not competitive

binder of thioflavin T. We have also tested the binding of ANS (8-anilino-1-naphthalenesulfonic acid) and its displacement by the selected compounds.35 As expected, the addition of the compounds decreased the fluorescence of the V30M-ANS complex, indicating the competitive displacement of ANS, which binds to the T4-binding site of TTR (Figure 1, Supporting Information Figure 5). For the additional inspection of the catechol and guaiacol groups, lignans, such as enterodiol, enterolactone, matairesinol, sesamin and capsaicin, were tested by thioflavin T assay (Supporting Information Table 2). The inhibition ratios when the compounds were used at concentrations of 20 μM were all less than 12%, and thus these compounds did not possess the inhibitory activities. Enterodiol has the same skeletal structure as NDGA (14) so that the lack of the inhibitory activities would C | J. Med. Chem. XXXX, XXX, XXX−XXX

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The methyl group of 3-OCH3 was surrounded by S117-Oγ, T119-Cγ2 and the mainchain of T118 at a distance of 3.2−3.8 Å. The higher inhibitory potency of 2 than CAPE could be accounted for by the addition of these van der Waals interactions. Since the hydrogen bond patterns were similar among CAPE, 8, 9, and 13, the differences of the inhibitory activities of CA alkyl esters would be derived from the alkyl ether moiety. The central oxycarbonyl and alkyl moieties of 8 and 9 were surrounded by K15, L17, T106, A108, and V121, forming hydrophobic interactions. The thioflavin T assays indicated that the larger the volume of an alkyl group is, the higher the inhibitory activity, and therefore hydrophobic interactions with these residues would be an important factor for the inhibitory activities. It is also natural that more hydrophobic molecules would be beneficial for the binding energy because of the lower desolvation penalty. Recently, we suggested that optimizing the hydrophobic contacts rather than the hydrogen bonds could be useful for the development of the amyloid fibril inhibitors.30 The same approach is also applicable to CA alkyl esters: the activities of CA alkyl esters depend on how the alkyl groups interact with K15, L17, T106, A108, and V121. As an exceptional case, compound 13 has a carboxylic group, which forms a salt bridge with K15 (Figure 2e). Binding of NDGA Derivatives. We also found that NDGA (14) and dihydroguaiaretic acid (15) bound to the T4-binding site (Figure 3a,b). The one catechol group occupies the inner cavity of the T4-binding site, while another catechol group points to the molecular surface. The hydrogen bonds between the catechol group and S117 were similar to those of the CA derivative complex. The central hydrocarbons and the outer catechol group were surrounded by K15, L17, T106, A108, and V121 without any hydrogen bond. Accordingly, the binding mode of 14 was similar to that of CA alkyl esters. However, the asymmetrical binding mode was observed only in the TTR−15 complex structure. Compound 15 in the AC site formed the hydrogen bonds with S117(A) and S117(C), connecting subunit A and C. Whereas the binding position of 15 in the AC site was similar to that of the other compound, 15 in the BD site sits more deeply into the center of the molecule (Figure 3a,b). This asymmetric binding mode was not found in the V30M-1, -2, -7, -8, -13, and -14 complex structures. Because of this binding mode, 15 in the BD site cannot form a hydrogen bond with S117. Thioflavin T assay showed that 15 was a more potent inhibitor than 14 (EC50 = 7.3 μM (14), 6.3 μM (15), p < 0.01), and therefore the loss of a hydrogen bond at the BD site did not decrease the inhibitory activity. It has been reported that the association constants for the first and second T4 molecule differ by a factor of about 100.12 The thermal B factors of 15 bound to the AC and BD sites were 32 and 38 Å2, respectively, and thus the AC site could be assigned as the first binding site and the BD site as the second binding site. It has also been demonstrated that occupancy of one of the two T4binding sites is sufficient to inhibit the tetramer dissociation of TTR.39 Finally, the novel binding mode found at the BD site does not affect its inhibitory activity but may affect the cooperative binding property. Inhibitory Potency of Propolis. A propolis from New Zealand is known to have a high CAPE (1) content, and therefore, we investigated whether Bio 30 liquid, a commercially available propolis from Manuka Health New Zealand, would inhibit the TTR amyloid formation. The thioflavin T assays revealed that Bio 30 showed the inhibitory activity with an EC50 of 24 μg/mL (Supporting Information

