Head-to-tail cyclization of a heptapeptide eliminates its cytotoxicity and significantly increases its inhibition effect on amyloid β-protein fibrillation and cytotoxicity

  • Shuai Ma
  • Huan Zhang
  • Xiaoyan DongEmail author
  • Linling Yu
  • Jie Zheng
  • Yan Sun
Research Article


Amyloid-β (Aβ) protein aggregation is the main hallmark of Alzheimer’s disease (AD). Inhibition of Aβ fibrillation is thus a promising therapeutic approach to the prevention and treatment of AD. Recently, we designed a heptapeptide inhibitor, LVFFARK (LK7). LK7 shows a promising inhibitory capability on Aβ fibrillation, but is prone to self-assembling and displays high cytotoxicity, which would hinder its practical application. Herein, we modified LK7 by a head-to-tail cyclization and obtained a cyclic LK7 (cLK7). cLK7 exhibits a different self-assembly behavior from LK7, and has higher stability against proteolysis than LK7 and little cytotoxicity to SHSY5Y cells. Thermodynamic analysis revealed that both LK7 and cLK7 could bind to Aβ40 by electrostatic interactions, hydrogen bonding and hydrophobic interactions, but the binding affinity of cLK7 for Aβ40 (KD = 4.96 μmol/L) is six times higher than that of LK7 (KD = 32.2 μmol/L). The strong binding enables cLK7 to stabilize the secondary structure of Aβ40 and potently inhibit its nucleation, fibrillation and cytotoxicity at extensive concentration range, whereas LK7 could only moderately inhibit Aβ40 fibrillation and cytotoxicity at low concentrations. The findings indicate that the peptide cyclization is a promising approach to enhance the performance of peptide-based amyloid inhibitors.


Alzheimer’s disease amyloid β-protein cyclic peptide inhibition protein aggregation 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.



This work was supported by the National Natural Science Foundation of China (Grant Nos. 21376172, 21406160, 21528601 and 21621004) and the Natural Science Foundation of Tianjin from Tianjin Municipal Science and Technology Commission (Contract No. 16JCZDJC32300).

Supplementary material

11705_2017_1687_MOESM1_ESM.pdf (370 kb)
Head-to-tail cyclization of a heptapeptide eliminates its cytotoxicity and significantly increases its inhibition effect on amyloid β-protein fibrillation and cytotoxicity


