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Table 2. IC50 values for the binding inhibitions of FGF-1 binding to immobilised heparins.HPLC purified peptides characterized by H 1 NMR.Peptide Structure: Ac-Peptide-K-NH 2 IC50A1 E S* N S* S* #* N Y* N #* >500 MA2 Q S* Q #* S* T* D S* Q #* >500 MA3 Q T* Q Y* S* S* N Y* Q S* >500 MA4 Q T* N #* S* S* N #* E #* >500 MA5 D S* Q S* Y* S* E S* Q #* 400 MA6 E T* Q S* S* T* D #* Q S* 325 MA7 Q S* Q S* S* T* Q Y* E #* 150 MA8 E T* E T* S* S* E S* E S* 4 MB LMW heparin 188 nM*Sulphated amino acids; # is HypResynthesized structures are presented in Table 2. The peptides were purified on HPLC andcharacterized by H 1 NMR (Figure 1). By comparing the spectra with non-sulphatedpeptides there is a significant shift in the -proton of amino acids after O-sulphatation.To test the hits from the screening of the anti-thrombin binding, a competition assay ofprotein binding to immobilized heparin was carried out using surface plasma resonance(SPR). The binding of anti-thrombin to the immobilized heparin in presence of varioussulphated peptides at different concentrations in solution was measured and onlyinsignificant binding inhibition was observed indicating that on beads the protein takesadvantage of more than one ligand. On the other hand, FGF1 showed significant interactionwith some of the sulphated peptides, e.g. A7 identified against anti-thrombin. FGF1binding inhibition (IC 50 ) of different sulphated peptides identified through antithrombinscreening are shown in Table 2. The binding affinity is less than that of the original peptideA8 [1], and active structures show high similarity with A8 in the central region.The results indicates that although binding to the arginine rich binding sites of heparinbinding proteins are obtained, it is still a challenge to identify close mimics of heparin withshort sulphated peptides. The affinity of sulphated peptides show significant sequencedependence and may be improved by screening of specifically designed targeted sulphatedpeptide libraries. Importantly, MPM encoded libraries of compounds, that otherwise aredifficult to analyse structurally, can be applied for fast screening of protein - ligandinteractions. Sulphated peptides can be conveniently synthesized using solid phaseapproach. In the present work H 1 NMR was used to fully characterize the structure of thesulphates peptides carrying numerous sulphate groups.AcknowledgmentsThis work was supported by the Danish National Research Foundation. Technical assistance was byPia Breddam (SPR) and Dana C. Tvermoes (SPS).References1. Vazquez-Campos, S., St.Hilaire, P.M., Damgaard, D., Meldal, M. QSAR Comb. Sci. 8, 923-942(2005).2. Meldal, M., Christisen F.S. Angew. Chem. Int. Ed. 49, 3473-3476 (2010).3. Rasmussen, J.E., Christisen, F.S., Nørskov-Lauritsen, L., Meldal, M., Jensen, K.J., St.Hilaire, P.M.Angew. Chem. Int. Ed. 49, 3477-3480 (2010).149