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In vitro investigation of the effect of mesalazine on amyloid fibril formation of hen egg-white lysozyme and defibrillation lysozyme fibrils
Journal Pre-proof In vitro investigation of the effect of mesalazine on amyloid fibril formation of hen eggwhite lysozyme and defibrillation lysozyme fibrils Masoumeh Faramarzian, Seifollah Bahramikia, Marzieh Dehghan Shasaltaneh PII:
S0014-2999(20)30103-5
DOI:
https://doi.org/10.1016/j.ejphar.2020.173011
Reference:
EJP 173011
To appear in:
European Journal of Pharmacology
Received Date: 17 October 2019 Revised Date:
2 February 2020
Accepted Date: 11 February 2020
Please cite this article as: Faramarzian, M., Bahramikia, S., Shasaltaneh, M.D., In vitro investigation of the effect of mesalazine on amyloid fibril formation of hen egg-white lysozyme and defibrillation lysozyme fibrils, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/ j.ejphar.2020.173011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
In vitro investigation of the effect of mesalazine on amyloid fibril formation of hen eggwhite lysozyme and defibrillation lysozyme fibrils Masoumeh Faramarzian a, Seifollah Bahramikia b, *, Marzieh Dehghan Shasaltaneh c a
Department of Biology, MSc of biology, University of Lorestan, Khorramabad, Iran
b
Department of Biology, Faculty of Science, University of Lorestan, Khorramabad, Iran
c
Department of Biology, Faculty of Science, University of Zanjan, Zanjan, Iran
* Corresponding author: Seifollah Bahramikia
Tel: +989166614082
E-mail address: [email protected]
Fax: +986633120100
Abstract In certain conditions (such as fever and stress) mutated lysozyme enzyme deposits in different tissues and organs in the form of amyloid fibrils (plaques). These aggregates lead to tissue destruction and the pathogenesis of a disease. In this study, we investigated the in vitro effects of mesalazine drug on both preventions of lysozyme aggregation and the removal of lysozyme fibrils. With this regard, hen egg-white lysozyme (HEWL) was incubated in the absence and presence of mesalazine in high temperature and acidic pH conditions. The influence of mesalazine was surveyed by Congo red (CR) absorbance, Circular dichroism spectroscopy (CD), Thioflavin T (ThT) fluorescence assay, 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence test, and Field-emission scanning electron microscopy (FE-SEM). Our results demonstrate that mesalazine, in all concentrations, especially in 1:1 and higher drug to protein ratios, has a strong inhibitory effect on protein fibrillation. Additionally, mesalazine does not show an acceptable impact on the reversibility of HEWL fibril in any of the related concentrations. Based on the obtained results, we conclude that mesalazine can be used as a drug for the prevention of amyloid-fibrils formation in hereditary lysozyme amyloidosis and other systemic non-neuropathic hereditary amyloidosis. Keywords: Amyloid fibrils, Hereditary lysozyme amyloidosis, Lysozyme, Mesalazine
1. Introduction A range of fatal and debilitating human diseases, including Alzheimer's disease, familial amyloidosis, and Parkinson's disease associated with high amounts of extracellular insoluble protein deposits in several organs such as brain, liver, and heart. These deposits are called plaques or amyloid fibrils. (Dobson, 1999; Chiti and Dobson, 2006, 2017). Inheritance, mutation, aging, stress, fever, increased protein concentration are all risk factors that influence amyloid fibrils formation in living organisms (Khurana et al., 2003). Systemic non-neuropathic hereditary amyloidosis is a group of autosomal dominant diseases in which high amounts, at times kilograms, of amyloid plaques accumulate in several tissues and viscera, causing disturbance and tissue disruption (Pepys et al., 1993). To date, 16 different amyloidogenic peptides or protein have been recognized that cause systemic nonneuropathic hereditary amyloidosis (Iadanza et al., 2018). In some families, the mutations in the lysozyme enzyme-encoding gene cause hereditary lysozyme amyloidosis (Pepys et al., 1993). On the pathway of amyloid fibrils formation, the first occurrence is the assembly of hexamer and pentamer units (oligomerization). These units then associate further and eventually form fibrils (Bitan et al., 2002). Disruption of the tertiary structure of a peptide and increased solvent accessibility of hydrophobic core leads to a collapse of the native structure of the peptide. When the native structure of the peptide collapses, it triggers intermolecular assembly and can form amyloid fibrils. Each molecule that cans withdrawal of hydrophobic patches can inhibit the formation of amyloid fibrils (Al-Shabib et al., 2019, Khan et al., 2019). Small molecular inhibitors are potential agents in the treatment of amyloid-related diseases. These compounds based on the mechanism of action divided into three classes: Class I that inhibit oligomerization but do not affect the process of fibrillation, Class II that block the oligomerization and fibrillation, and Class III that inhibit the fibrillation, but do not
influence oligomerization (Necula et al., 2007). The presence of aromatic rings and hydroxyl groups in the structure of small molecular compounds leads to an increase in the stability of the "inhibitor-protein" complex. It thus increases the anti-aggregation effects of these compounds (Porat et al., 2006). One of the small molecules that possess a ring and OH group is mesalazine. Mesalazine (5ASA, (5-amino-2-hydroxybenzoic acid) belongs to the salicylate family that is used to treat inflammation in Crohn's disease, Proctosigmoiditis, Ulcerative Colitis, and Ulcerative Proctitis (National Center for Biotechnology Information, 2016). Human and chicken lysozymes are two homologous proteins. Human lysozyme differs by 51 amino acids compared to HEWL. These two proteins have a very similar structure and conformation. The structural and sequence similarity of these two proteins is about 60% (Redfield and Dobson, 1990). In the present study, we investigated the effects of mesalazine on the formation and elimination of amyloid fibrils from lysozyme proteins, in vitro. HEWL was used as a protein model because of the similarity between HEWL and Human lysozyme.
2. Materials and Methods 2.1.Materials Hen’s egg-white lysozyme (EC: 3.2.1.17, C.N: L6876), anilinonaphthalene-8-sulfonic acid (ANS), Thioflavin T (ThT) and Congo red (CR) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All salts and organic solvents were obtained from Merck (Darmstadt, Germany). Mesalazine from Arya Pharmaceutical, Tehran, Iran. 2.2.Protein preparation to investigate the effects of mesalazine on amyloid fibril formation First HEWL was dissolved in 50 mM glycine–HCl buffer (pH 2.2, containing 0.02% (w/v) NaN3), in the presence and absence of different concentration of mesalazine (0.2, 1, 5 mM), to produce a 1 mM HEWL solution. The solutions were incubated in a water bath, without
agitation, at 60 ˚C for 15 days to investigate the effects of mesalazine on amyloid fibril formation (Arnaudov and De Vries, 2005). The ratios of mesalazine to protein were 0.2, 1, and 5 to 1 (mM). 2.3.Protein preparation for investigation of the effects of mesalazine on amyloid fibril elimination The amyloid fibrils were obtained after 15 days of HEWL incubation at 60 ˚C. Then, the pre-formed fibrils were incubated in the presence and absence of different concentration of mesalazine (0.2, 1, 5 mM) at 37˚C for 72 h to investigate the effects of mesalazine on amyloid fibril elimination (Arnaudov and De Vries, 2005). The ratios of mesalazine to protein were 0.2, 1, and 5 to 1 (mM). 2.4.Congo red (CR) absorbance test A 1 mM stock solution of CR was prepared in sodium phosphate buffer (10 mM, 0.15 M NaCl, 0.02% NaN3, pH 7.4). After passing the desired time for the incubation of protein samples (15 days for fibrillation tests and 3 days for defibrillation tests), 2.5 µl of each was added to 200 µl of diluted CR solution (20 µM). These solutions were incubated in a dark room for 30 min at room temperature. Then the solution was mixed by pipetting, and the CR spectrum was recorded (400-700 nm), using Epoch microplate reader (BioTek, USA), (Nilsson, 2004). 2.5.Thioflavin T (ThT) fluorescence test A 1 mM stock solution of ThT was prepared in sodium phosphate buffer (10 mM, 0.15 M NaCl, 0.02% NaN3, pH 7). After passing the desired time for the incubation of protein samples (15 days for fibrillation tests and 3 days for defibrillation tests),15 µl of each sample was added to 2985 µl of diluted ThT solution (15 µM). Then, the intensity of ThT fluorescence was recorded using the Cary-Eclipse fluorescence spectrophotometer (Agilent,
USA). The excitation and emission were set at 440 nm, and 460-600 nm, respectively. The excitation and emission slit widths were set at 5 nm and 10 nm, respectively (Nilsson, 2004). 2.6.1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence test A 1 mM stock solution of ANS was prepared in sodium phosphate buffer (10 mM, 0.15 M NaCl, 0.02% NaN3, pH 7). After passing the desired time for the incubation of protein samples (15 days for fibrillation tests and 3 days for defibrillation tests), 15 µl of each sample was added to 300 µl of diluted ANS solution (20 µM) and 2685 µl of Gly-HCL buffer (50 mM). Then, the intensity of ANS fluorescence was recorded using Cary-Eclipse fluorescence spectrophotometer (Agilent, USA). The excitation and emission were set at 380 nm, and 420– 600 nm, respectively. The excitation and emission slit widths were both set at 5 nm (Bahramikia, 2012).