  1. 1.
    Goedert M, Spillantini M G. A century of Alzheimer’s disease. Science, 2006, 314(5800): 777–781CrossRefPubMedGoogle Scholar
  2. 2.
    Hardy J, Selkoe D J. The amyloid hypothesis of Alzheimer’s disease: Progress and problems on the road to therapeutics. Science, 2002, 297(5580): 353–356CrossRefGoogle Scholar
  3. 3.
    Knowles T P, Vendruscolo M, Dobson C M. The amyloid state and its association with protein misfolding diseases. Nature Reviews. Molecular Cell Biology, 2014, 15(6): 384–396CrossRefPubMedGoogle Scholar
  4. 4.
    Selkoe D J. Soluble oligomers of the amyloid β-protein impair synaptic plasticity and behavior. Behavioural Brain Research, 2008, 192(1): 106–113CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Chimon S, Shaibat M A, Jones C R, Calero D C, Aizezi B, Ishii Y. Evidence of fibril-like β-sheet structures in a neurotoxic amyloid intermediate of Alzheimer’s β-amyloid. Nature Structural & Molecular Biology, 2008, 14(12): 1157–1164CrossRefGoogle Scholar
  6. 6.
    Härd T, Lendel C. Inhibition of amyloid formation. Journal of Molecular Biology, 2012, 421(4): 441–465CrossRefPubMedGoogle Scholar
  7. 7.
    Wang Q M, Yu X, Li L Y, Zheng J. Inhibition of amyloid-β aggregation in Alzheimer’s disease. Current Pharmaceutical Design, 2014, 20(8): 1223–1243CrossRefPubMedGoogle Scholar
  8. 8.
    Craik D J, Fairlie D P, Liras S, Price D. The future of peptide-based drugs. Chemical Biology & Drug Design, 2013, 81(1): 136–147CrossRefGoogle Scholar
  9. 9.
    Tjernberg L O, Näslund J, Lindqvist F, Johansson J, Karlström A R, Thyberg J, Terenius L, Nordstedt C. Arrest of β-amyloid fibril formation by a pentapeptide ligand. Journal of Biological Chemistry, 1996, 271(15): 8545–8548CrossRefPubMedGoogle Scholar
  10. 10.
    Liu F F, Du W J, Sun Y, Zheng J, Dong X Y. Atomistic characterization of binding modes and affinity of peptide inhibitors to amyloid-β protein. Frontiers of Chemical Science and Engineering, 2014, 8(4): 433–444CrossRefGoogle Scholar
  11. 11.
    Soto C, Kindy M S, Baumann M, Frangione B. Inhibition of Alzheimer’s amyloidosis by peptides that prevent β-sheet conformation. Biochemical and Biophysical Research Communications, 1996, 226(3): 672–680CrossRefPubMedGoogle Scholar
  12. 12.
    Bansal S, Maurya I K, Yadav N, Thota C K, Kumar V, Tikoo K, Chauhan V S, Jain R. C-terminal fragment, Aβ 32-37, analogues protect against Aβ aggregation-induced toxicity. ACS Chemical Neuroscience, 2016, 7(5): 615–623CrossRefPubMedGoogle Scholar
  13. 13.
    Fradinger E A, Monien B H, Urbanc B, Lomakin A, Tan M, Li H, Spring S M, Condron M M, Cruz L, Xie C W, Benedek G B, Bitan G. C-terminal peptides coassemble into Aβ 42 oligomers and protect neurons against Aβ 42-induced neurotoxicity. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(37): 14175–14180CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Takahashi T, Mihara H. Peptide and protein mimetics inhibiting amyloid β-peptide aggregation. Accounts of Chemical Research, 2008, 41(10): 1309–1318CrossRefPubMedGoogle Scholar
  15. 15.
    Turner J P, Lutzrechtin T, Moore K A, Rogers L, Bhave O, Moss M A, Servoss S L. Rationally designed peptoids modulate aggregation of amyloid-β 40. ACS Chemical Neuroscience, 2014, 5(7): 552–558CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Arai T, Sasaki D, Araya T, Sato T, Sohma Y, Kanai M. A cyclic KLVFF-derived peptide aggregation inhibitor induces the formation of less-toxic off-pathway amyloid-β oligomers. ChemBioChem, 2014, 15(17): 2577–2583CrossRefPubMedGoogle Scholar
  17. 17.