be caused by the different position of the hydroxyl groups. Although the compounds with the guaiacol group (2, 14) were strong inhibitors, matairesinol and capsaicin did not show the inhibitory activities. Crystal Structures of V30M Complexed with the Selected Compounds. In order to reveal the structure− activity relationship at an atomic level of detail, the crystal structures of V30M complexed with compound CAPE, 2, 8, 9, 13, 14, or 15 were solved at 1.8, 1.6, 1.8, 1.8, 1.5, 1.55, and 1.6 Å resolution, respectively (Supporting Information Table 3). We have selected CAPE (1) and NDGA (14) because they are demethylated derivatives of most potent inhibitors (2 and 15) (Tables 1 and 2). We have selected compounds 7, 8, and 13 because they are different from compound 1 in R3 group (Table 1), aiming the structural explanation for the contribution of R3 group to their inhibitory potency. The crystal structure of the V30M apo-form was also solved at 1.4 Å resolution as a reference. The omit difference Fourier maps clearly showed that these compounds bound to the T4-binding site of TTR (Figure 2, Figure 3, Supporting Information Figure

Figure 3. V30M crystal structures complexed with compounds 14 (a) and 15(b). Both of the AC (upper) and BD (lower) sites are shown. All representations are the same as in Figure 2

6, and Supporting Information Figure 7),36,37 and these results were in agreement with the ANS displacement experiments. There were two monomers (subunits A and B) in an asymmetric unit forming a tetramer by crystallographic symmetry operation. Since the T4-binding sites were located on the crystallographic 2-fold axis, the compounds were observed with the symmetry-related molecule at the corresponding site. Therefore, the compounds without such symmetry occupy this site with a 50% statistical disorder. The root-mean-square deviations of Cα atoms between the V30Mapo and V30M-compound complexes range from 0.30 to 0.56 Å, indicating that the binding to V30M did not induce any significant structural change. Binding of Caffeic Acid Derivatives. The guaiacol moiety of 2 and catechol moieties of CAPE, 8, and 9 were placed at the inner cavity of the T4-binding site, while the alkyl ether groups were turned to the molecular surface (Figure 2a−d). The hydrogen bonds with S117 were similar among CAPE, 2, 8, 9, and 13, indicating that the methylation of 3-OH did not influence the binding modes. These hydrogen bonds have also been observed in the TTR−flavonoids complex structures.38 D | J. Med. Chem. XXXX, XXX, XXX−XXX

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site of TTR, and these bindings were also confirmed by X-ray crystallography. The crystal structure of 2 and 15 complexed with V30M revealed that the 3-methoxy group was surrounded by A108, L110, S117, and T119 and hydrogen-bonded with S117. Since these hydrogen bonds were conserved among the complex structures of 8, 9, 13, and 14 which possess a 3-OH group, the higher potency of the compounds with 3-OCH3 could be explained by the increased hydrophobic interactions with these amino acid residues. On the other hand, the inhibitory potency of the caffeic acid ester derivatives (1, 6−11) matched the order of the volume of the ester groups, that is, the improved hydrophobic contacts with L17, A108, T106, and V121. The typical TTR stabilizers identified so far share the twoaromatic-ring substructure and the linkers that connect them, including biphenyl, stilbene, and biphenyl ether.16,41,42 Whereas CAPE and its derivatives (2−5) studied here also possess the two aromatic-ring substructure, the linkers are longer than those of the known inhibitors. The linker of CAPE consists of six atoms (−CC−C−O−C−C−), whereas the linkers of biphenyl, stilbene, and biphenyl ether are 0−2 atoms. These longer linkers of compounds 2 and 13 enable them to interact with T106 and V121 at the molecular surface (Figure 2). Curcumin, a component of the curry spice turmeric, is known as an inhibitor of TTR amyloid fibril formation,35 and its two guaiacol units are connected by 7 atoms (Supporting Information Figure 1). Although the inhibitory potency of curcumin could not be compared by thioflavin T assay because of the low affinity at low pH, curcumin has been shown to bind to the T4-binding site by ANS displacement assay.35 The structural similarity suggests that the binding mode of curcumin can be expected to be similar to that of compound 2: the innercatecol group forms hydrogen bonds with S117, the linker interacts with L17 and A108, and the outer catecol group interacts with T106 and V121. These studies indicate that a compound with a longer linker can also be used as a template for the TTR amyloid fibril inhibitor. Several natural products that inhibit the amyloid fibril formation of TTR have been identified so far.18,19,35,38 Among them, the flavonoids have been well-studied. Unfortunately, the flavonoids in aglycon form, such as quercetin, exhibit poor bioavailability and are not stable because of their antioxidant activities.43−45 However, various flavonoids have been found in natural foods, and most of them are very safe and cheap. Furthermore, several studies to improve the bioavailability are currently underway. If these problems could be overcome, natural products might be an alternative or auxiliary material for the amyloid fibril inhibitors such as tafamidis and diflunisal. Thus, our research has focused on the natural products that are included in dietary supplements, such as propolis. In the present study, the inhibitory activities of CAPE, its derivatives, and Bio 30 liquid on the amyloid fibril formation of TTR were investigated. The EC50 value of CAPE against 10 μM V30M was 8.6 μM, which is slightly higher than that of diflunisal (Table 1). The EC50 value of Bio 30 was 24 μg/mL, and the chemical cross-linking experiment showed that Bio30 ingredients bound to TTR and inhibited the dissociation of the TTR tetramer (Figure 4, Supporting Information Figure 9). The contribution of CAPE alone to the 24 μg/mL EC50 value of Bio 30 was only 0.28 μg/mL (0.98 μM). New Zealand propolis has also been shown to contain compounds 6, 7, 8, and 11,46 and Uruguayan propolis has been shown to contain compounds 2, 3, and 4.28 It is likely that some of these compounds with the

