Resistance of nepetin and its analogs on the fibril formation of human islet amyloid polypeptide
Graphical abstract
Introduction
Amyloid proteins and peptides can form toxic oligomers and fibrils by misfolding and self-assembly, which are closely correlated with those diseases caused by protein conformational changes, such as amyloid-β protein (Aβ) in Alzheimer's disease (AD), α-synuclein in Parkinson's disease, and human islet amyloid polypeptide (hIAPP) in type II diabetes mellitus (T2DM) [[1], [2], [3]]. T2DM is associated with pathologic insulin resistance and the nucleation, aggregation, and dysfunction of hIAPP [4,5]. hIAPP, a 37-residue hormone, is co-secreted with insulin and implicated in regulating blood sugar levels. However, it may cause a β-cell disorder in pancreatic islets under a disordered physiological condition [6]. The oligomerization and fibril formation of amyloid peptides, including hIAPP and Aβ, can damage cells to cause membrane leakage, oxidative stress disorder, and even cell death [[7], [8], [9], [10]].
Currently, the therapeutic strategy against T2DM and AD mainly focuses on the available approaches of traditional medicines [11,12]. However, the undesirable side effects of these traditional drugs impair insulin secretion, result in an inflexible blood–brain barrier, entail high costs, and pose organ risks [[13], [14], [15]]. Some promising compounds have potential values by taking amyloid peptides as targets, such as epigallocatechin gallate (EGCG), curcumin, and quercetin [[16], [17], [18]]. The inhibitors of amyloid peptides include organic aromatic molecules, short peptides, metal complexes, antibiotics, and nanoparticles, which have gained an increasing attention in inhibiting peptide aggregation against T2DM and AD [[19], [20], [21], [22], [23], [24]]. Genistein and tanshinones may reverse cytotoxicity and reduce membrane leakage by preventing the accumulation of amyloid peptides [19,20]. Curcumin, nepetin, ellagic acid, and naringin have been used to target T2DM [17,25]. Genkwanin regulates the activities of insulin-degrading enzymes and Aβ-degrading enzymes, maintains the blood–brain barrier integrity, and participates in neuroinflammation [26]. Myricetin upregulates the degradation of proteasomes and eliminates the aggregation of neurodegenerative proteins, such as α-synuclein, mutant of superoxide dismutase, ubiquitin, and β-actin [27]. Apigenin is applied to regulate insulin and inhibit inflammation, thereby impeding lipid accumulation in diabetic and AD mouse models [18,[28], [29], [30], [31]]. Luteolin is used to protect SK–N–SH cells against Aβ aggregation [32]. The ingenious structural characteristics of these flavones have beneficial effects on anti-aggregatory property and provide neuroprotection against hIAPP and Aβ aggregation. They may be the most effective inhibitors to prevent the self-assembly of amyloid peptides [33,34].
Nepetin, genkwanin, luteolin, and apigenin have not been reported to inhibit hIAPP aggregation though some of them affect the fibrillization of Aβ [31,35]. To explore the effects of these flavones on amyloid fibril formation and their peptide binding mechanism, we used these molecules to interact with hIAPP and compare the results with those of Aβ. The selected flavones in this work were nepetin (1), genkwanin (2), luteolin (3), and apigenin (4) as they have similar molecular structures and limited differences in substituent groups (Fig. 1). The effects of the four flavones against peptide aggregation morphology, their binding properties, and membrane protection behavior were explored with different methods. They inhibited the misfolding of two peptides and reduced their oligomerization and fibril formation. The interactions between the flavones and the peptides were verified, and the strong binding behavior of 1 and 3 with the two peptides was observed because of a favorable group substitution.
Section snippets
Materials
Flavones were purchased from RFS Biotechnology Co., Ltd. (Chengdu, China) without further purification. In this study, 5 mM flavones was dissolved in dimethyl sulfoxide (DMSO). 3-[4,5-Dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was procured from Sigma. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, ≥99%) and 1,2-dioleoyl-sn-glycero-3-phospho-(1-rac-glycerol) (DOPG, ≥98%) were obtained from the AVT Pharmaceutical Technology Co., Ltd. (Shanghai, China).
hIAPP and Aβ were
Inhibition of nepetin and its analogs on peptide aggregation
ThT can bind to peptide aggregates and emit intense fluorescence; therefore, it is used to monitor the aggregation extent in the presence of potential inhibitors [36,47]. Fig. 2 shows decreased fluorescence intensity at 484 nm with different molar ratio of flavone to peptide. The inhibitory activity of 1 and 3 against hIAPP and Aβ aggregation was better than that of 2 and 4. The results of 3 and 4 agreed with those reported previously for Aβ [31,32]. To strengthen the comparison, we still
Discussion
ThT assay and AFM images revealed that nepetin and its analogs displayed a strong inhibitory activity toward peptide misfolding. They could resist peptide misfolding and change the morphological characteristics of peptides. In addition, the fibrosis of the peptides was impeded, and the height distribution of the aggregates was low, as observed in the kinetic AFM morphology. Furthermore, 1 and 3 manifested remarkable inhibitory effects against peptide aggregation, as indicated by AFM images. The
Conclusion
In summary, this work revealed that four flavones effectively inhibited and caused depolymerization against amyloid fibril formation to protect the cell membrane and reduce oligomerization. The kinetic aggregation of hIAPP and Aβ was initially resisted because of the binding of flavones to peptides, and the inhibitory action against hIAPP was better than that against Aβ. Furthermore, Kd of 1 and 3 was lower than that of 2 and 4 because of distinct polyphenol structures as the former bound to
CRediT authorship contribution statement
Designed the studies: W. Du and J. Xu. Undertook the experimental work: J. Xu, T. Zheng, and X. Huang. Analyzed the data: J. Xu, C. Zhao, and W. Du. Contributed to figures and manuscript preparation: J. Xu, T. Zheng, and W. Du. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Declaration of competing interest
There is no conflict of interest.
Acknowledgements
The authors thank the resources supporting provided by high performance computing platform of Renmin University of China. This research was supported by the National Natural Science Foundation of China (No. 21473251).
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