748 ¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/cbic.200300631 ChemBioChem 2003, 4, 748 ± 753 Selection of D-Amino-Acid Peptides That Bind to Alzheimer's Disease Amyloid Peptide Ab 1±42 by Mirror Image Phage Display Katja Wiesehan, [a, b] Katrin Buder, [b] Reinhold P. Linke, [c] Stephan Patt, [d] Matthias Stoldt, [a, b, e] Eberhard Unger, [b] Bettina Schmitt, [b] Enrico Bucci, [b, f] and Dieter Willbold* [a, b, e] A mirror image phage display approach was used to identify novel and highly specific ligands for Alzheimer's disease amyloid peptide Ab(1 ± 42). A randomized 12-mer peptide library presented on M13 phages was screened for peptides with binding affinity for the mirror image of Ab(1 ± 42). After four rounds of selection and amplification the peptides were enriched with a dominating consensus sequence. The mirror image of the most representative peptide (D-pep) was shown to bind Ab(1 ± 42) with a dissociation constant in the submicromolar range. Furthermore, in brain tissue sections derived from patients that suffered from Alzheimer's disease, amyloid plaques and leptomeningeal vessels containing Ab amyloid were stained specifically with a fluorescence-labeled derivative of D-pep. Fibrillar deposits derived from other amyloi- dosis were not labeled by D-pep. Possible applications of this novel and highly specific Ab ligand in diagnosis and therapy of Alzheimer's disease are discussed. KEYWORDS: Alzheimer's disease ¥ amyloid peptide ¥ enantiomers ¥ ligand design ¥ phage display Introduction Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by memory loss, confusion, and a variety of cognitive disabilities. [1] The only reliable diagnosis to date of a patient with AD is through the post mortem identification of amyloid plaques and neurofibrillary tangles in the respective brain. A major component of the amyloid plaques is the Ab peptide, which consists of 39 ± 43 amino acid residues. Ab exists in a soluble overall helical structure [2] and an aggregated overall extended (b-sheet) structure. [3] Such a b- sheet conformation is found in the fibrils that build up amyloid plaques. Therefore, these plaques are stainable with chromo- phores, such as Congo red and thioflavin. These substances show high affinity for b-sheet structures irrespective of the identity of the peptides contained therein. The fact that no reliable method for pre mortem detection of plaques is available is not only unsatisfactory for people directly or indirectly affected by AD, but also hinders the search for therapeutic approaches because neither the time course of the disease nor any treatment success can be monitored in vivo and online. This applies to humans and animal models alike. Thus, a lot of effort is currently being invested in the development of novel diagnostic tools that enable online monitoring of amyloid plaque load in living brains. In particular, imaging techniques such as magnetic resonance tomography (MRT) [4] and positron emission tomography (PET) are under investigation for their use in AD diagnosis. Probes for use in any of these detection techniques are often based on Ab-binding substances like Congo red and thioflavin-T. [5±7] Very recently, novel substances have been invented that have been shown to cross the blood ± brain barrier. [8, 9] Even Ab itself has been used as a radio-labeled probe. [10] Still, a reliable diagnostic tool for AD does not exist. During the last two decades, methods were developed that allow the screening of very large randomized peptide libraries [a] Prof. Dr. D. Willbold, Dr. K. Wiesehan, Dr. M. Stoldt Forschungszentrum J¸lich IBI-2, 52425 J¸lich (Germany) Fax: ( 49) 2461-612023 E-mail : dieter.willbold@uni-duesseldorf.de [b] Prof. Dr. D. Willbold, Dr. K. Wiesehan, K. Buder, Dr. M. Stoldt, Prof. Dr. E. Unger, Dr. B. Schmitt, Dr. E. Bucci Institut f¸r Molekulare Biotechnologie Beutenbergstrasse 11, 07745 Jena (Germany) [c] Prof. Dr. R. P. Linke Max-Planck-Institut f¸r Biochemie Am Klopferspitz 18, 82152 Martinsried (Germany) [d] Prof. Dr. S. Patt Friedrich-Schiller-Universit‰t Institut f¸r Neuropathologie, 07743 Jena (Germany) [e] Prof. Dr. D. Willbold, Dr. M. Stoldt Institut f¸r Physikalische Biologie Heinrich-Heine-Universit‰t, 40225 D¸sseldorf (Germany) [f] Dr. E. Bucci Istituto di Biostrutture e Bioimmagini Via Mezzocannone 6/8, 80134 Naples (Italy)