Figure 8). Since Bio 30 contains 25% of a propolis lump, the EC50 values equivalent to the propolis lump is 5.9 μg/mL. On the other hand, the CAPE content of Bio 30 is approximately 1.2% 27 so that the EC50 value equivalent to the concentration of CAPE is only 0.28 μg/mL. The EC50 of CAPE alone is 2.4 μg/mL (8.6 μM), which is about 9 times higher than that of Bio 30. Since flavonoid ingredients such as chrysin and apigenin are known to inhibit TTR amyloid fibril formation,38 it is likely that CAPE and these flavonoids synergistically inhibit the amyloid fibril formation. The remarkable inhibitory activity of Bio 30 led to investigation of its ability to stabilize the TTR tetramer: the inhibitions of the tetramer dissociation mediated by acidic pH using Bio 30 and compound 2 were investigated. The TTR tetramer fraction was evaluated using glutaraldehyde cross-linking followed by SDS−PAGE (Figure 4).9,35,40

Figure 4. Effect of Bio 30 and compound 2 on the acid-mediated quaternary structural changes of TTR (7.3 μM). The + lane indicates the positive control incubated at pH 8.0 without the compounds. The − lane indicates the negative control incubated at pH 4.5 without the compounds. The concentrations of Bio 30 are 6.9, 34, 171, and 341 μg/mL. The concentrations of 2 are 7.3 and 36.5 μM (2.2 and 11 μg/ mL).

Interestingly, the presence of Bio 30 inhibited the acidmediated dissociation of the TTR tetramer, and the tetramer fractions were increased in a dose-dependent manner as with compound 2, indicating that Bio 30 ingredients including CAPE bind to TTR and stabilize the tetramer structure. It should be noted that the tetramer fractions observed by the glutaraldehyde cross-linking method using compounds 1, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16 and diflunisal were closely correlated with their EC50 values (Supporting Information Figure 9).

DISCUSSION The comparative thioflavin T assays and cocrystal structures presented herein revealed the structure−activity relationship. In the cases of both the CAPE and NDGA derivatives, 3-OCH3 was the most effective functional group for inhibiting the amyloid fibril formation among the selected compounds (Table 1 and Table 2). Our results suggested that our compounds reduce the amount of amyloid fibrils. The ANS displacement analysis showed that the compounds bound to the T4-binding E | J. Med. Chem. XXXX, XXX, XXX−XXX

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flavonoids such as chrysin and apigenin synergistically inhibited the amyloid fibril formation of TTR. It was noted that the poor bioavailability and water solubility of CAPE rendered it unusable clinically, but Bio 30, which contains large amounts of lipids that can dissolve CAPE, has been reported to induce nearly complete regression of human NF2 tumors in mice.24,27 In addition, attempts have been made to encapsulate Greek propolis ethanolic extracts in β-cyclodextrin in order to improve their solubility and bioavailability.47,48 These observations encourage us to speculate that Bio 30 liquid could be useful as a TTR amyloid fibril inhibitor. In summary, our results showed that Bio 30, a New Zealand propolis, and its ingredients, including caffeic acid phenethyl ester (1), inhibited the amyloid fibril formation of TTR and stabilized the TTR tetramer. Comprehensive thioflavin T assays revealed that phenethyl ferulate (2) and dihydroguaiaretic acid (15) were the most potent inhibitors among the CAPE and NDGA derivatives, respectively. The crystal structures complexed with the selected compounds provided structural insights into their inhibitory activities.

