Targeting protein aggregates by small organic molecules such as polyphenols is one of the most desirable and effective strategies to prevent and improve amyloid disease, which has received much attention in recent years. So far, the anti-amyloidogenic activity of many polyphenols, including quercetin (Wang et al. 2011), myricetin (Zelus et al. 2012), silibinin (Cheng et al. 2012), curcumin (Ono et al. 2004a; Pandey et al. 2008), tannic acid (Ono et al. 2004b), resveratrol (Feng et al. 2009), etc. have been investigated. Phenolic compounds in interaction with peptides and proteins, modify their structural properties and prevent amyloid aggregates and their toxicity. In addition, the antioxidant properties of these compounds play an important role in inhibiting amyloid fibrils (Sgarbossa 2012; Porat et al. 2006; Stefani and Rigacci 2013). It is said that the number of hydroxyl groups and the type of side-chain attached to the aromatic ring in the structure of polyphenols play an important role in the amount of possible binding to proteins so that with increasing the number of hydroxyl groups, the tendency of polyphenols to interact with proteins will be higher (Ozdal et al. 2018). In addition, reports indicate that prevention of π-π interaction and inhibition of fibrillation process is due to interactions between the phenolic rings of polyphenols and the aromatic residues of amyloidogenic proteins (Ngoungoure et al. 2015). Among the non-covalent interactions that may occur between polyphenols and proteins, hydrophobic interactions and hydrogen bonds are known as the main interactions and are of great importance (Ozdal et al. 2018). The results of molecular docking simulation in this study showed that Py was able to interact with HI through hydrophobic interactions and hydrogen bonding, thereby resulting in the inhibition of HI fibrillation. According to the results, it can be concluded that the presence of an aromatic ring and its attached hydroxyl groups in the Py structure is the reason for the potential of this phenolic compound to interact with the protein.
In this study, HI protein was exposed to pH 2.0 and a temperature of 50°C. These conditions lead to the opening of the folded structure of the protein and the formation of amyloid aggregates. The acidic pH makes the protein highly vulnerable and heat is the main factor in the dissociation of the dimer and the production of monomeric protein (Whittingham et al. 2002). It breaks hydrogen bonds, salt bridges, and disulfide bonds that play an important role in maintaining the native structure of the protein (Arora et al. 2004) and thus leads to exposure of hydrophobic regions. On the other hand, heating, by increasing the surface hydrophobicity of the protein, provides more binding sites for polyphenol, in which the hydrophobic bonds contribute significantly. It is said that the most interaction between protein and polyphenol is the hydrophobic interactions (Siebert et al. 1996).
Results of our experimental studies (CR absorbance, ThT and ANS fluorescence intensity) showed that Py has significant inhibitory potential against amyloid fibrils formation and is very effective in reducing the exposure of hydrophobic regions and thus reducing the tendency of the protein to form amyloid fibrils. This effect, like the inhibitory effect of many polyphenols, including gallic acid (Jayamani and Shanmugam 2014), silibinin (Katebi et al. 2018) and curcumin (Rabiee et al. 2013) was observed in a dose-dependent manner. In addition, our findings in the study of the effect of Py destabilizing on the process of HI defibrillation indicated the elimination of fibrillar structures and reduction of surface hydrophobicity of protein is due to the disappearance of hydrophobic regions. Also, in this process, with increasing Py concentration, the rate of defibrillation increased.
The inhibitory effect of Py on other amyloidogenic proteins has also been investigated. According to Di Giovanni et al. (2010), Py is effective in preventing the aggregation of α-synuclein and inhibiting the conversion of β-amyloid protofibrils to mature fibrils.
