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Table 1. Characteristics, K i and IC 50 values of azapeptidesMS [M] bCompound R t (min) a K i ( M) IC 50 ( M)calc. meas.Ac-TPLaGlySP-NH 2 18.3 612.3 612.4 - -Ac-TPLaGlySPPPS-NH 2 18.8 893.5 893.5 8.7 8.5H-TPLaGlySPPPSPR-NH 2 18.0 1104.6 1104.6 20.3 n.d.Ac-TPVaGlySPPPS-NH 2 19.0 879.4 879.8 - -Ac-TPTaGlySPPPS-NH 2 17.4 881.4 881.5 - -Ac-TSLaGlySPPPS-NH 2 20.0 883.4 883.7 3.5 9.5Ac-TWLaGlySPPPS-NH 2 27.1 982.4 982.5 5.8 9.8Ac-aGlySPPPS-NH 2 12.6 582.3 582.3 - -a Column:Phenomenex Jupiter C18, 250 x 4.6 mm, 5 m, 300Å; Gradient:0 min-0%, 5min 0%, 50 min 90%B, eluents 0.1% TFA/H 2 O (A), 0.1% TFA /acetonitrile-water(80:20,v/v) (B); flow rate: 1 ml/min, det.=220 nm; b ESI-MS7.5) contained 5 mM substrate, various inhibitor concentrations (10-100 M) and 3 mMCaCl 2 . The enzyme concentrations used were 0.2 µM m-calpain. Data were analyzed by theMicroCal Origin data analysis software to determine the initial slope of fluorescencechange. First, all peptides were studied at 100 M concentration. Peptides showinginhibition were examined at lower concentration and the K i and IC 50 values weredetermined (Table 1).Four peptides, Ac-TPLaGlySPPPS-NH 2 , H-TPLaGlySPPPSPR-NH 2 , Ac-TSLaGly-SPPPS-NH 2 and Ac-TWLaGlySPPPS-NH 2 , inhibited the m-calpain at 100 Mconcentration. The short peptides; Ac-aGlySPPPS-NH 2 and Ac-TPLaGlySP-NH 2 , had noinhibitory effect. It seems that a number of amino acid residues at the C- and N-terminal arenecessary for the inhibition. The data show that the Leu residue is highly important; thederivatives with other amino acid tested in the P 2 position did not exhibit inhibition.Contrarily, the amino acid in position P 3 might have no influence on the inhibitory activity,although only the best three amino acids in the preference matrix [3] were tested.In summary, new and effective azapeptide based calpain inhibitors were prepared andcharacterized. It turned out that the presence of amino acid residues at the C- and N-terminal could increase the inhibition, and also that the Leu residue in the P 2 position isimportant.AcknowledgmentsThis study was supported by grants: OTKA K-68285 and GVOP-3.2.1-2004-04-0352/3.0. Bánóczi, Z.acknowledges the support of Bolyai János Scholarship.References1. Huang, Y., Wang, K.K.W. Trends Mol. Med. 7, 355-362 (2001).2. Wells, G.J., Bihovsky, R. Exp. Opin. Ther. Patents 8, 1707-1727 (1998).3. Tompa, P., et al. J. Biol. Chem. 279, 20775-20785 (2004).607