    Xiong N, Dong X Y, Zheng J, Liu F F, Sun Y. Design of LVFFARK and LVFFARK-functionalized nanoparticles for inhibiting amyloid β-protein fibrillation and cytotoxicity. ACS Applied Materials & Interfaces, 2015, 7(10): 5650–5662CrossRefGoogle Scholar
  18. 18.
    Arai T, Araya T, Sasaki D, Taniguchi A, Sato T, Sohma Y, Kanai M. Rational design and identification of a non-peptidic aggregation inhibitor of amyloid-β based on a pharmacophore motif obtained from cyclo [-Lys-Leu-Val-Phe-Phe-]. Angewandte Chemie International Edition, 2014, 53(31): 8236–8239CrossRefPubMedGoogle Scholar
  19. 19.
    Luo J H, Otero J M, Yu C H, Wärmländer S K, Gräslund A, Overhand M, Abrahams J P. Inhibiting and reversing amyloid-β peptide (1–40) fibril formation with gramicidin S and engineered analogues. Chemistry (Weinheim an der Bergstrasse, Germany), 2013, 19(51): 17338–17348Google Scholar
  20. 20.
    Cho P Y, Joshi G, Boersma M D, Johnson J A, Murphy R M. A cyclic peptide mimic of the β-amyloid binding domain on transthyretin. ACS Chemical Neuroscience, 2015, 6(5): 778–789CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Zheng J, Baghkhanian A M, Nowick J S. A hydrophobic surface is essential to inhibit the aggregation of a Tau-protein-derived hexapeptide. Journal of the American Chemical Society, 2013, 135(18): 6846–6852CrossRefPubMedGoogle Scholar
  22. 22.
    Richman M, Wilk S, Chemerovski M, Wärmländer S K T S, Wahlström A, Gräslund A, Rahimipour S. In vitro and mechanistic studies of an antiamyloidogenic self-assembled cyclic D,L-a-peptide architecture. Journal of the American Chemical Society, 2013, 135 (9): 3474–3484CrossRefPubMedGoogle Scholar
  23. 23.
    Choi S J, Jeong W J, Kang S K, Lee M, Kim E, Ryu D Y, Lim Y B. Differential self-assembly behaviors of cyclic and linear peptides. Biomacromolecules, 2012, 13(7): 1991–1995CrossRefPubMedGoogle Scholar
  24. 24.
    Ziehm T, Brener O, Groen T, Kadish I, Frenzel D, Tusche M, Kutzsche J, Reiss K, Gremer L, Nagel-Steger L, et al. Increase of positive net charge and conformational rigidity enhances the efficacy of D-enantiomeric peptides designed to eliminate cytotoxic Aβ species. ACS Chemical Neuroscience, 2016, 7(8): 1088–1096CrossRefPubMedGoogle Scholar
  25. 25.
    March D R, Abbenante G, Bergman D A, Brinkworth R I, Wickramasinghe W, Begun J, Martin J L, Fairlie D P. Substratebased cyclic peptidomimetics of Phe-Ile-Val that Inhibit HIV-1 protease using a novel enzyme-binding mode. Journal of the American Chemical Society, 1996, 118(14): 3375–3379CrossRefGoogle Scholar
  26. 26.
    Rezai T, Yu B, Millhauser G L, Jacobson M P, Lokey R S. Testing the conformational hypothesis of passive membrane permeability using synthetic cyclic peptide diastereomers. Journal of the American Chemical Society, 2006, 128(8): 2510–2511CrossRefPubMedGoogle Scholar
  27. 27.
    Wang Q M, Shah N, Zhao J, Wang C, Zhao C, Liu L, Li L Y, Zhou F, Zheng J. Structural, morphological, and kinetic studies of β-amyloid peptide aggregation on self-assembled monolayers. Physical Chemistry Chemical Physics, 2011, 13(33): 15200–15210CrossRefPubMedGoogle Scholar
  28. 28.
    Gordon D J, Sciarretta K L, Meredith S C. Inhibition of β-amyloid (40) fibrillogenesis and disassembly of β-amyloid (40) fibrils by short β-amyloid congeners containing N-methyl amino acids at alternate residues. Biochemistry, 2001, 40(28): 8237–8245CrossRefPubMedGoogle Scholar
  29. 29.
    Ferrie J J, Gruskos J J, Goldwaser A L, Decker M E, Guarracino D A. A comparative protease stability study of synthetic macrocyclic peptides that mimic two endocrine hormones. Bioorganic & Medicinal Chemistry Letters, 2013, 23(4): 989–995CrossRefGoogle Scholar