2.7.Circular dichroism spectroscopy (CD) After passing the desired time for the incubation of protein samples (15 days for fibrillation tests and 3 days for defibrillation tests), each sample (HEWL alone, and HEWL in the presence of 1:1 ratio of drug to protein) was diluted to a final concentration of 0.2 mg/ml with the Gly-HCL buffer (50 mM). Then the CD spectrum was recorded in the far-UV region (190–260 nm) using an AVIV 215 spectropolarimeter (USA) with a 0.1 cm pathlength cuvette. The buffer scans were subtracted from the samples' spectra (Greenfield, 2007). 2.8.Field-emission scanning electron microscopy (FE-SEM) After passing the desired time for the incubation of protein samples (15 days for fibrillation tests and 3 days for defibrillation tests), each sample (HEWL alone, and HEWL in the presence of 0.2:1 ratio of drug to protein) was diluted 70-fold with distilled water. 5µl of each sample was placed on an aluminum foil, then air-dried and coated with a layer of gold. At that
point, electron microscopy images of each sample were taken using FE-SEM (TESCAN, Czech) at 15 Kev voltage and 90 kx magnification. 2.9.Docking studies and input files Input coordinates for lysozyme and the secondary structure of mesalazine were obtained from Protein Data Bank ((http://rcsb.org/) (PDB code: 3IJU) and PubChem webserver (ID: 4075), respectively. Protein docking was performed using Autodock Tool v1.5.6. Ligplus (Wallace et al., 1995). Epik server was applied to dramatize acidic conditions for ligand (Shelley et al., 2007; Greenwood et al., 2010; Schrödinger, 2018). Also, VMD v1.9.3 program (Humphrey W, 1996) was used to prepare 2D and 3D schematic diagrams of docking model to exhibit different interaction types between lysozyme and ligand.
3. Results 3.1.Investigation of the effects of mesalazine on protein fibrillation In order to evaluate the effects of mesalazine on amyloid fibril formation CR, ThT, ANS, CD, and FE-SEM assays were performed in the presence and absence of different mesalazine to protein ratios (0.2:1, 1:1 and 5:1 (mM)) following incubation of solutions for 15 days at 60˚C. 3.1.1.
CR absorbance and ThT fluorescence assays
The nearly flat β-sheet structures of amyloid fibrils differ from the twisted β-sheet structures of natural proteins. The CR and ThT molecules have specific bonding sites on almost flat β-sheet structures. By transforming the natural structure of the protein into amyloid fibrils, CR and ThT bind to the nearly flat β-sheet structures that increase the CRabsorption and ThT-fluorescence intensity. The increase of CR-absorption occurs with a redshift of the maximum absorbance from 490 to 500 nm (Biancalana and Koide, 2010; Groenning, 2010). The effects of mesalazine in the process of HEWL fibrillation are shown in Fig. 1.
As shown in Fig. 1; A, the CR absorption in the presence of mesalazine does not increase, compared with HEWL alone. CR absorption indicates the inhibitory effects of this drug on the process of lysozyme fibrillation that leads to the formation of CR-binding sites (rich-βsheet amyloid fibrils structures). The maximum absorption of 490 is observed in 1 mM and higher concentrations of mesalazine. The absence of a change in the peak area (from 490 to 500 nm) at these concentrations suggests that the 1- and 5-mM concentration of this drug has a stronger inhibitory effect than the 0.2 mM. According to this test, the best concentration of mesalazine for prevention of amyloid accumulation is at 1:1 ratio of protein to drug. As shown in Fig. 1; B, there is a small increase in ThT fluorescence intensity in the presence of different concentrations of mesalazine, compared to HEWL alone. ThT fluorescence indicates the inhibitory effects of this drug on the lysozyme fibrillation process. Through prevention of protein fibrillation, the THT binding is disrupted, and thereby ThT fluorescence intensity does not increase. As evident in Fig. 1; B, 1- and 5-mM concentration of the drug has a stronger inhibitory effect than the 0.2 mM concentration. However, increasing the concentration from 1 to 5 mM does not have a significant impact on ThT fluorescence intensity. Therefore, we can suggest that the protein is saturated in 1:1 drug to the protein ratio and by increasing the concentration of the drug more than 1mM, no significant effect will be observed. 3.1.2.
ANS fluorescence assay and CD spectrometry
The secondary structure and surface hydrophobicity of the amyloid fibrils differ from the natural proteins. In amyloid fibrils, unlike natural proteins, the hydrophobic regions are more exposed to the surface. So, ANS-fluorescence probe can attach these areas and increase fluorescence intensity. Also, amyloid fibrils are structurally rich in β-sheets (Bolognesi et al.,
2010; Groenning, 2010). The effects of mesalazine on the secondary structure and surface hydrophobicity of HEWL during the fibrillation process are shown in Fig. 2.
As shown in Fig. 2; A, all concentrations of mesalazine compared with the control sample, significantly prevent the exposure of hydrophobic regions of HEWL to the surface. The inhibitory effect of 1mM concentration of the drug on the exposure of the hydrophobic area of the protein is as strong as 5mM concentrations of the drug and more potent than 0.2 mM concentrations of the drug. As shown in Fig. 2; B, mesalazine at 1 mM concentration, compared to the control sample, significantly prevents the ellipticity in the 216 nm region and has an inhibitory effect on the formation of β-sheet structures in proteins that consequently leads to the protein fibrillation process. From the results obtained in CR, THT, and ANS assay may conclude that the effect of the 1- and 5-mM concentration of this drug has a stronger inhibitory effect on the formation of β-sheet structures than the 0.2 mM. 3.2.Investigation of the effects of mesalazine on amyloid defibrillation In order to evaluate the effects of mesalazine on elimination of amyloid fibrils CR, ThT, ANS, CD spectroscopy, and FE-SEM tests were performed in the presence and absence of different ratios of mesalazine to pre-formed fibrils (0.2:1, 1:1 and 5:1 (mM)) following incubation of solutions for 72 h at 37˚C. 3.2.1.
CR absorbance and ThT fluorescence assays
If the structure of the amyloid fibrils changes, then the binding positions of the CR and ThT dyes are lost, and the intensity of CR absorption and ThT fluorescence decreases. Fig. 3 shows the effects of mesalazine in the process of HEWL defibrillation.
As shown in Fig. 3; A, every concentration of mesalazine leads to a slight decrease in the intensity of CR absorption in pre-formed HEWL-fibrils solution. This drug does not affect the change in the maximum absorption region (500 nm) of CR. Therefore, it can be concluded that mesalazine has a little effect on the defibrillation process and reduction of the number of fibrils. Although, the concentration of 5 mM mesalazine has more effective to the elimination of amyloid fibrils than other concentrations (0.2- and 1-mM). As shown in Fig. 3; B, the concentration of 0.2 mM mesalazine does not reduce the ThT fluorescence intensity compared to non-drug HEWL-fibrils-control sample. Reduction of the fluorescence intensity is visible at 1:1 and 5:1 drug to protein ratios, but the amount of fluorescence reduction is not significant either. 3.2.2.
