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In vitro selection by phage display techniques

Phage display technologies allow the identification of peptide ligands for a given target molecule out of a huge library of different peptides expressed on the surface of bacteriophages. Presentation of the peptide library on the surface of bacteriophages ("phage display") as a fusion of peptide and a phage coat protein allows the physical link between the presented peptide and the DNA sequence coding for its amino acid sequence. Diversity of the peptides/proteins can be introduced by combinatorial mutagenesis of the fusion gene. Extremely large numbers of different peptides can be constructed, replicated, selected and amplified in a process called "biopanning". The libraries are incubated with a target molecule either as an immobilized target or prior to capture of the target-phage complex on a solid support. Similar to affinity chromatography, non-interacting peptides and proteins are washed off and the interacting molecules are subsequently eluted. The interacting phage displayed peptides or proteins can be amplified by bacterial infection to increase their copy number. The selection/amplification process can be repeated to further enrich those library members with relatively higher affinity to the target. The result is a final peptide population that is dominated by sequences that bind the target with highest affinity.

In therapeutic or diagnostic in vivo approaches, D-enantiomeric peptides have several advantages over L-enantiomeric peptides. Most importantly, they are resistant to proteases, which can dramatically increase serum and saliva half-life. A very elegant and attractive way to obtain D-peptides that bind to specific targets is mirror image phage display. During a common phage display approach, as invented in 1990, a L-peptide library is presented on the surface of phages and selected for those L-peptides (e.g. by biopanning) that bind to a given target protein. In mirror image phage display, the selection is carried out against the mirror image of the original target, which is in the case of proteins a protein with the same amino acid sequence but fully composed of D-enantiomeric amino acids (see figure). Mirror image phage display uses the full advantages of phage display, allowing the relatively easy and straight forward selection of ligands out of a huge biologically encoded library that consists of up to 1013 different peptide or protein variants, thereby also delivering a rich source of structural diversity.

  • Altendorf T, Gering I, Santiago-Schübel B, Aghabashlou Saisan S, Tamgüney G, Tusche M, Honold D, Schemmert S, Hoyer W, Mohrlüder J, Willbold D 
    Stabilization of monomeric Tau protein by all D-enantiomeric peptide ligands as therapeutic strategy for Alzheimer's disease and other tauopathies 
    Int J Mol Sci 24, 2161 (2023) 
  • Sevenich M, Honold D, Willuweit A, Kutzsche J, Mohrlüder J, Willbold D. 
    Development of an α-synuclein fibril and oligomer specific tracer for diagnosis of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy.  
    Neurochem Int. 161, 105422 (2022) 
  • Klein AN, Ziehm T, van Groen T, Kadish I, Elfgen A, Tusche M, Thomaier M, Reiss K, Brener O, Gremer L, Kutzsche J, Willbold D 
    Optimization of D-peptides for Aβ monomer binding specificity enhances their potential to eliminate toxic Aβ oligomers 
    ACS Chem Neurosci 8, 1889-1900 (2017) 
  • Mirecka EA, Feuerstein S, Gremer L, Schröder GF, Stoldt M, Willbold D, Hoyer W. 
    β-Hairpin of Islet Amyloid Polypeptide Bound to an Aggregation Inhibitor 
    Sci. Rep. 6, 33474 (2016) 
  • Orr AA, Wördehoff MM, Hoyer W, Tamamis P 
    Uncovering the binding and specificity of β-wrapins for amyloid-β and α-synuclein 
    J. Phys. Chem. B 120, 12781-12794 (2016) 
  • Rudolph S, Klein AN, Tusche M, Schlosser C, Elfgen A, Brener O, Teunissen C,Gremer L, Funke SA, Kutzsche J, Willbold D. 