  30. 30.
    Yu R, Seymour V A L, Berecki G, Jia X, Akcan M, Adams D J, Kaas Q, Craik D J. Less is more: Design of a highly stable disulfidedeleted mutant of analgesic cyclic α-conotoxin Vc1.1. Scientific Reports, 2015, 5(1): 13264CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Cheng P N, Liu C, Zhao M, Eisenberg D, Nowick J S. Amyloid β-sheet mimics that antagonize protein aggregation and reduce amyloid toxicity. Nature Chemistry, 2012, 4(11): 927–933CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Cabaleiro-Lago C, Szczepankiewicz O, Linse S. The effect of nanoparticles on amyloid aggregation depends on the protein stability and intrinsic aggregation rate. Langmuir, 2012, 28(3): 1852–1857CrossRefPubMedGoogle Scholar
  33. 33.
    Luo J H, Yu C H, Yu H X, Borstnar R, Kamerlin S C, Gräslund A, Abrahams J P, Wärmländer S K. Cellular polyamines promote amyloid-beta (Aβ) peptide fibrillation and modulate the aggregation pathways. ACS Chemical Neuroscience, 2013, 4(3): 454–462CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Micsonai A, Wien F, Kernya L, Lee Y H, Goto Y, Réfrégiers M, Kardos J. Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(24): 3095–3103CrossRefGoogle Scholar
  35. 35.
    Choi H J, Huber A H, Weis W I. Thermodynamics of β-cateninligand interactions: The roles of the N-and C-terminal tails in modulating binding affinity. Journal of Biological Chemistry, 2006, 281(2): 1027–1038CrossRefPubMedGoogle Scholar
  36. 36.
    Wiseman T, Williston S, Brandts J F, Lin L N. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Analytical Biochemistry, 1989, 179(1): 131–137CrossRefPubMedGoogle Scholar
  37. 37.
    Freyer M W, Lewis E A. Isothermal titration calorimetry: Experimental design, data analysis, and probing macromolecule/ ligand binding and kinetic interactions. Methods in Cell Biology, 2008, 84: 79–113CrossRefPubMedGoogle Scholar
  38. 38.
    Fotakis G, Timbrell J A. In vitro cytotoxicity assays: Comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicology Letters, 2006, 160(2): 171–177CrossRefPubMedGoogle Scholar
  39. 39.
    Gupta M, Bagaria A, Mishra A, Mathur P, Basu A, Ramakumar S, Chauhan V S. Self-assembly of a dipeptide-containing conformationally restricted dehydrophenylalanine residue to form ordered nanotubes. Advanced Materials, 2007, 19(6): 858–861CrossRefGoogle Scholar
  40. 40.
    Huang R L, Su R X, Qi W, Zhao J, He Z M. Hierarchical, interfaceinduced self-assembly of diphenylalanine: Formation of peptide nanofibers and microvesicles. Nanotechnology, 2011, 22(24): 245609CrossRefPubMedGoogle Scholar
  41. 41.
    Biancalana M, Koide S. Molecular mechanism of thioflavin-T binding to amyloid fibrils. Biochimica et Biophysica Acta (BBA)-Proteins Proteomics, 2010, 1804(7): 1405–1412CrossRefGoogle Scholar
  42. 42.
    Cohen S I, Linse S, Luheshi L M, Hellstrand E, White D A, Rajah L, Otzen D E, Vendruscolo M, Dobson C M, Knowles T P. Proliferation of amyloid-β 42 aggregates occurs through a secondary nucleation mechanism. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(24): 9758–9763CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Arosio P, Knowles T P, Linse S. On the lag phase in amyloid fibril formation. Physical Chemistry Chemical Physics, 2015, 17(12): 7606–7618CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Zhao Z J, Zhu L, Li H Y, Cheng P, Peng J X, Yin Y D, Yang Y, Wang C, Hu Z Y, Yang Y L. Antiamyloidogenic activity of Aβ42-binding peptoid in modulating amyloid oligomerization. Small, 2017, 13(1): 1602857CrossRefGoogle Scholar