ANS fluorescence assay and CD spectrometry
By changing the structure of the amyloid fibrils, the secondary structure and surface hydrophobicity of the amyloid fibrils together with the CD spectrum and intensity of ANS fluorescence alter as well. The effects of mesalazine on the secondary and surface hydrophobicity of the pre-formed HEWL fibrils, during the defibrillation process, are shown in Fig. 4.
As shown in Fig. 4; A, mesalazine does not have any effect on the alteration of the surface hydrophobicity of pre-formed HEWL fibrils in any of the tested concentrations. It can be saying that the surface of pre-formed HEWL fibrils in the presence of all concentrations of mesalazine is very hydrophobic. As shown in Fig. 4; B, after 72h, a change in CD spectra ellipticity region (from 216 to 209 nm) is observed in the pre-formed HEWL fibrils. CD spectra indicate changes in the structure of the amyloid fibrils during the fibril-reversibility period. As shown in this figure, mesalazine at 1:1 ratio of drug to pre-formed HEWL fibrils has a negligible impact on the
alteration of the amyloid fibrils secondary structure. From the results obtained in CR, THT, and ANS assay may conclude that the effect of the 5 mM concentration of this drug has a little inhibitory effect on the formation of β-sheet structures but the 0.2- and 1-mM concentrations of the drug have not any impact on it. 3.3.FE-SEM scanning One of the main characteristics of amyloid fibrils is having fibrillar morphology (Chiti and Dobson, 2017). The structural morphology of protein and the effects of mesalazine on it, during the fibrillation and defibrillation processes, are shown in Fig. 5.
With incubation of protein (in the absence of mesalazine) in acidic pH and high temperature, the globular structure of native HEWL moves toward the fibrillar form (Fig. 5; A). As shown in Fig. 5; B, HEWL in the presence of mesalazine at 0.2:1 ratio of drug to protein moves a little towards the amyloid fibrils formation. The presence of small amounts of amyloid fibrils in this concentration of the drug confirms the data from other tests. So, can conclude that a higher ratio of drug to protein (1- and 5- mM) can prevent the formation of amyloid fibrils completely. Therefore, mesalazine has a strong inhibitory effect on the formation of HEWL fibrils. The influence of mesalazine on the elimination of the pre-formed HEWL fibrils, during the defibrillation process, is shown in Fig. 5; C. We do not observe any decrease in the number of fibrillar structures in the 0.2 mM ratio of drug to protein. Hence, mesalazine does not affect the decreasing of the number of fibrils in 0.2 mM concentration. Also, based on the results of the other test, probably the other ratio of mesalazine to HEWL (1:1 and 5:1) does not have a significant effect on defibrillation too. The data from CR, ThT, ANS, CD spectroscopy and FE-SEM assays are consistent with each other and prove that all concentrations of mesalazine, especially in 1:1 and higher drug to protein ratios, have a strong inhibitory effect on protein fibrillation. Also, mesalazine does
not have an acceptable effect on the reversibility of pre-formed HEWL fibrils, in any of the concentrations. 3.4.Docking studies To survey the interaction of lysozyme with mesalazine, and determine the possible binding sites, AutoDock software was used. The results are shown in Fig. 6.
According to our results, the ligand binds into the hydrophobic core of the protein. In this complex, Tryptophan (Trp) 63, Aspartic acid (Asp) 52 and Glutamine (Gln) 57 that are involved in the hydrogen bonds surround the ligand (Fig. 6C & Table 1). Also, Asp 52, Gln 57, Isoleucine (Ile) 58, Asparagine (Asn) 59, Trp 63, Ile 98, Trp 108, Alanine (Ala) 107 show hydrophobic interaction with the ligand (Fig. 6; C). As shown in Fig. 6; D and 6; E, the complex is located inside the long tunnel of the active site interacting with the mentioned residues. The aromatic structure of the ligand is in an appropriate orientation, to bind to the surrounding indole rings of Trp 63, via π-π stacking interaction. In addition to hydrophobic bonds, several amino acids participate in the active site of proteins to form a strong interaction between ligand and protein. Our results show that the complex of the compound is bonded to protein and has higher interaction affinity. It suggests that the ligand can penetrate the protein with a suitable free binding energy.
4. Discussion Systemic non-neuropathic hereditary amyloidosis is a group of diseases that are caused by mutations in different proteins such as serum amyloid A protein, β2-microglobulin, Lysozyme, etc. In these diseases, proteins instead of folding to create natural soluble forms misfold to generate insoluble β-sheet-rich structures, called amyloid fibrils that accumulate in viscera. These accumulations, depending on the disease and tissues that are involved, cause disturbance, destruction of tissues, and pathogenesis of disorders (Chiti and Dobson, 2017).
We investigated the formation of amyloid fibrils and the elimination of the pre-formed fibrils in the presence of mesalazine. In high temperature and acidic pH, the hydrogen network in the active site of HEWL is destroyed, and this region of the protein is opened. So, the hydrophobic residues are exposed, and amyloid formation starts (Frare et al., 2006). Docking studies suggest that mesalazine binds to the active site of the protein and form hydrogen bonds plus hydrophobic interaction with HEWL. These bonds protect the active site of protein from surface exposure of its hydrophobic residues and therefore prevent amyloid formation. According to our results, mesalazine inhibits the formation of fibrils to a great extent but does not affect the pre-formed fibrils. Several studies have been done to investigate the effects of small molecular compounds, both on HEWL fibrillation and defibrillation processes. Two studies investigated the antiamyloidogenic and HEWL-fibril-destabilizing effects of manganese–salen derivatives (EUK8, EUK-108, EUK-122, EUK-134, EUK-113, EUK-115, EUK-15, and EUK189)(Bahramikia, 2012; Bahramikia and Yazdanparast, 2012). They found that manganese– salen derivatives are more effective with the inhibition of fibril formation than fibril destabilization. EUK-15 is the most effective compound. They determined that the two aromatic rings in these compounds produce the main effects and the extra groups that are attached to these rings cause side effects. The mentioned study maintains that H-bond acceptor groups on aromatic rings interact with fibril core and disturb the hydrogen bonds in the β-sheet structure. EUK-15 by having one methoxy group in para position has the best effect (Bahramikia, 2012; Bahramikia and Yazdanparast, 2012). Like manganese–salen derivatives, mesalazine has different effects on fibrillation and disaggregating processes. Similar to the manganese–salen derivatives, mesalazine is more productive with the inhibition of fibril formation than fibril destabilization (it has not any
disturbing effect on amyloid aggregates). Mesalazine has a lower inhibitory effect than EUK15, EUK-189, and EUK-113 on fibrillation process. The HEWL fibrillation process was investigated in the presence of phenol (phenyl ethyl alcohol), an aldehyde (cinnamaldehyde) and a diamine (N, N, N, N'Tetramethylethylenediamine; TEMED) (Seraj et al., 2018). All of these compounds cause a reduction of HEWL-amyloid formation. According to Seraj et al. (2018), phenolic and aldehyde compound partially inhibit the formation of amyloid fibrils, although diamine compound, as well as the inhibition of amyloid fibrils formation, leads to retaining the natural form of HEWL. Mentioned research concluded that the aromatic groups of phenyl ethyl alcohol and cinnamaldehyde prevent the fibril formation by stacking between beta-sheet structures. The strong inhibitory effect of TEMED on HEWL fibrillation was related to both its nitrogen atoms and alkyl chain. According to its hypothesis, nitrogen atoms are acceptors of H+ ions in acidic environments, therefore the water H-bonds get shorter, and HEWL can fold better. Mesalazine similar to TEMED has a strong inhibitory effect on HEWL fibrillation to the extent that lysozyme fibrillation process completely is inhibited in 1:1 and higher drug to protein ratios. However, in contrast to TEMED, it does not lead to retain the natural form of HEWL. Amyloid fibrillation of HEWL has been investigated in the presence of some phenolic compounds (phenol, resorcinol, catechol, hydroquinone, and benzoquinone) (Feng et al., 2012). The study maintains that catechol, hydroquinone, and benzoquinone have quinone intermediates that in oxidized form interact with the active sites of lysozyme via the covalent bond and form quinoprotein. Covalent bond changes the inter- and intra-interactions of protein and therefore inhibits the amyloid fibril formation. Resorcinol and phenol do not have
