Mesalazine Inhibits Amyloid Formation and Destabilizes Pre-formed Amyloid Fibrils in the Human Insulin

Amyloid formation due to protein aggregation is associated with several amyloid diseases (amyloidosis). The use of small organic ligands as inhibitors of protein aggregation is an attractive strategy for the treatment of these diseases. In the present study, we evaluated the in vitro inhibitory and destabilizing effects of Mesalazine on human insulin fibrillation. To induce fibrillation, human insulin was incubated in 50 mM glycine buffer (pH 2.0) at 50 °C. The effect of Mesalazine on insulin amyloid aggregation was studied using spectroscopic, imaging, and computational approaches. Based on the results, the Mesalazine in a concentration-dependent manner (different ratios (1:0.1, 1:0.5, 1:1, and 1:5) of the insulin to Mesalazine) prevented the formation of amyloid fibrils and destabilized pre-formed fibrils. In addition, our molecular docking study confirmed the binding of Mesalazine to insulin through hydrogen bonds and hydrophobic interactions. Our findings suggest that Mesalazine may have therapeutic potential in the prevention of insulin amyloidosis and localized amyloidosis.


Introduction
Amyloid-related disorders such as Alzheimer's, Parkinson's, type 2 diabetes, localized amyloidosis etc., the accumulation of amyloid aggregates from specific proteins [1,2]. The number of patients with protein misfolding diseases (PMDs) is increasing rapidly [3]. Several factors, such as the lack of effective drug options, increased life expectancy, and population growth, are the reasons for increasing attention to amyloid diseases worldwide [4].
Insulin, a small globular protein consisting of 51 amino acids, is one of the proteins that is used as an excellent model protein for studying protein aggregation inhibitors. Human insulin, as a blood sugar-reducing hormone, is widely used as an anti-diabetic medication [5]. On the other hand, different studies demonstrated that this protein causes localized amyloidosis after repeated subcutaneous injections [6]. This process can severely limit its therapeutic effect against type 2 diabetes [7]. In addition, according to a recent study, insulin aggregation has been identified at insulin injection sites and in diabetic patients with Parkinson's disease (PD). Such cases significantly reduce hormone activity and are often responsible for necrotic deposits in diabetic individuals [8]. Insulin fibrils also are associated with Parkinson's disease, as patients' sera show an autoimmune response to oligomers and insulin fibrils [9]. In the treatment of amyloidogenic disorders, inhibition of amyloid formation and disruption of pre-formed fibrils have been proposed as two important strategies [10]. Small molecule inhibitors are potential candidates for diseases caused by amyloid fibrillation [3,[11][12][13].
Mesalazine or 5-aminosalicylic acid (5-ASA) is one of the non-steroidal anti-inflammatory drugs (NSAIDs). This synthetic drug plays a role in the treatment of inflammatory bowel diseases and has anti-inflammatory, antioxidant, antifungal, antibacterial, anti-cancer, anti-diverticulosis, anti-amyloid, anti-ulcer and gastric protection (gastroprotective) properties [14]. Mesalazine can be very important therapeutically in the development of new molecules that prevent the accumulation of insulin and other localized amyloidosis. In previous study, we investigated the effects of Mesalazine on the formation and elimination of amyloid fibrils from lysozyme proteins, in vitro [15]. According to the potent anti-amyloidogenic properties of this compound on lysozyme protein, we decided to investigated the in vitro inhibitory and destabilizing effects of Mesalazine on human insulin protein fibrillation. Under experimental conditions (acidic pH and high temperature), insulin monomers change to relatively folded intermediates. Therefore, the mentioned factors are among the important triggers in the creation of amyloid fibrils [16,17].

Formation of Insulin Amyloid Fibrils and Anti-aggregating and Dis-aggregating Activities of Mesalazine
Using a 50 mM glycine buffer (pH 2.0) containing 0/02% NaN3, the protein solution was prepared at a concentration of 4 mg/ml. In order to amyloid fibril formation, 200 µl of native insulin (HI) solution was incubated at 50 °C without stirring. To evaluate the protein fibrillation process, insulin samples were incubated at 50 °C and pH 2.0 in the absence and the presence of different ratios of Mesalazine (1:0.01, 1:1, 0.05, 1:1, and 1:5). Then, to investigate the protein disaggregation process, the pre-formed insulin fibrils were incubated at 37 °C for 3 days without stirring in the absence and the presence of different ratios of the protein to Mesalazine (1:0.1, 1:0.5, 1:1 and 1:5).

Congo Red (CR)
In this test, CR dye stock solution (2 mM) was prepared by dissolving CR in 5 mM potassium phosphate buffer (pH 7.4) containing 150 mM NaCl and 0/02% NaN3, then passed through a 0.22 µm filter and kept in dark at temperature 4 °C. To measure the CR absorbance spectra of human insulin (incubated at 50 °C) in the absence and presence of Mesalazine, 195 µl of CR solution (final concentration 12.5 µM) was mixed with 5 µl of each sample and incubated at room temperature and kept for 30 min in dark [18,19]. CR spectra were recorded between 400 and 700 nm using Epoch microplate reader (BioTek, USA).