ANS and the compounds were sequentially added to the protein solution, stirred, and equilibrated for 5 min prior to the fluorescence measurements. In all cases, the maximal dilution was less than 0.4%. The fluorescence emission spectra (400−540 nm) were recorded by exciting at 280 nm.35 Crystallization. V30M-apo crystals were grown by mixing 2 μL of protein solution (10 mg/mL protein, 20 mM Tris-Cl, pH 8.0, 150 mM NaCl) with 2 μL of reservoir solution containing 24−34% polyethylene glycol (PEG) 400, 0.1 M HEPES, pH 7.5, and 0.4 M CaCl2 using the hanging-drop vapor diffusion method at 293 K. The stock solutions of the compounds for the crystallization were prepared using dimethyl sulfoxide. Prior to cocrystallization, the V30M solution (10 mg/mL protein, 20 mM Tris-Cl, pH 8.0, 150 mM NaCl) was incubated for 30 min at room temperature in the presence of 4 mM 2, 12 mM 8, 9 mM 9, 9 mM 13, 8 mM 14, or 6 mM 15. The crystals of V30M−ligand complexes suitable for X-ray diffraction experiments were grown from 10−30% PEG 400, 0.1 M HEPES, pH 7.0, and 0.2 M CaCl2. The typical crystal dimensions were 0.2 mm × 0.05 mm × 0.05 mm. The crystals were directly frozen in liquid nitrogen until X-ray data collection. X-ray Diffraction Data Collection, Processing, and Structure Refinement. X-ray diffraction data were collected at beamline NW12A or NE3A at the Photon Factory Advanced Ring in Japan. The diffraction data sets were processed with HKL2000 and SCALEPACK.51 The X-ray structure of WT-TTR (PDB code 4N85)30 was used as the initial model for V30M-apo data, and the refined V30M-apo structure was used for the complex data. The refined 3D structures and the dictionary data of the compounds were obtained from the PRODRG server.52,53 The protein structures were refined using PHENIX.REFINE54 with stepwise cycles of manual model building using COOT.55 The inhibitor molecules were refined with 50% of the occupancy in each site, since they sit on the crystallographic 2-fold axis. The final models were evaluated using the Protein Data Bank validation suite.56 The coordinates and structure factors of V30M-apo and V30M complexed with CAPE, 2, 8, 9, 13, 14, and 15 have been deposited in the Protein Data Bank under the accession codes 4PWE, 4QRF, 4PWF, 4PWG, 4PWH, 4PWI, 4PWJ, and 4PWK, respectively. Crystal and refinement data are listed in Supporting Information Table 3. All structure figures were created using PyMOL.57 Glutaraldehyde Cross-Linking and SDS−PAGE. The stock solutions of the selected compounds and Bio 30 liquid were prepared in ethanol. Prior to the acid-mediated tetramer dissociation, V30M in solution [20 mM Tris-Cl, pH 8.0, and 150 mM NaCl] was incubated with Bio 30 or the compounds at room temperature for 30 min. After the incubation period, a 4-fold volume of 0.1 M sodium acetate, pH 4.5, was added, and then the sample was incubated at 310 K for 8 days. At this stage, the protein concentration was 7.3 μM, the Bio 30 concentrations were 6.8, 34, 171, and 341 μg/mL, and the compound concentrations were 7.3 and 36.5 μM. After the incubation period, an amount of 50 μL of the incubated samples was mixed with 2.5 μL of 25% glutaraldehyde and incubated at room temperature for 5 min and the reactions were terminated by the addition of 5 μL of sodium borohydrate (7% (w/v) in 0.1 N NaOH). The aliquots were then mixed with SDS sample buffer, boiled, and resolved by SDS−PAGE (15% SDS−acrylamide gel). The protein bands were visualized with a silver staining kit (Silver Stain Kanto III).


