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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, 2010Assembly and Stimulatory Activity of Backbone to Side ChainCyclic Octapeptide-Ligands for the N-terminal SH2-domain ofthe Protein-tyrosine-phosphatase SHP-1Mohammad S. Zoda 1 , Martin Zacharias 2 , Franziska Mussbach 1,3 ,Buerk Schaefer 3 , and Siegmund Reissmann 1,3*1 Friedrich-Schiller-University Jena, Institute of Biochemistry and Biophysics,Philoso-phenweg 12, D-07743, Jena, Germany; 2 Technische Universität München,Physik-Department (T38), James Franck-Str. 1, D-85748, Garching, Germany;3 Jena-Bioscience GmbH, Loebstedter Str. 80, D-07749, Jena, GermanyIntroductionThe cytosolic protein-tyrosine phosphatase SHP-1 plays an important role in processing ofimmune cells, in prevention of cancer genesis, and in cell adhesion and proliferation. In thenon-stimulated form the catalytic domain it is shielded by the N-terminal SH2-domain.Activation by phosphotyrosine peptides occurs mainly through binding to this N-terminalSH2-domain [1,2]. As activating peptides would require a specific steric display of thephosphotyrosine side chain and backbone structure, a basic conformation was designedusing a docking model [3,4]. The purpose of our study was to stabilize such spatialarrangement by a specific backbone to side chain cyclization, containing N-functionalizedphosphotyrosine.Results and DiscussionAssembly: The amino acid sequence was derived from the activating physiological Roskinasesequence: Glu-Gly-Leu Asn-pTyr-Met-Asp-Leu. To reduce the syntheticdifficulties, Asn at position 4 was replaced by the isosteric α-amino-butyric acid (Abu) andMet at position 6 by Nle. Based on these concept peptides of the following generalstructure were synthesized:H-Glu-Gly-Leu-Aaa pTyr-Nle-Asp-Leu-NH 2CH 2 -X-NH Gly X: (CH 2 ) n , C 6 H 10Optimizing the synthesis of the designed phosphotyrosine containing cyclic peptides westudied the preparation of N-functionalized tyrosine derivatives, coupling of the next aminoacid to the N-alkylated tyrosine derivatives, and the assembly of the octapeptides [5].Activities: Table 1 shows the stimulating activities of linear and cyclic octapeptides onhighly purified SHP-1, free of any Tag. All linear and cyclic octapeptides stimulated thephosphatase activity of SHP-1. No reduction of the basal activity could be detected,indicating that no particular ligand has any inhibitory activity. Selected octapeptides (2,7,9)had no effect on the catalytic domain, even in high concentrations (250 µM). Thestimulatory activity seems to depend on ring size, flexibility and hydrophobicity of thelactam bridges. Obviously the cycles force the peptides to form a folded conformation andbind to the deep and hydrophobic pocket of the SH2-domain. The reduced activity of somecyclic (4,5,6) compared to the linear analogs (1,2) results from a missing H-bond to theSH2-domain as the N-alkylated residues are not able to form a H-bond. In contrast to theN-alkyl amide group, the reduced amide bond in the cyclic octapeptide 9 is not only moreflexible but also able to form H-bonds. Thus this compound shows the highest stimulatingactivity and furthermore represents an easier to synthesize structure. The found activitiesagree well with the docking model. By cyclization of the peptide ligands we achieved both,stabilization of bioactive conformation and stabilization against proteolytic degradation(cell homogenate, chymotrypsin and even proteinase K), respectively. Beside biologicalactivity this proteolytic stability remains an essential prerequisite for developing orallyapplicable drugs.To check the intracellular stimulation of SHP-1 we internalized certain octapeptideligandsinto transfected NIH 3T3-cells [1] using JBS-Proteoducin (a cocktail of cellpenetrating peptides). In contrast to their stimulatory activity on isolated SHP-1 the tested210