    Correction: Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity 
    PLoS One 11(7), (2016) 
  • Shaykhalishahi H, Mirecka EA, Gauhar A, Grüning CSR, Willbold D, Härd T, Stoldt M, Hoyer W 
    A beta-hairpin-binding protein for three different disease-related amyloidogenic proteins 
    ChemBioChem 16, 411-414 (2015) 
  • Gauhar A, Shaykhalishahi H, Gremer L, Mirecka EA, Hoyer W 
    Impact of subunit linkages in an engineered homodimeric binding protein to alpha-synuclein 
    Protein Eng. Des. Sel. 27, 473-479 (2014) 
  • Grüning CS, Mirecka EA, Klein AN, Mandelkow E, Willbold D, Marino SF, Stoldt M, Hoyer W 
    Alternative conformations of the tau repeat domain in complex with an engineered binding protein 
    J. Biol. Chem. 289, 23209-23218 (2014) 
  • Mirecka EA, Shaykhalishahi H, Gauhar A, Akgül S, Lecher J, Willbold D, Stoldt M, Hoyer W 
    Sequestration of a beta-hairpin for control of alpha-synuclein aggregation 
    Angew. Chem. Int. Ed. 53, 4227-4230 (2014) 
  • Widera M, Klein NA, Cinar Y, Funke SA, Willbold D, Schaal H 
    The D-amino acid peptide D3 reduces amyloid fibril boosted HIV-1 infectivity 
    AIDS Res. Therapy 11, 1 (2014) 
  • Pavlidou M, Hänel K, Möckel L, Willbold D 
    Nanodiscs allow phage display selection for ligands to non-linear epitopes on membrane proteins 
    PLoS ONE 9, e72272 (2013) 
  • Funke SA, Bartnik D, Glück JM, Pirkowska K, Wiesehan K, Weber U, Gulyas B, Halldin C, Pfeifer A, Spenger C, Muhs A, Willbold D 
    Development of a small D-enantiomeric Alzheimer's amyloid-beta binding peptide ligand for future in vivo imaging applications. 
    PLoS ONE 7, e41457 (2012) 
  • Sun N, Funke SA, Willbold D 
    Mirror image phage display - Generating stable therapeutically and diagnostically active peptides with biotechnological means. 
    J. Biotech. 161, 121-125 (2012) 
    full text  
  • Bartnik D, Funke SA, Andrei-Selmer L-C, Bacher M, Dodel R, Willbold D 
    Differently selected D-enantiomeric peptides act on different Aß species. 
    Rejuvenation Res. 13, 202-205 (2010) 
  • Batra-Safferling R, Granzin J, Moedder S, Hoffmann S, Willbold D 
    Structural studies of PI3K SH3 domain in complex with a peptide ligand: Role of anchor residue in ligand binding. 
    Biol. Chem. 391, 33-42 (2010) 
  • Funke SA, van Groen T, Kadish I, Bartnik D, Nagel-Steger L, Brener O, Sehl T, Batra-Safferling R, Moriscot C, Schoehn G, Horn AHC, Müller-Schiffmann A, Korth C, Sticht H, Willbold D 
    Oral Treatment with the D-Enantiomeric Peptide D3 Improves Pathology and Behavior of Alzheimer’s disease Transgenic Mice 
    ACS Chem. Neurosci. 1, 639-648 (2010) 
    Highlighted in: Chemical & Engineering News, 88(33), August 16, 2010 
  • Hoffmann S, Funke SA, Wiesehan K, Moedder S, Glück JM, Feuerstein S, Gerdts M, Moetter J, Willbold D 
    Competitively selected protein ligands pay theirs increase in specificity by a decrease in affinity. 
    Mol. BioSyst 6, 116-123 (2010) 
  • Liu HM, Funke SA, Willbold D 
    Transport of Alzheimer's disease amyloid-ß peptide binding D-amino acid peptides across a blood-brain barrier in vitro model. 
