Synthesis of d-amino acid peptides and their effect on beta-amyloid aggregation and toxicity in transgenic Caenorhabditis elegans

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Genetic, biochemical, and pathological evidence supports that aggregation of amyloid-beta (Aβ) peptide into fibrillar structures rich in beta-sheets is implicated as the cause of Alzheimer’s disease. Therefore, an attractive therapeutic strategy is to prevent or alter amyloid-beta aggregation. In this work we examine the effects of the short d-peptides pgklvya, kklvffarrrra, and kklvffa on Aβ aggregation in vitro and toxicity in vivo. These peptides are based on the central hydrophobic region of Aβ (residues 16–20), which is believed to be crucial in Aβ self-association. The effect of peptides on Aβ aggregation was examined by circular dichroism spectroscopy, Thioflavin T fluorescence, and ANS binding assay. Transgenic Caenorhabditis elegans model was used to evaluate the pharmacological effect of d-peptides on Aβ-initiated toxicity. The data suggested that d-peptides are very effective at inhibiting fibrillogenesis of Aβ. Among the three peptides tested, only pgklvya and kklvffa improved survival in the transgenic C. elegans. The activity of these peptides correlates with their ability to inhibit Aβ oligomerization. These suggest that d-peptides should be considered during future design of peptide-based inhibitors of amyloid deposition and toxicity.

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Amyloid beta


Amyloid precursor protein


Alzheimer’s disease


Thioflavin T




Trifluoroacetic acid




4,4′-Bis(1-anilinonaphthalene 8-sulfonate)


  1. Annaert W, De Strooper B (2002) A cell biological perspective on Alzheimer’s disease. Annu Rev Cell Dev Biol 18:25–51

  2. Austen BM, Paleologou KE, Ali SA, Qureshi MM, Allsop D, El-Agnaf OM (2008) Designing peptide inhibitors for oligomerization toxicity of Alzheimer’s beta-amyloid peptide. Biochemistry 47:1984–1992

  3. Ban T, Hoshino M, Takahashi S, Hamada D, Hasegawa K, Goto Y (2004) Direct observation of Abeta amyloid fibril growth inhibition. J Mol Biol 344:757–767

  4. Beher D, Graham SL (2005) Protease inhibitors as potential disease-modifying therapeutics for Alzheimer’s disease. Expert Opin Investig Drugs 14:1385–1409

  5. Bhattacharyya J, Sharma KK (2001) Conformational specificity of mini-alphaA crystallin as a molecular chaperone. J Peptide Res 57:428–434

  6. Bischofberger P, Han W, Feifel B, Schonfeld HJ, Christen P (2003) d-peptides as inhibitors of the DnaK/DnaJ/GrpE chaperone system. J Biol Chem 278:19044–19047

  7. Blanchard BJ, Konopka G, Russell M, Ingram VM (1997) Mechanism prevention of neurotoxicity caused by beta-amyloid peptides: relation to Alzheimer’s disease. Brain Res 776:40–50

  8. Brenneman DE, Spong CY, Hauser JM, Abebe D, Pinhasov A, Golian T, Gozes I (2004) Protective peptides that are orally active mechanistically nonchiral. J Pharmacol Exp Ther 309:1190–1197

  9. Chalifour RJ, McLaughlin RW, Lavoie L, Morissette C, Tremblay N, Boulé M, Sarazin P, Stéa D, Lacombe D, Tremblay P, Gervais F (2003) Stereoselective interactions of peptide inhibitors with the beta-amyloid peptide. J Biol Chem 278:34874–34881

  10. Chan WC, White PD (2000) F-moc solid phase peptide synthesis a practical approach. Oxford University Press Inc, New York

  11. Chen S, Wetzel R (2001) Solubilization disaggregation of polyglutamine peptides. Protein Sci 10:887–891

  12. Chen Y, Vasil AI, Rehaume L, Mant CT, Burns JL, Vasil ML, Hancock RE, Hodges RS (2006) Comparison of biophysical biologic properties of alpha-helical enantiomeric antimicrobial peptides. Chem Biol Drug Des 67:162–173

  13. Chiti F, Calamai M, Taddei N, Stefani M, Ramponi G, Dobson CM (2002) Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases. Proc Natl Acad Sci USA 99:16419–16426

  14. Chiti F, Stefani M, Taddei N, Ramponi G, Dobson CM (2003) Rationalization of the effects of mutations on peptide protein aggregation rates. Nature 424:805–808