a quinone moiety form; consequently, they do not have an inhibitory effect on fibril formation. The number of lysozyme fibrils that are formed in the presence of these compounds was as follows: phenol> resorcinol> catechol> hydroquinone> benzoquinone. The effect of mesalazine on HEWL fibril formation is similar to phenolic compounds that have quinone intermediates (catechol, hydroquinone, and benzoquinone). Our results are consistent with other studies. The effects of mesalazine on preventing the formation of the fibrils can be based on the possible mechanisms involved. Mesalazine, similar to manganese–salen derivatives and other aromatic compounds can interact with aromatic residues of the protein by its benzene ring. It reduces the hydrophobic and πstacking interactions, which lead to the formation of fibrils. The OH group attached to its benzene ring can interact with HEWL by hydrogen bond and increase its inhibitory effects on fibrillation. According to Bi et al. (2014), polyamines directly interact with the natural unfolded form of protein and by increasing positive charges on protein, electrostatic repulsion is increased, and hence intermolecular interaction is reduced (the vital force for the accumulation of amyloid fibrils). Considering that mesalazine is an aminobenzoic acid (P-amino phenol), the nitrogen atom of this compound can be the acceptor of the H+ in the acidic environment, therefore through the shortening of water H-bonds HEWL does not unfold completely. Mesalazine, like other polyamines, can interact by its amine group with the partially unfolded form of protein, and by increasing the charge of it causes an increase in electrostatic repulsion, thereby reducing intermolecular interactions. When unfolded proteins in β-strand regions cannot interact with each other, the process of fibrils accumulation will be inhibited. So, the binding site of ThT and CR will not be created, and the intensity of CR absorbance and ThT fluorescence decreases. On the other hand, with the inhibition of fibrillation, the CD
spectrum of the protein remains in roughly unfolded structure form until it has the desire to form structures rich in β-sheet. Surface hydrophobicity of the protein will not increase any further; hence, ANS fluorescence intensity does not increase. Mesalazine can form intermediate quinine (quinone-imine) that interacts with the active site of lysozyme via covalence bond. The quinoprotein changes inter- and intramolecular interactions of HEWL and ultimately leads to the inhibition of fibrillation. Mesalazine most likely inhibits lysozyme fibrillation through a series of hydrophobic interactions, hydrogen bonds, covalent interactions, and π- stacking interactions. As a result, ThT, ANS fluorescence intensity, and CR absorbance do not increase. Besides, the secondary structure of protein maintains its partially unfolded form. As pointed out in the introduction, the Class I of small molecular inhibitors inhibit oligomerization but do not affect the process of fibrillation (Necula et al., 2007). Therefore, it can be concluded that mesalazine is a part of Class I molecular inhibitors. The effects of mesalazine on inhibition of protein fibrillation and its lack of effect on defibrillation confirm the results mentioned from other studies that the paths affecting the processes of fibrillation and defibrillation differ from each other. A research study reported about a family with the lysozyme amyloidosis variant (substitution of Trp by Arg at the amino acid position 82 (Jean et al., 2014). A review of the clinical history of the family showed that two family members with rectal hemorrhage had amyloidosis in the gastrointestinal tract but were misdiagnosed with Ulcerative Colitis, and was treated with mesalazine. Bleeding has been seen in cases with amyloid fibrils deposits in the blood vessels (Tan and Pepys, 1994; Pepys, 2001). Maybe rectal hemorrhage of these family members was caused by amyloid fibrils deposits in the rectum and mesalazine had a therapeutic effect on these fibrils that led to a reduction in bleeding by a decrease in the amount of amyloid deposits. Based on Jean et al. (2014) article, we investigated in vitro, the
effects of mesalazine on amyloid fibrils formation and elimination of the pre-formed fibrils. Established on the results originated from the present study, we find that mesalazine has a strong effect on the prevention of amyloid deposits in vitro. Further researches in vivo and in vitro are required to investigate the role of mesalazine in hereditary lysozyme amyloidosis. In this experiment, we have used the drug-to-protein ratio, and its results can be generalized in different concentrations of the drug. Two issues must be considering about this drug: one that the drug has very low bioavailability because it is rapidly & extensively metabolized by the intestinal mucosal wall and the liver (National Center for Biotechnology Information, 2016). The other point is that the drug contains carboxylate moiety in its structure that limits its cell penetration. Thus far, a myriad of oral mesalazine preparations have been formulated with various drug delivery methods to minimize systemic absorption and maximize drug availability at the inflamed colonic epithelium (Emmrich, 1999; Schäfer-Korting, 2009; Ye, 2015). Because in systemic non-neuropathic hereditary amyloidosis amyloid plaques accumulate in gastrointestinal areas extensively, various formulations and other drug carriers can be used and be developed in an attempt to optimize drug delivery to the active regions of disease. Declaration of competing interest The authors declare that there are no conflicts to declare. Acknowledgments The authors appreciate the financial support of Lorestan University for this investigation.
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Fig. 1. To understanding the effect of mesalazine on prevention of amyloid diseases, HEWL samples in the absence and presence of mesalazine at different mesalazine to protein ratios (0.2:1, 1:1, and 5:1), incubated in 50 mM glycine buffer (pH 2.2) at 60 ˚C for 15 days. After 15 days, the formation of fibrillar structures and the effects of mesalazine on this process were investigated using CR test (a), and ThT test (b). Fig. 2. HEWL samples, in the absence and presence of mesalazine at different mesalazine to protein ratios (0.2:1, 1:1, and 5:1), incubated in 50 mM glycine buffer (pH 2.2) at 60˚C for 15 days. After 15 days, the secondary structure and surface hydrophobicity of the amyloid fibrils and the effects of mesalazine on them were investigated by measuring ANS fluorescence intensity (a), and CD spectrum (b). Fig. 3. Pre-formed HEWL fibrils, in the absence and presence of mesalazine at different mesalazine to protein ratios, incubated at 37 ˚C for 72 h. After 72 h, amyloid defibrillation and the effects of mesalazine on this process were investigated using CR (a), and ThT test (b). Fig. 4. Pre-formed HEWL fibrils, in the absence and presence of mesalazine at different mesalazine to protein ratios, incubated at 37˚C for 72 h. After 72 h, the secondary structure and surface hydrophobicity of the amyloid fibrils and the effects of mesalazine on these structures were investigated by measuring ANS fluorescence intensity (a), and CD spectrum (b). Fig. 5. Evaluation of the effects of mesalazine on HEWL morphology using FE-SEM. Scale bar is 500 nm. Microscopic image of HEWL fibrils in the absence of mesalazine, during fibrillation process (after 15 days incubation in glycine buffer (pH 2.2) at 60˚C) (a). Microscopic image of HEWL in the presence of mesalazine at 0.2:1 mesalazine to protein ratio (mM), during fibrillation process (b). Microscopic image of pre-formed HEWL fibrils in the presence of mesalazine at 0.2:1 mesalazine to fibrils ratio (mM), during defibrillation process (after 72 h incubation at 37˚C) (c).
Fig. 6. Determination of the possible interaction between lysozyme and mesalazine using AutoDock software. Schematic structure of mesalazine (a). 3IJU (HEWL) ligand and protein structure are shown using VMD software (b). Binding mode of the protein-ligand complex with Ligplot. Hydrogen bonds have been shown as green lines (c). Schematic structure of the protein-ligand complex (d & e). Both protein and ligand have been shown using VMD and AutoDock tools.