    Rejuvenation Res. 13, 210-213 (2010) 
  • Luheshi LM, Hoyer W, de Barros TP, van Dijk-Härd I, Brorsson AC, Macao B, Persson C, Crowther DC, Lomas DA, Ståhl S, Dobson CM, Härd T 
    Sequestration of the Abeta peptide prevents toxicity and promotes degradation in vivo 
    PLoS Biol. 8, e1000334 (2010) 
  • Müller-Schiffmann A, März-Berberich J, Andrjevna A, Rönicke R, Bartnik D, Brener O, Kutzsche J, Horn AHC, Hellmert M, Polkowska J, Gottmann K, Reymann K, Funke SA, Nagel-Steger L, Moriscot C, Schoehn G, Sticht H, Willbold D, Schrader T, Korth C 
    Combining independent drug classes into superior, synergistically acting hybrid molecules. 
    Angew. Chem. Int. Ed. Engl. 49, 8743-8746 (2010) 
  • Funke SA, Willbold D 
    Mirror image phage display - a method to generate D-peptide ligands for use in diagnostic or therapeutical applications? 
    Mol. BioSyst. 5, 783-786 (2009) 
  • van Groen T, Kadish I, Wiesehan K, Funke, SA, Willbold D 
    In vitro and in vivo staining characteristics of small, fluorescent, Aß42 binding D-enantiomeric peptides in transgenic AD mouse models. 
    ChemMedChem 4, 276-282 (2009) 
  • van Groen T, Wiesehan K, Funke SA, Kadish I, Nagel-Steger L, Willbold D 
    Reduction of Alzheimer's disease amyloid plaque load in transgenic mice by D3, a D-enantiomeric peptide identified by mirror image phage display. 
    ChemMedChem 3, 1848-1852 (2008) 
    [This article was evaluated by the faculty of 1000 Biology as a "must read"]  
  • Wiesehan K, Stöhr J, Nagel-Steger K, van Groen T, Riesner D and Willbold D 
    Inhibition of cytotoxicity and fibril formation by a D-amino acid peptide that specifically binds to Alzheimer's disease amyloid peptide 
    Prot. Eng. Des. Sel. 21, 241-246 (2008) 
  • Mohrlüder J, Hoffmann Y, Stangler T, Hänel K, Willbold D 
    Identification of clathrin heavy chain as a direct interaction partner for the gamma-aminobutyric acid type A receptor associated protein 
    Biochemistry 46, 14537-14543 (2007) 
  • Mohrlüder J, Stangler T, Wiesehan K, Hoffmann Y, Mataruga A, Willbold D 
    Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide library 
    FEBS J 274, 5543-5555 (2007) 
  • Schmidt H, Hoffmann S, Tran T, Stoldt M, Stangler T, Wiesehan K, Willbold D 
    Solution structure of a Hck SH3 domain ligand complex reveals novel interaction modes. 
    J. Mol. Biol. 365, 1517-1532 (2007) 
  • Stangler T, Tran T, Hoffmann S, Schmidt H, Jonas E, Willbold D 
    Competitive displacement of full-length HIV-1 Nef from Hck SH3 domain by a high affinity artificial peptide. 
    Biol. Chem. 388, 611-615 (2007) 
  • Tran T, Hoffmann S, Wiesehan K, Jonas E, Luge C, Aladag A, Willbold D 
    Insights into human Lck SH3 domain binding specificity: different binding modes of artificial and native ligands. 
    Biochemistry 44, 15042-15052 (2005) 
  • Wiesehan K, Buder K, Linke RP, Patt S, Stoldt M, Unger E, Schmitt B, Bucci E and Willbold D 
    Selection of D-amino-acid peptides that bind to Alzheimer's disease amyloid peptide Abeta(1-42) by mirror image phage display. 
    ChemBioChem 4, 748-753 (2003) 
  • Wiesehan K, Willbold D 
    Mirror image phage display: Aiming at the mirror. 
    ChemBioChem 4, 811-815 (2003) 
  • Jonas G, Hoffmann S, Willbold D 
    Binding of phage displayed HIV-1 Tat to TAR RNA in the presence of Cyclin T1. 
    J. Biomed. Sci. 8, 340-346 (2001) 
  • Willbold D & Wiesehan K 
    Peptid zur Diagnose und Therapie der Alzheimer-Demenz National: DE 10117281A1, international: PCT/EP02/03862 
    , (2001) 
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