  45. 45.
    Sugiura Y, Ikeda K, Nakano M. High membrane curvature enhances binding, conformational changes, and fibrillation of amyloid-β on lipid bilayer surfaces. Langmuir, 2015, 31(42): 11549–11557CrossRefPubMedGoogle Scholar
  46. 46.
    Nagarathinam A, Höflinger P, Bühler A, Schäfer C, Mcgovern G, Jeffrey M, Staufenbiel M, Jucker M, Baumann F. Membraneanchored Aβ accelerates amyloid formation and exacerbates amyloid-associated toxicity in mice. Journal of Neuroscience, 2013, 33(49): 19284–19294CrossRefPubMedGoogle Scholar
  47. 47.
    Bartolini M, Bertucci C, Bolognesi M L, Cavalli A, Melchiorre C, Andrisano V. Insight into the kinetic of amyloid-β (1–42) peptide self-aggregation: Elucidation of inhibitors’ mechanism of action. ChemBioChem, 2007, 8(17): 2152–2161CrossRefPubMedGoogle Scholar
  48. 48.
    Ehrnhoefer D E, Bieschke J, Boeddrich A, Herbst M, Masino L, Lurz R, Engemann S, Pastore A, Wanker E E. EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers. Nature Structural & Molecular Biology, 2008, 15(6): 558–566CrossRefGoogle Scholar
  49. 49.
    Du W J, Guo J J, Gao M T, Hu S Q, Dong X Y, Han Y F, Liu F F, Jiang S, Sun Y. Brazilin inhibits amyloid β-protein fibrillogenesis, remodels amyloid fibrils and reduces amyloid cytotoxicity. Scientific Reports, 2015, 5(1): 7992CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Kumar S, Udgaonkar J B. Mechanisms of amyloid fibril formation by proteins. Current Science, 2010, 98(5): 639–656Google Scholar
  51. 51.
    Tu Y L, Ma S, Liu F F, Sun Y, Dong X Y. Hematoxylin inhibits amyloid β-protein fibrillation and alleviates amyloid-induced cytotoxicity. Journal of Physical Chemistry B, 2016, 120(44): 11360–11368CrossRefGoogle Scholar
  52. 52.
    Qiang W, Yau W M, Luo Y, Mattson M P, Tycko R. Antiparallel β-sheet architecture in iowa-mutant β-amyloid fibrils. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(12): 4443–4448CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Acerra N, Kad N M, Griffith D A, Ott S, Crowther D C, Mason J M. Retro-inversal of intracellular selected β-amyloid-interacting peptides: Implications for a novel Alzheimer’s disease treatment. Biochemistry, 2014, 53(13): 2101–2111CrossRefPubMedGoogle Scholar
  54. 54.
    Liu J, Wang W, Zhang Q, Zhang S H, Yuan Z. Study on the efficiency and interaction mechanism of a decapeptide inhibitor of β-amyloid aggregation. Biomacromolecules, 2014, 15(3): 931–939CrossRefPubMedGoogle Scholar
  55. 55.
    Ross P D, Subramanian S. Thermodynamics of protein association reactions: Forces contributing to stability. Biochemistry, 1981, 20 (11): 3096–3102CrossRefPubMedGoogle Scholar
  56. 56.
    Cairo C W, Strzelec A, Murphy R M, Kiessling L L. Affinity-based inhibition of β-amyloid toxicity. Biochemistry, 2002, 41(27): 8620–8629CrossRefPubMedGoogle Scholar
  57. 57.
    Liao Y H, Chang Y J, Yoshiike Y, Chang Y C, Chen Y R. Negatively charged gold nanoparticles inhibit Alzheimer’s amyloid-β fibrillization, induce fibril dissociation, and mitigate neurotoxicity. Small, 2012, 8(23): 3631–3639CrossRefPubMedGoogle Scholar

Copyright information

© Higher Education Press and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Shuai Ma
    • 1
  • Huan Zhang
    • 1
  • Xiaoyan Dong
    • 1
    Email author
  • Linling Yu
    • 1
  • Jie Zheng
    • 2
  • Yan Sun
    • 1
  1. 1.Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and TechnologyTianjin UniversityTianjinChina
  2. 2.Department of Chemical and Biomolecular EngineeringThe University of AkronAkronUSA

Personalised recommendations