  15. Dahlgren KN, Manelli AM, Stine WB Jr, Baker LK, Krafft GA, LaDu MJ (2002) Oligomeric fibrillar species of amyloid-beta peptides differentially affect neuronal viability. J Biol Chem 277:32046–32053

  16. El-Agnaf OMA, Guthrie DJ, Walsh DM, Irvine GB (1998) The influence of the central region containing residues 19–25 on the aggregation properties secondary structure of Alzheimer’s β-amyloid peptide. Eur J Biochem 256:560–569

  17. Feifel B, Schonfeld HJ, Christen P (1998) d-peptide ligands for the co-chaperone DnaJ. J Biol Chem 273:11999–12002

  18. Fields GB, Noble RL (1990) Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int J Peptide Protein Res 35:161–214

  19. Findeis MA (2007) The role of amyloid beta peptide 42 in Alzheimer’s disease. Pharmacol Ther 116:266–286

  20. Findeis MA, Musso GM, Arico-Muendel CC, Benjamin HW, Hundal AM, Lee JJ, Chin J, Kelley M, Wakefield J, Hayward NJ, Molineaux SM (1999) Modified-peptide inhibitors of amyloid β-peptide polymerization. Biochemistry 38:6791–6800

  21. Fink AL (1998) Protein aggregation: folding aggregates inclusion bodies amyloid. Fold Des 3:R9–R23

  22. Fonte V, Kipp DR, Yerg J 3rd, Merin D, Forrestal M, Wagner E, Roberts CM, Link CD (2008) Suppression of in vivo beta-amyloid peptide toxicity by overexpression of the HSP-162 small chaperone protein. J Biol Chem 283:784–791

  23. Giasson BI, Murray IV, Trojanowski JQ, Lee VM (2001) A hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly. J Biol Chem 276:2380–2386

  24. Glabe CG (2006) Common mechanisms of amyloid oligomer pathogenesis in degenerative disease. Neurobiol Aging 27:570–575

  25. Goedert M, Spillantini MG (2006) A century of Alzheimer’s disease. Science 314:777–781

  26. Goldsbury CS, Wirtz S, Muller SA, Sunderji S, Wicki P, Aebi U, Frey P (2000) Studies on the in vitro assembly of a beta 1–40: implications for the search for a beta fibril formation inhibitors. J Struct Biol 130:217–223

  27. Gong Y, Chang L, Viola KL, Lacor PN, Lambert MP, Finch CE, Krafft GA, Klein WL (2003) Alzheimer’s disease-affected brain: presence of oligomeric A beta ligands (ADDLs) suggests a molecular basis for reversible memory loss. Proc Natl Acad Sci USA 100:10417–10422

  28. Gordon DJ, Sciarretta KL, Meredith SC (2001) Inhibition of β amyloid(40) fibrillogenesis disassembly of β-amyloid(40) fibrils by short β-amyloid congeners containing N-methyl amino acids at alternate residues. Biochemistry 40:8237–8245

  29. Hardy J (2006) A hundred years of Alzheimer’s disease research. Neuron 52:3–13

  30. Harper JD, Wong SS, Lieber CM, Lansbury PT Jr (1999) Assembly of a beta amyloid protofibrils: an in vitro model for a possible early event in Alzheimer’s disease. Biochemistry 38:8972–8980

  31. Hartley DM, Walsh DM, Ye CP, Diehl T, Vasquez S, Vassilev PM, Teplow DB, Selkoe DJ (1999) Protofibrillar intermediates of amyloid beta-protein induce acute electrophysiological changes progressive neurotoxicity in cortical neurons. J Neurosci 19:8876–8884

  32. Hoozemans JJ, Chafekar SM, Baas F, Eikelenboom P, Scheper W (2006) Always around never the same: pathways of amyloid beta induced neurodegeneration throughout the pathogenic cascade of Alzheimer’s disease. Curr Med Chem 13:2599–2605

  33. Hsia AY, Masliah E, McConlogue L, Yu GQ, Tatsuno G, Kang H, Kholodenenko D, Malenka RC, Nicoll RA, Mucke L (1999) Plaque-independent disruption of neural circuits in Alzheimer’s disease mouse models. Proc Natl Acad Sci USA 96:3228–3233

  34. Hutton M, Perez-Tur J, Hardy J (1998) Genetics of Alzheimer’s disease. Essays Biochem 33:117–131

  35. Kaiser E, Colescott RL, Bossinger CD, Cook PI (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal Biochem 34:595–598

