Proceedings book download - 5Z.com

Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Stability of CLIPS Peptides in Human SerumWim Schaaper, Peter van Dijken, and Peter TimmermanPepscan Therapeutics B.V., Zuidersluisweg 2, 8243RC, Lelystad, The NetherlandsIntroductionSmall peptides generally do not express high biological activity due to their flexible randomstructure. Many methods have been developed to introduce a more rigid conformation. Ourproprietary CLIPS TM technology [1,2] (Chemical LInkage of Peptides onto Scaffolds) wasused to develop a bicyclic enzyme inhibitor specific for human plasma kallikrein [3]. ThisCLIPS peptide (S905/T3, IC 50 = 1.7 nM) was > 5000-fold more active than the linear peptide.Here, we present the results of an enzyme degradation study in human serum of the bicyclicS905/T3 peptide. We also tested both monocyclic loops, either as CLIPS construct, as cyclicdisulfide, or as the Cys-blocked linear analogue. Furthermore, the bicyclic retro-inversopeptide, based on D-amino acids and inversed sequence, was also tested.The CLIPS peptides, especially the bicyclic products, were found to be superior instability against enzymatic degradation compared to their linear analogues.Results and DiscussionFour peptides (S905: ACSDRFRNCPADEALCG-amide, S906: ACSDRFRNC, S907:CPADEALCG-amide, and P431: Gclaedapcnrfrdsca-amide) were synthesized using Fmocchemistry and purified using preparative RP-HPLC. The peptides were modified by one of thefollowing routes: a) cyclisation by overnight air oxidation at pH 8 (for S906 and S907), b)blocking of the free cysteines by reaction with iodoacetamide at pH 8 for 2 hours (allpeptides), c) CLIPS reaction (Figure 1) by reaction with 1,3,5-tris(bromomethyl)benzene (forS905 and P431) or with , ’-dibromo-m-xylene (for S906 and S907) in 0.02M ammoniumbicarbonate/acetonitrile for 1 hour. All products were purified using preparative RP-HPLC toyield a final purity of >95%.In the stability study, the CLIPSpeptides (Figure 1) and their linearcounterparts in which the cysteines wereblocked with iodoacetamide weredissolved in PBS, pH 7.2 [4] at aconcentration of 2 mg/mL. This solutionwas mixed with human serum (Sigma)1:1 (v/v) or, as reference samples, withPBS 1:1 (v/v) to a final concentration of1 mg/mL. All samples were stored at37 o C and 50 µL samples were taken atseveral time intervals for analysis.These samples were mixed with 50 µLof 10% TFA/water. Precipitatedproteins in the serum samples wereseparated by centrifugation.Fig. 1. CLIPS constructs: S905/T3: bicyclic T3,S906/T2, cyclic N-terminal T2, S907/T2: cyclicC-terminal T2, P431/T3: bicyclic retro-inversoT3.Stability of bicyclic T3 constructs:in S905/T3 the N-terminal Ala, whichwas outside the ring structure, wasslowly cleaved in serum (t ½ = 113 hr),however, the ring structure itself was farmore stable (t ½ = 240 hr). Ring cleavageoccurred slowly at the carboxyl group of Arg-7 and at the amino group of Asp-12. Finally,after 1060 hours, most of the peptide was digested to yield Cys 3 /T3. The linear Cys-blockedpeptide was digested very rapidly (t ½ = 0.9 hr).As expected, the retro-inverso peptides were extremely resistant against enzymaticdegradation, since they are composed of D-amino acids. Nevertheless, the T3 CLIPS construct(P431/T3), is even more stable (t ½ = 5165 hr) than the linear Cys-blocked product (t ½ = 2174hr). For the retro-inverso products no specific degradation products could be identified by LC-MS within 1060 hours.612