Resistance of nepetin and its analogs on the fibril formation of human islet amyloid polypeptide

https://doi.org/10.1016/j.ijbiomac.2020.10.202Get rights and content

Highlights

  • Nepetin and its analogs impeded the aggregation of human islet amyloid polypeptide.

  • These flavones bound to the peptide by hydrogen bonding and hydrophobic interactions.

  • These flavones reduced peptide-induced cytotoxicity by decreasing oligomerization.

  • These flavones performed better on hIAPP than on Aβ in many aspects.

Abstract

The self-aggregation of human islet amyloid polypeptide (hIAPP) into toxic oligomers and fibrils is closely linked to the pathogenesis of type II diabetes mellitus. Inhibitors can resist hIAPP misfolding, and the resistance can be considered an alternative therapeutic strategy for this disease. Flavones have been applied in the field of diabetes research, however, the inhibition mechanism of many compounds on the fibril formation of related pathogenic peptides remains unclear. In this work, four flavones, namely, nepetin (1), genkwanin (2), luteolin (3), and apigenin (4), were used to impede the peptide aggregation of hIAPP and compared with that on protein, which is correlated with Alzheimer's disease. Results indicated that the four flavones effectively inhibited the aggregation of the two peptides and mostly dispersed the mature fibrils to monomers. The interactions of flavones with the two peptides demonstrated a spontaneous and exothermic reaction through predominant hydrophobic and hydrogen bonding interactions. The binding affinities of 1 and 3 were stronger than those of 2 and 4 possibly because of the difference in the substituent groups of these molecules. These flavones could also decrease membrane leakage and upregulate cell viability by reducing the formation of toxic oligomers. Moreover, the performance of these flavones in terms of binding affinity, cellular viability, and decreased oligomerization was better on hIAPP than on Aβ. This work offered valuable data about these flavones as prospective therapeutic agents against relevant diseases.

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).

References (86)

  • C. Wu et al.

    Binding of Congo red to amyloid protofibrils of the alzheimer Aβ9-40 peptide probed by molecular dynamics simulations

    Biophys. J.

    (2012)
  • F. Bellia et al.

    Ubiquitin binds the amyloid β peptide and interferes with its clearance pathways

    Chem. Sci.

    (2019)
  • B.N. Ratha et al.

    Insulin-eukaryotic model membrane interaction: mechanistic insight of insulin fibrillation and membrane disruption

    Biochim. Biophys. Acta (BBA)-Biomembranes

    (2018)
  • M.F. Tomasello et al.

    Molecular and cytotoxic properties of hIAPP17–29 and rIAPP17–29 fragments: a comparative study with the respective full-length parent polypeptides

    Eur. J. Med. Chem.

    (2014)
  • A.L. Clos et al.

    Role of oligomers in the amyloidogenesis of primary cutaneous amyloidosis

    J. Am. Acad. Dermatol.

    (2011)
  • M.F.M. Sciacca et al.

    Amyloid growth and membrane damage: current themes and emerging perspectives from theory and experiments on Aβ and hIAPP

    Biochim. Biophys. Acta (BBA)-Biomembranes

    (2018)
  • C. Bai et al.

    Influences of fullerenol on hIAPP aggregation: amyloid inhibition and mechanistic aspects

    Phys. Chem. Chem. Phys.

    (2019)
  • L. Gao et al.

    Fibrillation of human islet amyloid polypeptide and its toxicity to pancreatic β-cells under lipid environment

    Biochim. Biophys. Acta (BBA)-General Subjects

    (2020)
  • M. Tornquist et al.

    Secondary nucleation in amyloid formation

    Chem. Commun.

    (2018)
  • D.C. Rodriguez Camargo et al.

    hIAPP forms toxic oligomers in plasma

    Chem. Commun.

    (2018)
  • D.H.J. Lopes et al.

    Molecular tweezers inhibit islet amyloid polypeptide assembly and toxicity by a new mechanism

    ACS Chem. Biol.

    (2015)
  • M.A. Wright et al.