Materials. V30M mutated TTR (V30M) was prepared from an Escherichia coli expression system as previously described.49 V30MTTR samples were purified using Ni-NTA and then dialyzed against the buffer [20 mM Tris-HCl, pH 8.0, and 150 mM NaCl]. The single purification step was enough to obtain high-purity sample because of its high level of the expression. The purity has been assessed by SDS− PAGE/CBB staining and estimated to be greater than 90%. Compounds 1, 8, and 10 were purchased from Enzo Life Sciences (catalog nos. 270-244-M010, ALX-270-480-M050, and ALX-350-278M005). Compounds 2, 3, and 4 were purchased from LKT Laboratories (P1917, P2918, and P2410). Compounds 5 and 6 were purchased from Tokyo Chemical Industry (P2007 and C0002). Compounds 13, 14, and 16 were purchased from Cayman Chemical (20283-92-5, 500-38-9, and 24150-24-1). Compounds 7, 9, 11, 12, and 15 were purchased from Santa Cruz Biotechnology (SC-204664), Fluka (40785-50MG), Analyticon Discovery (NP-001972), Wako Pure Chemical Industries (033-14241), and BioBioPha (BBP00124), respectively. All chemicals were supplied in greater than at least 95% purity as determined by HPLC (e.g., 3 at >99.5% and 13 at >95.0%). Acid-Mediated Aggregation Experiments (Thioflavin T Assay). The thioflavin T assay was performed as described previously.30 Amyloid fibril inhibitory curves were fitted using the four-parameter logistic model50 by the least-squares methods, and at least four ligand concentrations (2.5−80 μM) were used. EC50 was estimated according to this model. The EC50 values, standard deviations (SD), coefficients of determination (r2), and numbers of measurements are listed in Table 1, Table 2, and Supporting Information Table 1. The competitive binding at fibrils between the compounds and thioflavin T was tested using preformed amyloid fibrils. The amyloid fibrils were prepared from 800 μL of 10 μM V30M containing 66.8 μM thioflavin T incubated at 37 °C for 72 h. After the incubation period, 20 μL of compounds, 500 μL of 1 M Tris-HCl, pH 8.0, and 1230 μL of H2O were added into the 750 μL of preformed fibrils. The final concentrations of thioflavin T, compounds, Tris-HCl, pH 8.0, were 20 μM, 0−80 μM, and 200 mM, respectively. Fluorescence emission spectra (460−700 nm) were obtained with excitation wavelength of 440 nm. For the analysis of the concentration dependence, the fluorescence intensities at 484 nm were used. The standard deviations and p values were calculated from triplicate experiments. ANS Competitive Binding Assay. The binding of ANS (8anilino-1-naphthalenesulfonic acid) and its displacement by the selected compounds were analyzed in 2 μM V30M, 50 mM TrisHCl, pH 7.4, 150 mM NaCl at 298 K. The concentrated solutions of


S Supporting Information *

Chemical structures of CAPE and the TTR stabilizers, semilog plots of the thioflavin T assays of the selected compounds, fluorescence emission spectra (competitive binding assay between thioflavin T and compounds), detailed fluorescence intensities for the competitive binding assay with thioflavin T, ANS displacement experiments using the selected compounds, electron density maps of inhibitors, the semilog plot of the thioflavin T assay of propolis, chemical cross-linking experiF | J. Med. Chem. XXXX, XXX, XXX−XXX

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ments using the selected compounds, a complete list of inhibitory activities, the inhibition ratios for 20 μM compounds, and the crystal data and refinement data statistics. This material is available free of charge via the Internet at


Corresponding Author

*Phone: +81 (0)76-434-7595. Fax: +81 (0)76-434-7872. Email: [email protected]. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We gratefully acknowledge access to the synchrotron radiation facility at PF, Japan. This work was supported by the Takeda Science Foundation, Japan, and by a grant for the Amyloidosis Research Committee from the Ministry of Health, Labour, and Welfare, Japan.

ABBREVIATIONS USED TTR, transthyretin; WT, wild type transthyretin; V30M, V30M mutated transthyretin; FAP, familial amyloid polyneuropathy; FAC, familial amyloid cardiomyopathy; SSA, senile systemic amyloidosis; CAPE, caffeic acid phenethyl ester; CA, caffeic acid; NDGA, nordihydroguaiaretic acid; PEG, polyethylene glycol; ANS, 8-anilino-1-naphthalenesulfonic acid


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H | J. Med. Chem. XXXX, XXX, XXX−XXX

Inhibitory activities of propolis and its promising component, caffeic acid phenethyl ester, against amyloidogenesis of human transthyretin.

Transthyretin (TTR) is a homotetrameric serum protein associated with amyloidoses such as familial amyloid polyneuropathy and senile systemic amyloido...
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