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Table 1. Half lives of the tested peptide constructs in human serumproducthalf life (t 1/2 in hours)CLIPS-T3 CLIPS-T2 linear Cyclic SSS905 113/240* - 0.9 -S907 - 9.6 0.5 12.4S906 - 1.9 0.5 1.2P431 5165 - 2174 -* half-life of the des-Ala-1 peptideFig. 2. Concentration of T3 (left) and T2 (right) CLIPS constructs, compared to theirlinear, Cys-blocked counterparts in human serum in time. Starting concentration was1 mg/mL in 50% human serum/PBS, time in hours.Stability of cyclic T2 constructs (Table 1, Figure 2): in S906/T2, the N-terminal Ala, whichwas outside the ring, again was the first to be cleaved (t ½ = 1.9 hr). However, shortly after, thering was opened at Arg-7 and this resulted after 168 hours in an almost complete digestion toCys 2 /T2. The disulfide loop S906/ox was digested somewhat faster (t ½ = 1.2 hr) and the linearCys-blocked peptide almost completely disappeared within 6 hours (t ½ = 0.5 hr).In S907/T2 and S907/ox, the ring was preferentially cleaved at Ala-3, and both productscould not be detected anymore at 48 hours (t ½ = 9.6 resp. 12.4 hr). For the T2 construct againCys 2 /T2 was the final product. From the linear Cys-blocked peptide, only 3% was left after 3hours (t ½ = 0.5 hr). An intramolecular cyclization reaction of the N-terminal S-carbamoylmethyl-cysteine [5] in this peptide generated a more enzyme-resistant productwhich could be identified by LC-MS up to 24 hrs.The CLIPS technology was developed in order to constrain a variety of peptides in a“loop-like” conformation that adequately mimics -turns, hairpins or -sheets. Here weshowed that the CLIPS technology is also very effective in stabilizing peptides againstenzymatic degradation. The T3 scaffold, which forms bicyclic peptides, is superior ingenerating stability in human serum, even in retro-inverso peptides. The T2 scaffold peptidesshow similar stability as cyclic disulfide peptides, but are more stable than their linearanalogues. After ring opening, the CLIPS constructs are ultimately digested to Cys 3 /T3 orCys 2 /T2.References1. Timmerman, P., Beld, J., Puijk, W.C., Meloen, R.H. ChemBioChem 6, 821-824 (2005).2. Timmerman, P., Puijk, W.C., et al. Open Vacc. J. 2, 56-67 (2009).3. Heinis, C., Rutherford, T., Freund, S., Winter, G. Nat. Chem. Biol. 5, 502-507 (2009).4. Tugyi, R., Uray, K., Ivan, D., Fellinger, E., Perkins, A., Hudecz, F. PNAS 102, 413-418 (2005).5. Geoghegan, K.F., Hoth, L.R., Tan, D.H., Borzilleri, K.A., Withka, J.M., Boyd, J.G., J. ProteomeRes. 1, 181-187 (2002).613