    Biophysical approaches for the study of interactions between molecular chaperones and protein aggregates

    Chem. Commun.

    (2015)
  • R. Butbul et al.

    Chiral modulation of amyloid beta fibrillation and cytotoxicity by enantiomeric carbon dots

    Chem. Commun.

    (2018)
  • T. Schrader et al.

    Molecular tweezers for lysine and arginine-powerful inhibitors of pathologic protein aggregation

    Chem. Commun.

    (2016)
  • C.J. Bailey et al.

    Future glucose-lowering drugs for type 2 diabetes

    Lancet Diabetes Endo

    (2016)
  • Y.J. Hong et al.

    Effectiveness of anti-dementia drugs in extremely severe alzheimer's disease: a 12-week, multicenter, randomized, single-blind study

    J. Alzheimers Dis.

    (2018)
  • A.R. Meloni et al.

    GLP-1 receptor activated insulin secretion from pancreatic β-cells: mechanism and glucose dependence

    Diabetes Obes. Metab.

    (2013)
  • R.A. Defronzo et al.

    Pathophysiologic approach to therapy in patients with newly diagnosed type 2 diabetes

    Diabetes Care

    (2013)
  • R.A. DeFronzo et al.

    Revitalization of pioglitazone: the optimum agent to be combined with a sodium-glucose co-transporter-2 inhibitor

    Diabetes Obes. Metab.

    (2016)
  • Z. Xu et al.

    Inhibitory mechanism of epigallocatechin gallate on fibrillation and aggregation of amidated human islet amyloid polypeptide

    Chem. Phys. Chem.

    (2017)
  • M. Daval et al.

    The effect of curcumin on human islet amyloid polypeptide misfolding and toxicity

    Amyloid

    (2010)
  • Z. Dhouafli et al.

    Inhibition of protein misfolding and aggregation by natural phenolic compounds

    Cell. Mol. Life Sci.

    (2018)
  • B. Ren et al.

    Genistein: a dual inhibitor of both amyloid β and human islet amylin peptides

    ACS Chem. Neurosci.

    (2018)
  • B. Ren et al.

    Tanshinones inhibit hIAPP aggregation, disaggregate preformed hIAPP fibrils, and protect cultured cells

    J. Mater. Chem. B

    (2018)
  • Y.S. Eisele et al.

    Targeting protein aggregation for the treatment of degenerative diseases

    Nat. Rev. Drug Discov.

    (2015)
  • J. Xu et al.

    Inhibitory effects of oxidovanadium complexes on the aggregation of human islet amyloid polypeptide and its fragments

    J. Inorg. Biochem.

    (2019)
  • X. Wang et al.

    Inhibitory effects of NAMI-A-like ruthenium complexes on prion neuropeptide fibril formation

    Metallomics

    (2015)
  • S.K. Panigrahy et al.

    Reactive oxygen species: sources, consequences and targeted therapy in type 2 diabetes

    J. Drug Target.

    (2016)
  • H. Li et al.

    In silico investigation of the pharmacological mechanisms of beneficial effects of ginkgo biloba L. on alzheimer's disease

    Nutrients

    (2018)
  • V. Joshi et al.

    Polyphenolic flavonoid (myricetin) upregulated proteasomal degradation mechanisms: eliminates neurodegenerative proteins aggregation

    J. Cell. Physiol.

    (2019)
  • A. Natalicchio et al.

    Effects of extra virgin olive oil polyphenols on pancreatic beta-cell function and survival

    Diabetes

    (2018)
  • N. Courtney et al.

    Apigenin blocks lipopolysaccharide-induced lethality in vivo and proinflammatory cytokines expression by inactivating NF-kappaB through the suppression of p65 phosphorylation

    J. Immunol.

    (2007)
  • M. Zang et al.

    Polyphenols stimulate AMP-activated protein kinase, lower lipids, and inhibit accelerated atherosclerosis in diabetic LDL receptor–deficient mice

    Diabetes

    (2006)
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