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Sidechain-to-backbone cyclized peptides were obtained by using Boc-Lys(Fmoc)-OH. Thebicyclic peptides were made by introducing two cysteine-residues in the sequence.The K D values for binding of the upain-1 analogues to uPA were determined bydisplacement of the fluorescent p-aminobenzamidine from uPA (Table 1).Table 1. K D values for monocyclic (even entries) and bicyclic (uneven entries > 1) upain-1analoguesEntry Sequence K D (µM)1 Ac-CSWRGLENHAAC-NH 2 162 ASWRGLENHAAA >2003 ASWRGCENHAAC N.D.4 PSWRGLENHAAA >2005 PSWRGCENHAAC 8726 PSWRGLENHAAAA 1177 PSWRGCENHCAAA 6768 H-KSWRGLENHAAP 1739 H-KSWRGCENHAPC >20010 H-KSWRGLENHAAA 36311 H-KSWRGCENHAAC 31N.D. = not determinedAll monocyclic analogues show a reduced affinity for uPA. The bicyclic peptides show afurther reduced affinity, except for entry 11 (Table 1). This bicyclic peptide binds ~10times better than its monocyclic precursor and shows similar affinity as the syntheticupain-1 analogue (entry 1).We conclude that remodeling the structure of upain-1 to form head-to-tail andsidechain to-backbone cyclized analogues has reduced the binding affinity for uPA.Bicyclization of these analogues by introducing a disulfide bridge has improved the affinityof a single analogue by a factor ~10, resulting in a similar affinity as synthetic upain-1.Introducing rigidity can thus improve binding affinity, but only if the binding enthalpy ispreserved.AcknowledgmentsWe gratefully acknowledge Anni Christensen, Århus University, for performing the displacementstudies. The Danish-Chinese Centre for Proteases and Cancer and KU LIFE are thanked for funding.References1. Retrieved from: http://www.who.int/mediacentre/factsheets/fs297/en/ (August 5, 2010).2. Andreasen, P.A., Egelund, R., Petersen, H.H. Cell. Mol. Life Sci. 57, 25-40 (2000).3. Schmitt, M., et al. Fibrinilysis & Proteolysis 14, 114-132 (2000).4. Henneke, I., et al. Am. J. Respir. Crit. Care Med. 181, 611-619 (2010).5. Hansen, M., et al. J. Bio. Chem. 280, 38424-38437 (2005).6. Hosseini, M., et al. manuscript in preparation.7. Zhao, G., et al. J. Struct. Bio. 160, 1-10 (2007).555