It has been reported that main factor in the inhibitory properties of polyphenols with similar chemical structure is the phenolic hydroxyl group (Liu et al. 2013). Our results confirmed this claim. The samples containing HI + Ga(1:5) and the sample containing HI + Py(1:5) showed the same inhibitory effect, so the structural difference between the two compounds (carboxyl group) has no effect on inhibiting the HI fibrillation process. These results support the Jayamani and Shanmugam (2014) hypothesis that the carboxyl group is not effective in inhibiting fibril formation. In fact, the trihydroxy benzene group in Ga is the major component of the inhibitory effect in this compound. In a study by Konar et al. (2018), the inhibitory effect of several phenolic compounds including benzoic acid (BA), 4-hydroxy benzoic acid (4-HBA), 3,4-dihydroxy benzoic acid (3,4-DHBA), 3,5-dihydroxy benzoic acid (3,5-DHBA), and Py, which their structures are close to the structure of Ga, were investigated on HEWL fibrillation. It was reported that Ga and Py had the greatest inhibitory effect compared to other compounds. The presence of more hydroxyl groups in Ga and Py is the most significant reason for the high inhibitory effect of these two compounds, compared to other phenolic compounds. On the other hand, the similar inhibitory effect of Ga and Py, as well as the ineffectiveness of benzoic acid (which has only one carboxyl group in its structure) in the process of preventing the formation of HEWL fibrils, were other results obtained in this study which led to the conclusion that the carboxyl group had no significant effect on the inhibitory properties of polyphenols.
In another study, the effect of hydroxyl groups of three polyphenolic compounds, myricetin, morin, and flavone, on the elimination of amyloid fibrils was investigated (Gargari et al. 2018). These three compounds have a similar structure and the only difference is in the number of hydroxyl groups. The results of this study demonstrated that myricetin, with six hydroxyl groups, and morin, with five hydroxyl groups, were more effective at destabilizing amyloid fibrils than flavone, which lacked the hydroxyl group. According to Ono et al. (2006), the number of hydroxyl groups is directly related to the anti-amyloidogenic effects of polyphenolic compounds.
Investigation of microscopic images of the anti-amyloidogenic effects of small organic molecules on amyloid aggregates in many studies showed that some compounds belonging to this group of inhibitors, in addition to preventing fibril formation, can also redirect amyloidogenic proteins toward the creation of non-fibrillar aggregates. Wang et al. (2012) in the study of the effect of Epigallocatechin-3-gallate (EGCG) on human insulin fibrillation found that EGCG in physiological conditions, in addition to having high inhibitory effects, leads to a change in the path of aggregation and the formation of globular aggregates. In a study on inhibitory and destabilizing effects of quercetin against bovine insulin fibrillation (at acidic pH and high temperature) and its defibrillation it was shown that this compound leads to the formation of amorphous aggregates both during inhibition of fibrillation and in the process of elimination of amyloid fibrils (Wang et al. 2011). In other studies, the effect of silibinin (Katebi et al. 2018), curcumin (Rabiee et al. 2013) and ferulic acid (Jayamani et al. 2014) on the formation of bovine insulin fibril also indicated the presence of amorphous species instead of fibrillar morphology in samples containing these compounds. Our observations in this study are consistent with the above reports, as in the microscopic images obtained, the presence of non-fibrillar species and similar to amorphous aggregates in both the inhibitory and destabilizing effects of Py were clearly observed. The results of FTIR spectroscopy also indicate a high probability of amorphous aggregation formation during inhibition and treatment of HI amyloid fibrils by Py.
Totally, based on various studies it can be concluded that polyphenols prevent the formation of amyloid species in different ways. For example, a group of polyphenols, despite being successful in inhibiting the formation of oligomers, lead to the formation of fibrils. Another group prevents the formation of fibrils but has no effect on inhibiting the formation of oligomers. There are also polyphenols that are capable of preventing the formation of both oligomeric and fibrillar forms. Many of them cause a change of the direction of fibrillation and the creation of non-fibrillar oligomers instead of fibrillar forms (Ngoungoure et al. 2015). Regarding these facts, the structure of the compound is very important in determining how it affects the process of amyloid formation.