  36. Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW, Glabe CG (2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300:486–489

  37. Kelly JW (1996) Alternative conformations of amyloidogenic proteins govern their behavior. Curr Opin Struct Biol 6:11–17

  38. Koo EH, Lansbury PT Jr, Kelly JW (1999) Amyloid diseases: abnormal protein aggregation in neurodegeneration. Proc Natl Acad Sci USA 96:9989–9990

  39. Kremer JJ, Pallitto MM, Sklansky DJ, Murphy RM (2000) Correlation of β-amyloid aggregate size hydrophobicity with decreased bilayer fluidity of model membranes. Biochemistry 39:10309–10318

  40. Ladiwala AR, Dordick JS, Tessier PM (2011a) Aromatic small molecules remodel toxic soluble oligomers of amyloid beta through three independent pathways. J Biol Chem 286(5):3209–3218

  41. Ladiwala AR, Mora-Pale M, Lin JC, Bale SS, Fishman ZS, Dordick JS, Tessier PM (2011b) Polyphenolic glycosides and aglycones utilize opposing pathways to selectively remodel and inactivate toxic oligomers of amyloid β. ChemBioChem 12(11):1749–1758

  42. Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, Liosatos M, Morgan TE, Rozovsky I, Trommer B, Viola KL, Wals P, Zhang C, Finch CE, Kraft GA, Klein WL (1998) Diffusible nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc Natl Acad Sci USA 95:6448–6453

  43. Lansbury PT Jr (1999) Evolution of amyloid: what normal protein folding may tell us about fibrillogenesis disease. Proc Natl Acad Sci USA 96:3342–3344

  44. LeVine H (1999) Quantification of beta-sheet amyloid fibril structures with thioflavin T methods. Enzymol 309:274–284

  45. Link CD (1995) Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans. Proc Natl Acad Sci USA 92:9368–9372

  46. Link CD (2006) Caenorhabditis elegans models of age-associated neurodegenerative diseases: lessons from transgenic worm models of Alzheimer’s disease. Exp Gerontol 41:1007–1013

  47. Link CD, Johnson CJ, Fonte V, Paupard M, Hall DH, Styren S, Mathis CA, Klunk WE (2001) Visualization of fibrillar amyloid deposits in living transgenic Caenorhabditis elegans animals using the sensitive amyloid dye X-34. Neurobiol Aging 22:217–226

  48. Lorenzo A, Yankner BA (1994) Beta-amyloid neurotoxicity requires fibril formation is inhibited by congo red. Proc Nat Acad Sci USA 91:12243–12247

  49. Lowe TL, Strzelec A, Kiessling LL, Murphy RM (2001) Structure-function relationships for inhibitors of -amyloid toxicity containing the recognition sequence KLVFF. Biochemistry 40:7882–7889

  50. Milton RC, Milton SC, Kent SB (1992) Total chemical synthesis of a d-enzyme: the enantiomers of HIV-1 protease show reciprocal chiral substrate specificity. Science 256:1445–1448

  51. Mucke L, Masliah E, Yu GQ, Mallory M, Rockenstein EM, Tatsuno G, Hu K, Kholodenko D, Johnson-Wood K, McConlogue L (2000) High-level neuronal expression of abeta 1–42 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation. J Neurosci 20:4050–4058

  52. Naiki H, Higuchi K, Hosokawa M, Takeda T (1989) Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye thioflavin T1. Anal Biochem 177:244–249

  53. Naiki H, Higuchi K, Matsushima K, Shimada A, Chen WH, Hosokawa M, Takeda T (1990) Fluorometric examination of tissue amyloid fibrils in murine senile amyloidosis: use of the fluorescent indicator thioflavine T. Lab Invest 62:768–773

  54. Otzen DE, Kristensen O, Oliveberg M (2000) Designed protein tetramer zipped together with a hydrophobic Alzheimer homology: a structural clue to amyloid assembly. Proc Natl Acad Sci USA 97:9907–9912

  55. Pappenheimer JR, Dahl CE, Karnovsky ML, Maggio JE (1994) Intestinal absorption excretion of octapeptides composed of d amino acids. Proc Natl Acad Sci USA 91:1942–1945

  56. Pappenheimer JR, Karnovsky ML, Maggio JE (1997) Absorption excretion of undegradable peptides: role of lipid solubility net charge. J Pharmacol Exp Ther 280:292–300