Table 1 Hydrogen bonds between mesalazine and HEWL. Interaction type
Hydrogen bonds
Interaction site
Length of hydrogen bond (Å)
Trp 63
O1
3.12
Asp 52
N
3.21
Gln 57
O3
2.87
S0014-2999(20)30103-5
DOI:
https://doi.org/10.1016/j.ejphar.2020.173011
Reference:
EJP 173011
To appear in:
European Journal of Pharmacology
Received Date: 17 October 2019 Revised Date:
2 February 2020
Accepted Date: 11 February 2020
Please cite this article as: Faramarzian, M., Bahramikia, S., Shasaltaneh, M.D., In vitro investigation of the effect of mesalazine on amyloid fibril formation of hen egg-white lysozyme and defibrillation lysozyme fibrils, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/ j.ejphar.2020.173011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
In vitro investigation of the effect of mesalazine on amyloid fibril formation of hen eggwhite lysozyme and defibrillation lysozyme fibrils Masoumeh Faramarzian a, Seifollah Bahramikia b, *, Marzieh Dehghan Shasaltaneh c a
Department of Biology, MSc of biology, University of Lorestan, Khorramabad, Iran
b
Department of Biology, Faculty of Science, University of Lorestan, Khorramabad, Iran
c
Department of Biology, Faculty of Science, University of Zanjan, Zanjan, Iran
* Corresponding author: Seifollah Bahramikia
Tel: +989166614082
E-mail address: [email protected]
Fax: +986633120100
Abstract In certain conditions (such as fever and stress) mutated lysozyme enzyme deposits in different tissues and organs in the form of amyloid fibrils (plaques). These aggregates lead to tissue destruction and the pathogenesis of a disease. In this study, we investigated the in vitro effects of mesalazine drug on both preventions of lysozyme aggregation and the removal of lysozyme fibrils. With this regard, hen egg-white lysozyme (HEWL) was incubated in the absence and presence of mesalazine in high temperature and acidic pH conditions. The influence of mesalazine was surveyed by Congo red (CR) absorbance, Circular dichroism spectroscopy (CD), Thioflavin T (ThT) fluorescence assay, 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence test, and Field-emission scanning electron microscopy (FE-SEM). Our results demonstrate that mesalazine, in all concentrations, especially in 1:1 and higher drug to protein ratios, has a strong inhibitory effect on protein fibrillation. Additionally, mesalazine does not show an acceptable impact on the reversibility of HEWL fibril in any of the related concentrations. Based on the obtained results, we conclude that mesalazine can be used as a drug for the prevention of amyloid-fibrils formation in hereditary lysozyme amyloidosis and other systemic non-neuropathic hereditary amyloidosis. Keywords: Amyloid fibrils, Hereditary lysozyme amyloidosis, Lysozyme, Mesalazine
1. Introduction A range of fatal and debilitating human diseases, including Alzheimer's disease, familial amyloidosis, and Parkinson's disease associated with high amounts of extracellular insoluble protein deposits in several organs such as brain, liver, and heart. These deposits are called plaques or amyloid fibrils. (Dobson, 1999; Chiti and Dobson, 2006, 2017). Inheritance, mutation, aging, stress, fever, increased protein concentration are all risk factors that influence amyloid fibrils formation in living organisms (Khurana et al., 2003). Systemic non-neuropathic hereditary amyloidosis is a group of autosomal dominant diseases in which high amounts, at times kilograms, of amyloid plaques accumulate in several tissues and viscera, causing disturbance and tissue disruption (Pepys et al., 1993). To date, 16 different amyloidogenic peptides or protein have been recognized that cause systemic nonneuropathic hereditary amyloidosis (Iadanza et al., 2018). In some families, the mutations in the lysozyme enzyme-encoding gene cause hereditary lysozyme amyloidosis (Pepys et al., 1993). On the pathway of amyloid fibrils formation, the first occurrence is the assembly of hexamer and pentamer units (oligomerization). These units then associate further and eventually form fibrils (Bitan et al., 2002). Disruption of the tertiary structure of a peptide and increased solvent accessibility of hydrophobic core leads to a collapse of the native structure of the peptide. When the native structure of the peptide collapses, it triggers intermolecular assembly and can form amyloid fibrils. Each molecule that cans withdrawal of hydrophobic patches can inhibit the formation of amyloid fibrils (Al-Shabib et al., 2019, Khan et al., 2019). Small molecular inhibitors are potential agents in the treatment of amyloid-related diseases. These compounds based on the mechanism of action divided into three classes: Class I that inhibit oligomerization but do not affect the process of fibrillation, Class II that block the oligomerization and fibrillation, and Class III that inhibit the fibrillation, but do not
influence oligomerization (Necula et al., 2007). The presence of aromatic rings and hydroxyl groups in the structure of small molecular compounds leads to an increase in the stability of the "inhibitor-protein" complex. It thus increases the anti-aggregation effects of these compounds (Porat et al., 2006). One of the small molecules that possess a ring and OH group is mesalazine. Mesalazine (5ASA, (5-amino-2-hydroxybenzoic acid) belongs to the salicylate family that is used to treat inflammation in Crohn's disease, Proctosigmoiditis, Ulcerative Colitis, and Ulcerative Proctitis (National Center for Biotechnology Information, 2016). Human and chicken lysozymes are two homologous proteins. Human lysozyme differs by 51 amino acids compared to HEWL. These two proteins have a very similar structure and conformation. The structural and sequence similarity of these two proteins is about 60% (Redfield and Dobson, 1990). In the present study, we investigated the effects of mesalazine on the formation and elimination of amyloid fibrils from lysozyme proteins, in vitro. HEWL was used as a protein model because of the similarity between HEWL and Human lysozyme.
2. Materials and Methods 2.1.Materials Hen’s egg-white lysozyme (EC: 3.2.1.17, C.N: L6876), anilinonaphthalene-8-sulfonic acid (ANS), Thioflavin T (ThT) and Congo red (CR) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All salts and organic solvents were obtained from Merck (Darmstadt, Germany). Mesalazine from Arya Pharmaceutical, Tehran, Iran. 2.2.Protein preparation to investigate the effects of mesalazine on amyloid fibril formation First HEWL was dissolved in 50 mM glycine–HCl buffer (pH 2.2, containing 0.02% (w/v) NaN3), in the presence and absence of different concentration of mesalazine (0.2, 1, 5 mM), to produce a 1 mM HEWL solution. The solutions were incubated in a water bath, without
agitation, at 60 ˚C for 15 days to investigate the effects of mesalazine on amyloid fibril formation (Arnaudov and De Vries, 2005). The ratios of mesalazine to protein were 0.2, 1, and 5 to 1 (mM). 2.3.Protein preparation for investigation of the effects of mesalazine on amyloid fibril elimination The amyloid fibrils were obtained after 15 days of HEWL incubation at 60 ˚C. Then, the pre-formed fibrils were incubated in the presence and absence of different concentration of mesalazine (0.2, 1, 5 mM) at 37˚C for 72 h to investigate the effects of mesalazine on amyloid fibril elimination (Arnaudov and De Vries, 2005). The ratios of mesalazine to protein were 0.2, 1, and 5 to 1 (mM). 2.4.Congo red (CR) absorbance test A 1 mM stock solution of CR was prepared in sodium phosphate buffer (10 mM, 0.15 M NaCl, 0.02% NaN3, pH 7.4). After passing the desired time for the incubation of protein samples (15 days for fibrillation tests and 3 days for defibrillation tests), 2.5 µl of each was added to 200 µl of diluted CR solution (20 µM). These solutions were incubated in a dark room for 30 min at room temperature. Then the solution was mixed by pipetting, and the CR spectrum was recorded (400-700 nm), using Epoch microplate reader (BioTek, USA), (Nilsson, 2004). 2.5.Thioflavin T (ThT) fluorescence test A 1 mM stock solution of ThT was prepared in sodium phosphate buffer (10 mM, 0.15 M NaCl, 0.02% NaN3, pH 7). After passing the desired time for the incubation of protein samples (15 days for fibrillation tests and 3 days for defibrillation tests),15 µl of each sample was added to 2985 µl of diluted ThT solution (15 µM). Then, the intensity of ThT fluorescence was recorded using the Cary-Eclipse fluorescence spectrophotometer (Agilent,
USA). The excitation and emission were set at 440 nm, and 460-600 nm, respectively. The excitation and emission slit widths were set at 5 nm and 10 nm, respectively (Nilsson, 2004). 2.6.1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence test A 1 mM stock solution of ANS was prepared in sodium phosphate buffer (10 mM, 0.15 M NaCl, 0.02% NaN3, pH 7). After passing the desired time for the incubation of protein samples (15 days for fibrillation tests and 3 days for defibrillation tests), 15 µl of each sample was added to 300 µl of diluted ANS solution (20 µM) and 2685 µl of Gly-HCL buffer (50 mM). Then, the intensity of ANS fluorescence was recorded using Cary-Eclipse fluorescence spectrophotometer (Agilent, USA). The excitation and emission were set at 380 nm, and 420– 600 nm, respectively. The excitation and emission slit widths were both set at 5 nm (Bahramikia, 2012).
