In vitro binding and in vivo biodistribution studies of the neuroprotective peptide humanin using [ 125 I]humanin derivatives Alexandra Evangelou a , Christos Zikos a,b , Dimitra Benaki c , Maria Pelecanou c , Penelope Bouziotis a , Minas Papadopoulos a , Lenka Borovickova d , Iva Vesela d , Tomas Elbert d , Gabriela Kunes ˇova ´ e , Ioannis Pirmettis a , Maria Paravatou-Petsotas a , Jirina Slaninova ´ d , Evangelia Livaniou a, * a Institute of Radioisotopes and Radiodiagnostic Products, NCSR ‘‘Demokritos’’, 153 10 Athens, Greece b Biomedica Life Sciences, Athens, Greece c Institute of Biology, NCSR ‘‘Demokritos’’, 153 10 Athens, Greece d Institute of Organic Chemistry and Biochemistry AVC ˇ R, v.v.i., Flemingovo nam. 2, 166 10 Prague 6, Czech Republic e Department of Toxicology, University of Defense, Trˇebesˇska ´ 1575, 500 01 Hradec Kra ´love ´, Czech Republic 1. Introduction Alzheimer’s disease (AD) is the most prevalent neurodegen- erative disorder that causes dementia. So far no fundamental therapy for this devastating disease has been established. Humanin (‘‘HN’’) is a 24-residue peptide discovered in 2001 with unique neuroprotective activity. More specifically, HN suppresses neuronal cell death caused by a wide spectrum of early onset Familial Alzheimer’s Disease (FAD) genes, the b-amyloid (Ab) peptide (main constituent of the toxic amyloid plaques) and the anti-Amyloid Precursor Protein (anti-APP) antibody [14]. High potency and specificity of its neuroprotective action suggest that HN and its derivatives are appropriate starting points for the effort to generate new AD therapeutics targeting neurons. The relationship of primary structure with HN function has been extensively investigated. The hydrophobic region Leu9– Leu12 is known to have a crucial role in biological activity [36] and substitution of certain amino acids dramatically increases neuroprotective potency; for example, substitution of Ser14 by Gly (‘‘HNG’’) enhances the in vitro neuroprotective activity of HN 1000-fold, i.e., to 10 nM [14]. The core domain required for rescue activity comprises the 17 residues from Pro3 to Pro19 [36]. The derivative of the core domain of HNG, PAGASRLLLLTGEIDLP (‘‘RG- PAGA’’), is even more potent than HN and HNG [6]. In 2005, a HN- derivative called colivelin (primary structure: SALLRSIPAPA- GASRLLLLTGEIDLP), which was formed by attaching the N- terminal nonapeptide of the activity-dependent neurotrophic factor (‘‘ADNF-9’’) to the N-terminus of RG-PAGA, was found to exert in vitro neuroprotective action through a broad range of concentrations, i.e., from 100 fM up to >1 nM [6]. ADNF-9 itself shows in vitro protective action against Ab toxicity and other Peptides 30 (2009) 2409–2417 ARTICLE INFO Article history: Received 17 June 2009 Received in revised form 30 July 2009 Accepted 30 July 2009 Available online 8 August 2009 Keywords: Neuroprotective peptides Humanin derivatives [ 125 I]Radiolabeling In vitro cell binding In vivo biodistribution Metabolic stability ABSTRACT Humanin (HN) and HN-derivatives are a family of peptides first reported in the last decade with potent in vitro and in vivo neuroprotective activity, which is mediated through a not completely elucidated mechanism. Recently, our group has evaluated the effect of various HN-derivatives on the 3- quinuclidinyl benzilate (QNB)-induced impairment of spatial orientation and memory in rats, by employing the T-maze test. In the present work four new, tyrosine containing HN-derivatives were synthesized (Y-PAGASRLLLTGEIDLP, peptide I; Y-PAGASRLLLLTGEIDLP, peptide II; Y-SALLRSIPAPA- GASRLLLTGEIDLP, peptide III; Y-SALLRSIPAPAGASRLLLLTGEIDLP, peptide IV). The neuroprotective action of these peptides was evaluated in the T-maze test and the most active among them (peptides I and III) was radiolabeled with 125 I. The pure monoradioiodinated peptides were used in: (i) in vitro binding studies with various neuronal cell lines and with brain and stomach membranes from rats and mice and (ii) in vivo biodistribution studies in rats and mice. Moreover, the metabolic stability of the above radiolabeled peptides was studied. Under the experimental conditions used, our data do not confirm the existence of specific binding sites for HN on the neuronal tissue. Nevertheless, they are setting the basis for further relevant studies aiming at the clarification of the mode of the neuroprotective action of HN- peptides. ß 2009 Elsevier Inc. All rights reserved. * Corresponding author at: Immunopeptide Chemistry Lab., Institute of Radio- isotopes & Radiodiagnostic Products, National Centre for Scientific Research ‘‘Demokritos’’, Patriarchou Grigoriou & Neapoleos, Aghia Paraskevi Attikis, Athens 153 10, Greece. Tel.: +30 210 6548416/6503839; fax: +30 210 6545695. E-mail address: livanlts@rrp.demokritos.gr (E. Livaniou). Contents lists available at ScienceDirect Peptides journal homepage: www.elsevier.com/locate/peptides 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.07.028