  57. Petrushina I, Ghochikyan A, Mktrichyan M, Mamikonyan G, Movsesyan N, Davtyan H, Patel A, Head E, Cribbs DH, Agadjanyan MG (2007) Alzheimer’s disease peptide epitope vaccine reduces insoluble but not soluble/oligomeric Abeta species in amyloid precursor protein transgenic mice. J Neurosci 27:12721–12731

  58. Pike CJ, Walencewicz AJ, Glabe CG, Cotman CW (1991) Aggregation-related toxicity of synthetic beta-amyloid protein in hippocampal cultures. Eur J Pharmacol 207:367–368

  59. Pike CJ, Burdick D, Walencewicz AJ, Glabe CG, Cotman CW (1993) Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state. J Neurosci 13:1676–1687

  60. Pritsker M, Jones P, Blumenthal R, Shai Y (1998) A synthetic all d-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 recognizes the wild-type fusion peptide in the membrane inhibits HIV-1 envelope glycoprotein-mediated cell fusion. Proc Natl Acad Sci U S A 95:7287–7292

  61. Reddy KRC, Lilie H, Rudolph R, Lange C (2005) l-Arginine increases the solubility of unfolded species of hen egg white lysozyme. Protein Sci 14:929–935

  62. Rivière C, Richard T, Vitrac X, Mérillon JM, Valls J, Monti JP (2008) New polyphenols active on beta-amyloid aggregation. Bioorg Med Chem Lett 18:828–831

  63. Samuel D, Kumar TKS, Jayaraman G, Yang PW, Yu C (1997) Proline is a protein stabilizing solute. Biochem Mol Biol Int 41:235–242

  64. Schobert B, Tschessche H (1978) Unusual solution properties of proline its interaction with proteins. Biochim Biophys Acta 541:270–277

  65. Selkoe DJ (2004) Cell biology of protein misfolding: the examples of Alzheimer’s Parkinson’s diseases. Nat Cell Bio 6:1054–1061

  66. Shiraki K, Kudou M, Fujiwara S, Imanaka T, Takagi M (2002) Biophysical effect of amino acids on the prevention of protein aggregation. J Biochem (Tokyo) 132:591–595

  67. Simmons LK, May PC, Tomaselli KJ (1994) Secondary structure of amyloid β-peptide correlates with neurotoxic activity in vitro. Mol Pharmacol 45:373–379

  68. Solomon B (2004) Alzheimer’s disease immunotherapy. Curr Alzheimer Res 1:149–163

  69. Soto C, Estrada LD (2008) Protein misfolding neurodegeneration. Arch Neurol 65:184–189

  70. Soto C, Sigurdsson EM, Morelli L, Kumar RA, Castano EM, Frangione B (1998) β-sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: implications for Alzheimer’s therapy. Nat Med 4:822–826

  71. Soto C, Estrada L, Castilla J (2006) Amyloids prions the inherent infectious nature of misfolded protein aggregates. Trends Biochem Sci 31:150–155

  72. Srinivas V, Balasubramanian D (1995) Proline is a compatible hydrotrope. Langmuir 11:2830–2833

  73. Stackman RW, Eckenstein F, Frei B, Kulhanek D, Nowlin J, Quinn JF (2003) Prevention of age-related spatial memory deficits in a transgenic mouse model of Alzheimer’s disease by chronic Ginkgo biloba treatment. Exp Neurol 184:510–520

  74. Stryer L (1965) The interaction of a naphthalene dye with apomyoglobin apohemoglobin. A fluorescent probe of non-polar binding sites. J Mol Biol 13:482–495

  75. Taylor JP, Hardy J, Fischbeck KH (2002) Toxic proteins in neurodegenerative disease. Science 296:1991–1995

  76. Tjernberg LO, Na¨slund J, Lindqvist F, Johansson J, Karlstro¨m AR, Thyberg J, Terenius L, Nordstedt C (1996) Arrest of β-amyloid fibril formation by a pentapeptide ligand. J Biol Chem 271:8545–8548

  77. Tjernberg LO, Lilliehook C, Callaway DJ, Naslund J, Hahne S, Thyberg J (1997) Controlling amyloid beta-peptide fibril formation with protease- stable ligands. J Biol Chem 272:12601–12605

  78. Tjernberg LO, Hosia W, Bark N, Thyberg J, Johansson J (2002) Charge attraction beta propensity are necessary for amyloid fibril formation from tetrapeptides. J Biol Chem 277:43243–43246