2.7.Circular dichroism spectroscopy (CD) After passing the desired time for the incubation of protein samples (15 days for fibrillation tests and 3 days for defibrillation tests), each sample (HEWL alone, and HEWL in the presence of 1:1 ratio of drug to protein) was diluted to a final concentration of 0.2 mg/ml with the Gly-HCL buffer (50 mM). Then the CD spectrum was recorded in the far-UV region (190–260 nm) using an AVIV 215 spectropolarimeter (USA) with a 0.1 cm pathlength cuvette. The buffer scans were subtracted from the samples' spectra (Greenfield, 2007). 2.8.Field-emission scanning electron microscopy (FE-SEM) After passing the desired time for the incubation of protein samples (15 days for fibrillation tests and 3 days for defibrillation tests), each sample (HEWL alone, and HEWL in the presence of 0.2:1 ratio of drug to protein) was diluted 70-fold with distilled water. 5µl of each sample was placed on an aluminum foil, then air-dried and coated with a layer of gold. At that
point, electron microscopy images of each sample were taken using FE-SEM (TESCAN, Czech) at 15 Kev voltage and 90 kx magnification. 2.9.Docking studies and input files Input coordinates for lysozyme and the secondary structure of mesalazine were obtained from Protein Data Bank ((http://rcsb.org/) (PDB code: 3IJU) and PubChem webserver (ID: 4075), respectively. Protein docking was performed using Autodock Tool v1.5.6. Ligplus (Wallace et al., 1995). Epik server was applied to dramatize acidic conditions for ligand (Shelley et al., 2007; Greenwood et al., 2010; Schrödinger, 2018). Also, VMD v1.9.3 program (Humphrey W, 1996) was used to prepare 2D and 3D schematic diagrams of docking model to exhibit different interaction types between lysozyme and ligand.
3. Results 3.1.Investigation of the effects of mesalazine on protein fibrillation In order to evaluate the effects of mesalazine on amyloid fibril formation CR, ThT, ANS, CD, and FE-SEM assays were performed in the presence and absence of different mesalazine to protein ratios (0.2:1, 1:1 and 5:1 (mM)) following incubation of solutions for 15 days at 60˚C. 3.1.1.
CR absorbance and ThT fluorescence assays
The nearly flat β-sheet structures of amyloid fibrils differ from the twisted β-sheet structures of natural proteins. The CR and ThT molecules have specific bonding sites on almost flat β-sheet structures. By transforming the natural structure of the protein into amyloid fibrils, CR and ThT bind to the nearly flat β-sheet structures that increase the CRabsorption and ThT-fluorescence intensity. The increase of CR-absorption occurs with a redshift of the maximum absorbance from 490 to 500 nm (Biancalana and Koide, 2010; Groenning, 2010). The effects of mesalazine in the process of HEWL fibrillation are shown in Fig. 1.
As shown in Fig. 1; A, the CR absorption in the presence of mesalazine does not increase, compared with HEWL alone. CR absorption indicates the inhibitory effects of this drug on the process of lysozyme fibrillation that leads to the formation of CR-binding sites (rich-βsheet amyloid fibrils structures). The maximum absorption of 490 is observed in 1 mM and higher concentrations of mesalazine. The absence of a change in the peak area (from 490 to 500 nm) at these concentrations suggests that the 1- and 5-mM concentration of this drug has a stronger inhibitory effect than the 0.2 mM. According to this test, the best concentration of mesalazine for prevention of amyloid accumulation is at 1:1 ratio of protein to drug. As shown in Fig. 1; B, there is a small increase in ThT fluorescence intensity in the presence of different concentrations of mesalazine, compared to HEWL alone. ThT fluorescence indicates the inhibitory effects of this drug on the lysozyme fibrillation process. Through prevention of protein fibrillation, the THT binding is disrupted, and thereby ThT fluorescence intensity does not increase. As evident in Fig. 1; B, 1- and 5-mM concentration of the drug has a stronger inhibitory effect than the 0.2 mM concentration. However, increasing the concentration from 1 to 5 mM does not have a significant impact on ThT fluorescence intensity. Therefore, we can suggest that the protein is saturated in 1:1 drug to the protein ratio and by increasing the concentration of the drug more than 1mM, no significant effect will be observed. 3.1.2.
ANS fluorescence assay and CD spectrometry
The secondary structure and surface hydrophobicity of the amyloid fibrils differ from the natural proteins. In amyloid fibrils, unlike natural proteins, the hydrophobic regions are more exposed to the surface. So, ANS-fluorescence probe can attach these areas and increase fluorescence intensity. Also, amyloid fibrils are structurally rich in β-sheets (Bolognesi et al.,
2010; Groenning, 2010). The effects of mesalazine on the secondary structure and surface hydrophobicity of HEWL during the fibrillation process are shown in Fig. 2.
As shown in Fig. 2; A, all concentrations of mesalazine compared with the control sample, significantly prevent the exposure of hydrophobic regions of HEWL to the surface. The inhibitory effect of 1mM concentration of the drug on the exposure of the hydrophobic area of the protein is as strong as 5mM concentrations of the drug and more potent than 0.2 mM concentrations of the drug. As shown in Fig. 2; B, mesalazine at 1 mM concentration, compared to the control sample, significantly prevents the ellipticity in the 216 nm region and has an inhibitory effect on the formation of β-sheet structures in proteins that consequently leads to the protein fibrillation process. From the results obtained in CR, THT, and ANS assay may conclude that the effect of the 1- and 5-mM concentration of this drug has a stronger inhibitory effect on the formation of β-sheet structures than the 0.2 mM. 3.2.Investigation of the effects of mesalazine on amyloid defibrillation In order to evaluate the effects of mesalazine on elimination of amyloid fibrils CR, ThT, ANS, CD spectroscopy, and FE-SEM tests were performed in the presence and absence of different ratios of mesalazine to pre-formed fibrils (0.2:1, 1:1 and 5:1 (mM)) following incubation of solutions for 72 h at 37˚C. 3.2.1.
CR absorbance and ThT fluorescence assays
If the structure of the amyloid fibrils changes, then the binding positions of the CR and ThT dyes are lost, and the intensity of CR absorption and ThT fluorescence decreases. Fig. 3 shows the effects of mesalazine in the process of HEWL defibrillation.
As shown in Fig. 3; A, every concentration of mesalazine leads to a slight decrease in the intensity of CR absorption in pre-formed HEWL-fibrils solution. This drug does not affect the change in the maximum absorption region (500 nm) of CR. Therefore, it can be concluded that mesalazine has a little effect on the defibrillation process and reduction of the number of fibrils. Although, the concentration of 5 mM mesalazine has more effective to the elimination of amyloid fibrils than other concentrations (0.2- and 1-mM). As shown in Fig. 3; B, the concentration of 0.2 mM mesalazine does not reduce the ThT fluorescence intensity compared to non-drug HEWL-fibrils-control sample. Reduction of the fluorescence intensity is visible at 1:1 and 5:1 drug to protein ratios, but the amount of fluorescence reduction is not significant either. 3.2.2.