Thioflavin T Fluorescence (ThT)
In this assay, ThT dye stock solution (2 mM) was prepared by dissolving ThT in 10 mM sodium phosphate buffer (pH 7) containing 150 mM NaCl and 0/02% NaN3, then passed through a 0.22 µm filter and kept in dark at temperature 4 °C. To measure the fluorescence intensity, 15 µl of each sample was added to 585 µl of ThT solution (dye final concentration of 20 µM) and incubated for 2 min at room temperature [18,19]. ThT fluorescence intensities were recorded using a Cary-Eclipse fluorescence spectrophotometer (Agilent, USA) with excitation at 440 nm and emission at 488 nm. Excitation and emission slit widths were set at 5 and 10 nm, respectively. In order to obtain the kinetic curve and the lag phase time, ThT fluorescence intensity of incubated samples was measured over time at 485 nm according to the above method, then the data were fitted according to the following equation [20]: where, F is the fluorescence intensity at time t, F m ax is the fluorescence intensity at the end of experiment, and t m is the time it takes to reach 50% of maximum fluorescence. Nonlinear regression was used to calculate the value of the fibril growth time constant (τ). 1/τ, the fibril growth rate constant and t m -2τ express the early lag time.

8-anilinonaphthalene-1-sulfonic Acid (ANS)
The method of preparation and storage of concentrated stock solution of ANS is the same as ThT. To measure the ANS fluorescence intensity of the sample, 15 µl of it was added to 585 µl of ANS solution with a final concentration of 20 µM and mixed and incubated for 1 min at room temperature [18,19]. ANS fluorescence intensities were recorded using a Cary-Eclipse fluorescence spectrophotometer (Agilent, USA) with excitation at 380 nm and emission between 420 to 600 nm. Excitation and emission slit widths were set at 5 and 10 nm, respectively.

Atomic Force Microscopy (AFM)
A volume of 5 μL of each sample was diluted 50 times with deionized water and was dried on a mica plate in the air to study the effect of Mesalazine on the morphology of insulin fibrils. After drying, the samples were imaged using Full Plus AFM in the non-contact AFM (NC-AFM) imaging mode at a scan frequency of 0.5 Hz (Ara Pajoohesh, Iran).

Molecular Docking
AutoDock tool v1.5.6 was used to investigate the interaction between insulin and Mesalazine. The crystalline structure of human insulin was extracted from the Protein Database (PDB id code: 1GUJ) (http:// rcsb. org/) and the three-dimensional structure of Mesalazine (CID: 4075) was extracted from the PubChem database. Discovery studio and VMD v1.9.3 software was used to prepare the two-dimensional (2D) and three-dimensional (3D) schematic geometric shapes of the docking model and also to show different orientations between the ligand and the protein.

Statistical Analysis
Data analysis was performed in GraphPad Prism software version 9.2.0 with one-way ANOVA test. Data obtained from 3 repetitions of experiments were displayed as mean ± deviation from the standard and the value of P value < 0.05 was considered as a significant difference.

CR Binding Assay
Detection of amyloid fibrils using Congo red is associated with increased Congo red absorption and shift to wavelength above 490 nm (red shift) [11,21]. Congo red absorption in the presence of amyloid fibrils increased and shifted from 490 to 510 nm (Fig. 1). We showed that the effects of Mesalazine on fibrillation (Fig. 1a) and disaggregation (Fig. 1b) of insulin. Mesalazine in different concentrations reduced the absorption of Congo red and shift the maximum absorption peak to a shorter wavelength (blue shift). The largest decrease is related to the ratio of 1:5 (insulin: Mesalazine). These changes confirm the ability of Mesalazine to inhibit and destabilize amyloid fibrillation [22,23].

ThT Fluorescence Analyses
One of the methods to identify amyloid fibrils and study the fibrillation kinetics is to bind to the fluorescent dye ThT, which increases the fluorescence emission [5]. Identifying the key stages of insulin fibril formation can show the important information to prevent fibril formation [24]. Changes in the ThT fluorescence intensity of insulin solutions in the presence and absence of different concentrations of Mesalazine for different time intervals are shown in Fig. 2a. Co-incubation of different concentrations of Mesalazine with insulin show a decrease in ThT intensity (Fig. 2b), which is more evident in the ratio of 1:5 (insulin: Mesalazine). Addition of Mesalazine in different ratios to pre-formed fibrils decreased the fluorescence intensity, as shown in Fig. 2c.