  79. Turner DC, Brand L (1968) Quantitative estimation of protein binding site polarity fluorescence of N-arylaminonapthalenesulfonates. Biochemistry 7:3381–3390

  80. Vassar R (2005) Beta-secretase APP Abeta in Alzheimer’s disease. Subcell Biochem 38:79–103

  81. Villegas V, Zurdo J, Filimonov VV, Aviles FX, Dobson CM, Serrano L (2000) Protein engineering as a strategy to avoid formation of amyloid fibrils. Protein Sci 9:1700–1708

  82. Walsh DM, Selkoe DJ (2007) A beta oligomers—a decade of discovery. J Neurochem 101:1172–1184

  83. Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, Wolfe MS, Rowan MJ, Selkoe DJ (2002) Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416:535–539

  84. Ward RV, Jennings KH, Jepras R, Neville W, Owen DE, Hawkins J, Christie G, Davis JB, George A, Karran EH, Howlett DR (2000) Fractionation characterization of oligomeric protofibrillar fibrillar forms of beta-amyloid peptide. Biochem J 348:137–144

  85. Weggen S, Rogers M, Eriksen J (2007) NSAIDs: small molecules for prevention of Alzheimer’s disease or precursors for future drug development? Trends Pharmacol Sci 28:536–543

  86. Wiesehan K, Buder K, Linke RP, Patt S, Stoldt M, Unger E (2003) Selection of d-amino-acid peptides that bind to Alzheimer’s disease amyloid peptide abeta1–42 by mirror image phage display. ChemBioChem 4:748–753

  87. Wolfe MS (2007) Gamma-secretase modulators. Curr Alzheimer Res 4:571–573

  88. Wu Y, Luo Y (2005) Transgenic Caenorhabditis elegans as a model in Alzheimer’s research. Curr Alzheimer Res 2:37–45

  89. Wu Y, Wu Z, Butko P, Christen Y, Lambert MP, Klein WL, Link CD, Luo Y (2006) Amyloid-β-induced pathological behaviors are suppressed by ginkgo biloba extract EGb 761 ginkgolides in transgenic Caenorhabditis elegans. J Neurosci 26:13102–13113

  90. Yang F, Lim GP, Begum AN, Ubeda OJ, Simmons MR, Ambegaokar SS, Chen PP, Kayed R, Glabe CG, Frautzchy SA, Cole GM (2005) Curcumin inhibits formation of amyloid β oligomers fibrils binds plaques reduces amyloid in vivo. J Biol Chem 280:5892–5901

  91. Yankner BA (1996) Mechanisms of neuronal degeneration in Alzheimer’s disease. Neuron 16:921–932

  92. Zhou N, Luo MZW, Luo JS, Fan XJ, Cayabyab M, Hiraoka M, Liu DX, Han XB, Pesavento J, Dong CZ, Wang YL, An J, Kaji H, Sodroski JG, Huang ZW (2002) Exploring the stereochemistry of CXCR4-peptide recognition inhibiting HIV-1 entry with d-peptides derived from chemokines. J Biol Chem 277:17476–17485

  93. Zou K, Kim D, Kakio A, Byun K, Gong JS, Kim J, Kim M, Sawamura N, Nishimoto S, Matsuzaki K, Lee B, Yanagisawa K, Michikawa M (2003) Amyloid beta-protein (Abeta)1–40 protects neurons from damage induced by Abeta1-42 in culture in rat brain. J Neurochem 87:609–619

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We are thankful to Dr. Asit Baran Mandal, Director, Central Leather Research Institute, Chennai for his kind support for this work. The author thanks the Council of Scientific and Industrial Research (CSIR), India, for the award of Junior and Senior Research Fellowships. We are thankful to Man-Wah Tan, Stanford University for allowing us to do C.elegans work in his lab. We are also thankful to Prof. T. Pradeep, IIT Madras for doing Maldi-MS.

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Correspondence to Seema Jagota.

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Jagota, S., Rajadas, J. Synthesis of d-amino acid peptides and their effect on beta-amyloid aggregation and toxicity in transgenic Caenorhabditis elegans . Med Chem Res 22, 3991–4000 (2013) doi:10.1007/s00044-012-0386-2

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  • Amyloid-beta peptide
  • Caenorhabditis elegans
  • Bis-ANS
  • Thioflavin T
  • d-amino acids