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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Synthesis, Characterization and Activity of Antamanide and itsAnalogues as Inhibitors of the Mitochondrial PermeabilityTransition PoreAndrea Calderan 1 , Nicola Antolini 1 , Luca Azzolin 2 , Paolo Bernardi 2 ,Andrea Rasola 2 , Paolo Ruzza 1 , and Stefano Mammi 11 Institute of Biomolecular Chemistry of CNR, Padova Unit and Department of ChemicalSciences, University of Padova, Padova, 35131, Italy; 2 Department of Biomedical Sciences,University of Padova, Padova, 35121, ItalyIntroductionAntamanide (AA) is a non-toxic cyclic decapeptide which has the following amino acidsequence: c(Val-Pro-Pro-Ala-Phe-Phe-Pro-Pro-Phe-Phe). It was isolated in 1968 from thefungus Amanita phalloides. AA acts as an antidote in poisoning by this fungus, preventingthe cellular accumulation of toxins, such as Phalloidin. It was also reported that AA inhibitstumor cell growth in vitro [1], displays an antitumor action in an animal model [2], andattenuates IL-2-induced multisystem organ edema [3]. Like Cyclosporin A (CsA) andCyclolinopeptide A, it has a powerful immunosuppressant activity [4]. Moreover, as CsAand Cyclolinopeptide A, it interacts favorably with Cyclophilin-D (Cyp-D). Cyp-D is amitochondrial protein that has a high sequence homology and essentially identical foldingwith other Cyclophilins. Cyp-D plays a key role in regulating the mitochondrialpermeability transition pore (PTP), but it is not a component of the pore. The fact that theadministration of CsA or the genetic ablation of Cyp-D leads to inhibition of PTP openingsuggested Cyp-D as a potential pharmacological target for the treatment of diseases causedby dysregulation of the pore, such as muscular dystrophy [5]. Studies on mice showed thatablation of Cyp-D protects the animal from cerebral ischemia and reperfusion injury andpromotes recovery in diseases such as muscular dystrophy, autoimmune encephalomyelitis,and Alzheimer's disease. All these factors suggest that inhibiting Cyp-D can be a valuabletherapeutic strategy [6]. To assess its affinity for Cyp-D and to obtain valuable informationabout the structure-activity relationships, we synthesized AA and several of its monosubstitutedderivatives and studied their effect on the PTP on isolated mitochondria or inwhole cells.Results and DiscussionPeptides (Table 1) were synthesized by classical solution methodology. The synthetic routewas based on the preparation of three different fragments corresponding to the sequences1-4 (containing ab initio a C-terminal tert-butyloxycarbonyl-protected hydrazide moiety),5-6 and 7-10. These fragments were subsequently assembled by the azide method followingthe Rudinger procedure obtaining the linear 5-4 sequence of AA. The same method wasused for the cyclization reaction exploiting the possibility to separate the activation fromthe condensation, which was carried out at concentrations not exceeding 10 -3 M in thepresence of an inorganic base (K 2 HPO 4 ).Table 1. Sequences of Antamanide and its derivatives (substitutions are underlined)Antamanide (AA) c(Val-Pro-Pro-Ala-Phe-Phe-Pro-Pro-Phe-Phe)AAGly 6c(Val-Pro-Pro-Ala-Phe-Gly-Pro-Pro-Phe-Phe)AAGly 9c(Val-Pro-Pro-Ala-Phe-Phe-Pro-Pro-Gly-Phe)AATyr 6c(Val-Pro-Pro-Ala-Phe-Tyr-Pro-Pro-Phe-Phe)AATyr 9c(Val-Pro-Pro-Ala-Phe-Phe-Pro-Pro-Tyr-Phe)desPhe 5,6 AAGly 9 c(Val-Pro-Pro-Ala-des-des-Pro-Pro-Gly-Phe)desPhe 5,6 AATyr 9 c(Val-Pro-Pro-Ala-des-des-Pro-Pro-Tyr-Phe)328