ANS fluorescence assay and CD spectrometry
By changing the structure of the amyloid fibrils, the secondary structure and surface hydrophobicity of the amyloid fibrils together with the CD spectrum and intensity of ANS fluorescence alter as well. The effects of mesalazine on the secondary and surface hydrophobicity of the pre-formed HEWL fibrils, during the defibrillation process, are shown in Fig. 4.
As shown in Fig. 4; A, mesalazine does not have any effect on the alteration of the surface hydrophobicity of pre-formed HEWL fibrils in any of the tested concentrations. It can be saying that the surface of pre-formed HEWL fibrils in the presence of all concentrations of mesalazine is very hydrophobic. As shown in Fig. 4; B, after 72h, a change in CD spectra ellipticity region (from 216 to 209 nm) is observed in the pre-formed HEWL fibrils. CD spectra indicate changes in the structure of the amyloid fibrils during the fibril-reversibility period. As shown in this figure, mesalazine at 1:1 ratio of drug to pre-formed HEWL fibrils has a negligible impact on the
alteration of the amyloid fibrils secondary structure. From the results obtained in CR, THT, and ANS assay may conclude that the effect of the 5 mM concentration of this drug has a little inhibitory effect on the formation of β-sheet structures but the 0.2- and 1-mM concentrations of the drug have not any impact on it. 3.3.FE-SEM scanning One of the main characteristics of amyloid fibrils is having fibrillar morphology (Chiti and Dobson, 2017). The structural morphology of protein and the effects of mesalazine on it, during the fibrillation and defibrillation processes, are shown in Fig. 5.
With incubation of protein (in the absence of mesalazine) in acidic pH and high temperature, the globular structure of native HEWL moves toward the fibrillar form (Fig. 5; A). As shown in Fig. 5; B, HEWL in the presence of mesalazine at 0.2:1 ratio of drug to protein moves a little towards the amyloid fibrils formation. The presence of small amounts of amyloid fibrils in this concentration of the drug confirms the data from other tests. So, can conclude that a higher ratio of drug to protein (1- and 5- mM) can prevent the formation of amyloid fibrils completely. Therefore, mesalazine has a strong inhibitory effect on the formation of HEWL fibrils. The influence of mesalazine on the elimination of the pre-formed HEWL fibrils, during the defibrillation process, is shown in Fig. 5; C. We do not observe any decrease in the number of fibrillar structures in the 0.2 mM ratio of drug to protein. Hence, mesalazine does not affect the decreasing of the number of fibrils in 0.2 mM concentration. Also, based on the results of the other test, probably the other ratio of mesalazine to HEWL (1:1 and 5:1) does not have a significant effect on defibrillation too. The data from CR, ThT, ANS, CD spectroscopy and FE-SEM assays are consistent with each other and prove that all concentrations of mesalazine, especially in 1:1 and higher drug to protein ratios, have a strong inhibitory effect on protein fibrillation. Also, mesalazine does
not have an acceptable effect on the reversibility of pre-formed HEWL fibrils, in any of the concentrations. 3.4.Docking studies To survey the interaction of lysozyme with mesalazine, and determine the possible binding sites, AutoDock software was used. The results are shown in Fig. 6.
According to our results, the ligand binds into the hydrophobic core of the protein. In this complex, Tryptophan (Trp) 63, Aspartic acid (Asp) 52 and Glutamine (Gln) 57 that are involved in the hydrogen bonds surround the ligand (Fig. 6C & Table 1). Also, Asp 52, Gln 57, Isoleucine (Ile) 58, Asparagine (Asn) 59, Trp 63, Ile 98, Trp 108, Alanine (Ala) 107 show hydrophobic interaction with the ligand (Fig. 6; C). As shown in Fig. 6; D and 6; E, the complex is located inside the long tunnel of the active site interacting with the mentioned residues. The aromatic structure of the ligand is in an appropriate orientation, to bind to the surrounding indole rings of Trp 63, via π-π stacking interaction. In addition to hydrophobic bonds, several amino acids participate in the active site of proteins to form a strong interaction between ligand and protein. Our results show that the complex of the compound is bonded to protein and has higher interaction affinity. It suggests that the ligand can penetrate the protein with a suitable free binding energy.
4. Discussion Systemic non-neuropathic hereditary amyloidosis is a group of diseases that are caused by mutations in different proteins such as serum amyloid A protein, β2-microglobulin, Lysozyme, etc. In these diseases, proteins instead of folding to create natural soluble forms misfold to generate insoluble β-sheet-rich structures, called amyloid fibrils that accumulate in viscera. These accumulations, depending on the disease and tissues that are involved, cause disturbance, destruction of tissues, and pathogenesis of disorders (Chiti and Dobson, 2017).
We investigated the formation of amyloid fibrils and the elimination of the pre-formed fibrils in the presence of mesalazine. In high temperature and acidic pH, the hydrogen network in the active site of HEWL is destroyed, and this region of the protein is opened. So, the hydrophobic residues are exposed, and amyloid formation starts (Frare et al., 2006). Docking studies suggest that mesalazine binds to the active site of the protein and form hydrogen bonds plus hydrophobic interaction with HEWL. These bonds protect the active site of protein from surface exposure of its hydrophobic residues and therefore prevent amyloid formation. According to our results, mesalazine inhibits the formation of fibrils to a great extent but does not affect the pre-formed fibrils. Several studies have been done to investigate the effects of small molecular compounds, both on HEWL fibrillation and defibrillation processes. Two studies investigated the antiamyloidogenic and HEWL-fibril-destabilizing effects of manganese–salen derivatives (EUK8, EUK-108, EUK-122, EUK-134, EUK-113, EUK-115, EUK-15, and EUK189)(Bahramikia, 2012; Bahramikia and Yazdanparast, 2012). They found that manganese– salen derivatives are more effective with the inhibition of fibril formation than fibril destabilization. EUK-15 is the most effective compound. They determined that the two aromatic rings in these compounds produce the main effects and the extra groups that are attached to these rings cause side effects. The mentioned study maintains that H-bond acceptor groups on aromatic rings interact with fibril core and disturb the hydrogen bonds in the β-sheet structure. EUK-15 by having one methoxy group in para position has the best effect (Bahramikia, 2012; Bahramikia and Yazdanparast, 2012). Like manganese–salen derivatives, mesalazine has different effects on fibrillation and disaggregating processes. Similar to the manganese–salen derivatives, mesalazine is more productive with the inhibition of fibril formation than fibril destabilization (it has not any
disturbing effect on amyloid aggregates). Mesalazine has a lower inhibitory effect than EUK15, EUK-189, and EUK-113 on fibrillation process. The HEWL fibrillation process was investigated in the presence of phenol (phenyl ethyl alcohol), an aldehyde (cinnamaldehyde) and a diamine (N, N, N, N'Tetramethylethylenediamine; TEMED) (Seraj et al., 2018). All of these compounds cause a reduction of HEWL-amyloid formation. According to Seraj et al. (2018), phenolic and aldehyde compound partially inhibit the formation of amyloid fibrils, although diamine compound, as well as the inhibition of amyloid fibrils formation, leads to retaining the natural form of HEWL. Mentioned research concluded that the aromatic groups of phenyl ethyl alcohol and cinnamaldehyde prevent the fibril formation by stacking between beta-sheet structures. The strong inhibitory effect of TEMED on HEWL fibrillation was related to both its nitrogen atoms and alkyl chain. According to its hypothesis, nitrogen atoms are acceptors of H+ ions in acidic environments, therefore the water H-bonds get shorter, and HEWL can fold better. Mesalazine similar to TEMED has a strong inhibitory effect on HEWL fibrillation to the extent that lysozyme fibrillation process completely is inhibited in 1:1 and higher drug to protein ratios. However, in contrast to TEMED, it does not lead to retain the natural form of HEWL. Amyloid fibrillation of HEWL has been investigated in the presence of some phenolic compounds (phenol, resorcinol, catechol, hydroquinone, and benzoquinone) (Feng et al., 2012). The study maintains that catechol, hydroquinone, and benzoquinone have quinone intermediates that in oxidized form interact with the active sites of lysozyme via the covalent bond and form quinoprotein. Covalent bond changes the inter- and intra-interactions of protein and therefore inhibits the amyloid fibril formation. Resorcinol and phenol do not have