ANS Fluorescence Analyses
ANS is a hydrophobic fluorescence probe that increases its intensity by binding to hydrophobic surfaces of proteins [25,26]. It is used to study the surface hydrophobic changes in amyloid fibrils. In amyloid fibrils, hydrophobic areas are more exposed to the surface. As shown in Fig. 3, the fluorescence intensity in the fibrils samples increases significantly, and the emission maximum shifts to shorter wavelengths. These changes indicate the formation of amyloid fibrils due to the exposure of hydrophobic surfaces and the interaction of ANS with these regions. In the presence of different concentrations of Mesalazine, (Fig. 3a, b) the fluorescence intensity, especially at a ratio of 1:5 (insulin: Mesalazine) decreased. Figure 4a and d shows the elongated appearance and unbranching of Insulin amyloid fibrils [9] formed after incubation at 50 °C and pH 2.0. AFM images of insulin samples incubated in the presence of Mesalazine did not show any visible fibrillar structures ( Fig. 4b and e), in comparison to fibril samples. In addition, the preformed insulin fibrils were significantly reduced in the presence of Mesalazine (Fig. 4c and f).

In Silico Study
Molecular docking is used as a key virtual search tool in drug discovery [27]. Based on the results, Mesalazine binds to the hydrophobic nucleus of the protein as a ligand (Fig. 5a, b). At this binding, the ligand is surrounded by Tyr19 and Thr27 form regular hydrogen bonds with these amino acids (3.16 and 3.20 Å, respectively). On the other hand, the amino acids Ile2, Tyr19, Phe25, Tyr26, Thr27 also form hydrophobic bonds with the ligand (Fig. 5c).

Discussion
One of the main problems that occur due to long term repeated subcutaneous injections of insulin in diabetic patients is localized amyloidosis at the injection sites. This event causes many problems related to the process of insulin treatment in these patients. It is well accepted that hydrophobic interactions such as π-π stacking between aromatic rings as well as hydrogen bonds between polypeptide chains play an important role in intermolecular interactions in fibrils and aggregation-prone structures [28][29][30][31][32]. using of small molecules that interfere with the formation of insulin protein aggregates or destabilize these pre-formed fibrils can be as an important strategy to prevent amyloidosis in these sites. In one study, it is proved that ibuprofen was able to inhibit human insulin amyloid formation in the nucleation phase. Following the binding of ibuprofen to the native protein, its secondary and tertiary structures are almost completely protected. In addition, it is also able to maintain the native structure of insulin by reducing the hydrophobicity of the protein surface [33]. In another study, researchers shown that minocycline can bind hydrophobic regions in the sheet rich structures and destabilize the insulin amyloids composed of human insulin in diabetic patients treated with insulin [34]. Our results were in line with these studies. In the presence of Mesalazine the ThT fluorescence intensity decreased and the lag phase was delayed, indicating its anti-aggregation potential. Moreover, other studies have shown prolongation of the lag phase of insulin aggregation [35,36]. Our previous study demonstrate that Mesalazine, in all concentrations, especially in 1:1 ratio and higher (drug to protein), had a greater inhibitory effect on lysozyme protein fibrillation It has been shown a direct role of aromatic ring and OH groups around the ring in the inhibitory activity of Mesalazine [15]. After confirming the inhibitory effect of Mesalazine on insulin fibril formation, the effects of the compound on preformed amyloid fibrils was investigated. Our results demonstrated that Mesalazine is able to destabilized the preformed fibrils, dose-dependently. Since aromatic residues are present in the structure of insulin, it's thought that Mesalazine, as a small molecular compound with an aromatic ring, was able to interact with aromatic residues and impede interaction between amino acid side chains, resulting in fibrillar aggregates destabilize. These results were in contrast to our previous study, which showed that Mesalazine had no effect on the destabilization of preformed fibrils [15]. This contradiction can be related to the difference in the structure of insulin and lysozyme and the type of fibrils formed by these two proteins. Docking studies also confirmed the experimental results. As above mentioned, hydrophobic interaction and hydrogen bonding have been identified as the key driving factors in promethazine anti-amyloid activity [37]. Based on biophysical studies, B chain or part of it may be the main determinant of insulin fibrillation [38]. The results of a study showed that aggregation begins with dimerization and therefore surface and non-polar residues (RGFFYTPKT, B22-30) are critical for the nucleation stage [39]. In molecular docking, we showed that Mesalazine has the potential to prevent the exposure to hydrophobic core surface of the protein, due to its binding to the structure of insulin through hydrogen bonding and hydrophobic interactions. It is assumed, this ability of Mesalazine is thought to stabilize insulin and inhibit the process of protein fibrillogenesis.

Conclusions
The results of CR, THT, ANS analyses, and AFM images were consistent with each other, indicating the high ability of Mesalazine to inhibit and destabilize the process of protein fibrillation, in all concentrations tested and especially in the ratio of 1:5 insulin to Mesalazine. Therefore, Mesalazine can largely prevent the formation of amyloid fibrils and has a great effect on the removal of preformed plaques. Our research is also consistent with the results of studies conducted by other researchers. Therefore, Mesalazine may be able to contribute to the development of potential therapeutic strategies and the design of drug molecules against the formation of amyloid fibrils as a drug candidate.