a quinone moiety form; consequently, they do not have an inhibitory effect on fibril formation. The number of lysozyme fibrils that are formed in the presence of these compounds was as follows: phenol> resorcinol> catechol> hydroquinone> benzoquinone. The effect of mesalazine on HEWL fibril formation is similar to phenolic compounds that have quinone intermediates (catechol, hydroquinone, and benzoquinone). Our results are consistent with other studies. The effects of mesalazine on preventing the formation of the fibrils can be based on the possible mechanisms involved. Mesalazine, similar to manganese–salen derivatives and other aromatic compounds can interact with aromatic residues of the protein by its benzene ring. It reduces the hydrophobic and πstacking interactions, which lead to the formation of fibrils. The OH group attached to its benzene ring can interact with HEWL by hydrogen bond and increase its inhibitory effects on fibrillation. According to Bi et al. (2014), polyamines directly interact with the natural unfolded form of protein and by increasing positive charges on protein, electrostatic repulsion is increased, and hence intermolecular interaction is reduced (the vital force for the accumulation of amyloid fibrils). Considering that mesalazine is an aminobenzoic acid (P-amino phenol), the nitrogen atom of this compound can be the acceptor of the H+ in the acidic environment, therefore through the shortening of water H-bonds HEWL does not unfold completely. Mesalazine, like other polyamines, can interact by its amine group with the partially unfolded form of protein, and by increasing the charge of it causes an increase in electrostatic repulsion, thereby reducing intermolecular interactions. When unfolded proteins in β-strand regions cannot interact with each other, the process of fibrils accumulation will be inhibited. So, the binding site of ThT and CR will not be created, and the intensity of CR absorbance and ThT fluorescence decreases. On the other hand, with the inhibition of fibrillation, the CD
spectrum of the protein remains in roughly unfolded structure form until it has the desire to form structures rich in β-sheet. Surface hydrophobicity of the protein will not increase any further; hence, ANS fluorescence intensity does not increase. Mesalazine can form intermediate quinine (quinone-imine) that interacts with the active site of lysozyme via covalence bond. The quinoprotein changes inter- and intramolecular interactions of HEWL and ultimately leads to the inhibition of fibrillation. Mesalazine most likely inhibits lysozyme fibrillation through a series of hydrophobic interactions, hydrogen bonds, covalent interactions, and π- stacking interactions. As a result, ThT, ANS fluorescence intensity, and CR absorbance do not increase. Besides, the secondary structure of protein maintains its partially unfolded form. As pointed out in the introduction, the Class I of small molecular inhibitors inhibit oligomerization but do not affect the process of fibrillation (Necula et al., 2007). Therefore, it can be concluded that mesalazine is a part of Class I molecular inhibitors. The effects of mesalazine on inhibition of protein fibrillation and its lack of effect on defibrillation confirm the results mentioned from other studies that the paths affecting the processes of fibrillation and defibrillation differ from each other. A research study reported about a family with the lysozyme amyloidosis variant (substitution of Trp by Arg at the amino acid position 82 (Jean et al., 2014). A review of the clinical history of the family showed that two family members with rectal hemorrhage had amyloidosis in the gastrointestinal tract but were misdiagnosed with Ulcerative Colitis, and was treated with mesalazine. Bleeding has been seen in cases with amyloid fibrils deposits in the blood vessels (Tan and Pepys, 1994; Pepys, 2001). Maybe rectal hemorrhage of these family members was caused by amyloid fibrils deposits in the rectum and mesalazine had a therapeutic effect on these fibrils that led to a reduction in bleeding by a decrease in the amount of amyloid deposits. Based on Jean et al. (2014) article, we investigated in vitro, the
effects of mesalazine on amyloid fibrils formation and elimination of the pre-formed fibrils. Established on the results originated from the present study, we find that mesalazine has a strong effect on the prevention of amyloid deposits in vitro. Further researches in vivo and in vitro are required to investigate the role of mesalazine in hereditary lysozyme amyloidosis. In this experiment, we have used the drug-to-protein ratio, and its results can be generalized in different concentrations of the drug. Two issues must be considering about this drug: one that the drug has very low bioavailability because it is rapidly & extensively metabolized by the intestinal mucosal wall and the liver (National Center for Biotechnology Information, 2016). The other point is that the drug contains carboxylate moiety in its structure that limits its cell penetration. Thus far, a myriad of oral mesalazine preparations have been formulated with various drug delivery methods to minimize systemic absorption and maximize drug availability at the inflamed colonic epithelium (Emmrich, 1999; Schäfer-Korting, 2009; Ye, 2015). Because in systemic non-neuropathic hereditary amyloidosis amyloid plaques accumulate in gastrointestinal areas extensively, various formulations and other drug carriers can be used and be developed in an attempt to optimize drug delivery to the active regions of disease. Declaration of competing interest The authors declare that there are no conflicts to declare. Acknowledgments The authors appreciate the financial support of Lorestan University for this investigation.
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Fig. 1. To understanding the effect of mesalazine on prevention of amyloid diseases, HEWL samples in the absence and presence of mesalazine at different mesalazine to protein ratios (0.2:1, 1:1, and 5:1), incubated in 50 mM glycine buffer (pH 2.2) at 60 ˚C for 15 days. After 15 days, the formation of fibrillar structures and the effects of mesalazine on this process were investigated using CR test (a), and ThT test (b). Fig. 2. HEWL samples, in the absence and presence of mesalazine at different mesalazine to protein ratios (0.2:1, 1:1, and 5:1), incubated in 50 mM glycine buffer (pH 2.2) at 60˚C for 15 days. After 15 days, the secondary structure and surface hydrophobicity of the amyloid fibrils and the effects of mesalazine on them were investigated by measuring ANS fluorescence intensity (a), and CD spectrum (b). Fig. 3. Pre-formed HEWL fibrils, in the absence and presence of mesalazine at different mesalazine to protein ratios, incubated at 37 ˚C for 72 h. After 72 h, amyloid defibrillation and the effects of mesalazine on this process were investigated using CR (a), and ThT test (b). Fig. 4. Pre-formed HEWL fibrils, in the absence and presence of mesalazine at different mesalazine to protein ratios, incubated at 37˚C for 72 h. After 72 h, the secondary structure and surface hydrophobicity of the amyloid fibrils and the effects of mesalazine on these structures were investigated by measuring ANS fluorescence intensity (a), and CD spectrum (b). Fig. 5. Evaluation of the effects of mesalazine on HEWL morphology using FE-SEM. Scale bar is 500 nm. Microscopic image of HEWL fibrils in the absence of mesalazine, during fibrillation process (after 15 days incubation in glycine buffer (pH 2.2) at 60˚C) (a). Microscopic image of HEWL in the presence of mesalazine at 0.2:1 mesalazine to protein ratio (mM), during fibrillation process (b). Microscopic image of pre-formed HEWL fibrils in the presence of mesalazine at 0.2:1 mesalazine to fibrils ratio (mM), during defibrillation process (after 72 h incubation at 37˚C) (c).
Fig. 6. Determination of the possible interaction between lysozyme and mesalazine using AutoDock software. Schematic structure of mesalazine (a). 3IJU (HEWL) ligand and protein structure are shown using VMD software (b). Binding mode of the protein-ligand complex with Ligplot. Hydrogen bonds have been shown as green lines (c). Schematic structure of the protein-ligand complex (d & e). Both protein and ligand have been shown using VMD and AutoDock tools.
Table 1 Hydrogen bonds between mesalazine and HEWL. Interaction type
Hydrogen bonds
Interaction site
Length of hydrogen bond (Å)
Trp 63
O1
3.12
Asp 52
N
3.21
Gln 57
O3
2.87