A series of dipeptidyl and tripeptidyl alpha-keto esters, alpha-keto amides, and alpha-keto acids having leucine in the P2 position were synthesized and evaluated as inhibitors for the cysteine proteases calpain I, calpain II, cathepsin B, and papain. In general, peptidyl alpha-keto acids were more inhibitory toward calpain I and II than alpha-keto amides, which in turn were more effective than alpha-keto esters. In the series Z-Leu-AA-COOEt, the inhibitory potency decreased in the order: Met (lowest KI) > Nva > Phe > 4-Cl-Phe > Abu > Nle (highest KI) with calpain I, while almost the reverse order was observed for calpain II. Extending the dipeptide alpha-keto ester to a tripeptide alpha-keto ester yielded significant enhancement in the inhibitory potency toward cathepsin B, but smaller changes toward the calpains. Changing the ester group in the alpha-keto esters did not substantially decrease KI values for calpain I and calpain II. N-Monosubstituted alpha-keto amides were better inhibitors than the corresponding alpha-keto esters. alpha-Keto amides with hydrophobic alkyl groups or alkyl groups with an attached phenyl group had the lower KI values. N,N-Disubstituted alpha-keto amides were much less potent inhibitors than the corresponding N-monosubstituted peptide alpha-keto amides. The peptide alpha-keto acid Z-Leu-Phe-COOH was the best inhibitor for calpain I (KI = 0.0085 microM) and calpain II (KI = 0.0057 microM) discovered in this study. It is likely that the inhibitors are transition-state analogs and form tetrahedral adducts with the active site cysteine of cysteine proteases and form hydrogen bonds with the active site histidine and possibly another hydrogen bond donor in the case of monosubstituted amides. Several inhibitors prevented spectrin degradation in a platelet membrane permeability assay and may be useful for the treatment of diseases which involve neurodegeneration.

A series of new dipeptidyl alpha-keto amides of the general structure R1-L-Leu-D,L-AA-CONH-R2 were synthesized and evaluated as inhibitors for the cysteine proteases calpain I, calpain II, and cathepsin B. They combine 10 different N-protecting groups (R1), 3 amino acids residues in P1 (AA), and 44 distinct substituents on the alpha-keto amide nitrogen (R2). In general, calpain II was more sensitive to these inhibitors than calpain I, with a large number of inhibitors displaying dissociation constants (Ki) in the 10-100 nM range. Calpain I was also effectively inhibited, but very low Ki values were observed with a smaller number of inhibitors than with calpain II. Cathepsin B was weakly inhibited by most compounds in this study. The best inhibitors for calpain II were Z-Leu-Abu-CONH-CH2-CHOH-C6H5 (Ki = 15 nM), Z-Leu-Abu-CONH-CH2-2-pyridyl (Ki = 17 nM), and Z-Leu-Abu-CONH-CH2-C6H3(3,5(OMe)2) (Ki = 22 nM). The best calpain I inhibitor in this study was Z-Leu-Nva-CONH-CH2-2-pyridyl (Ki = 19 nM). The peptide alpha-keto amide Z-Leu-Abu-CONH-(CH2)2-3-indolyl was the best inhibitor for cathepsin B (Ki = 31 nM). Some compounds acted as specific calpain inhibitors, with comparable activity on both calpains I and II and a lack of activity on cathepsin B (e.g., 40, 42, 48, 70). Others were specific inhibitors for calpain I (e.g., 73) or calpain II (e.g., 18, 19, 33, 35, 56). Such inhibitors may be useful in elucidating the physiological and pathological events involving these proteases and may become possible therapeutic agents.

Changes in neocortex and striatum were characterized over time following focal ischemia to the brain. Rats were subjected to permanent middle cerebral artery occlusion (MCA-O) and sacrificed 1, 3, 6, 12, or 24 h later. The affected tissue was processed for tetrazolium chloride (TTC) and cresyl violet staining, as well as for Western blots to detect calpain-induced spectrin proteolysis. Significant changes in cell size and spectrin breakdown occurred within the first hour of occlusion, with further, dramatic changes in these two early markers continuing over time. Initial evidence of cell loss was noted at 1 h postocclusion in the striatum and at 3 h in the neocortex. However, even in the center of the most affected portion of the neocortex, the majority of cells appeared to be intact through 6 h. By this time, a significant TTC-defined infarct also emerged. These quantitative data indicate that calpain-induced proteolysis occurs very soon after the ischemic insult, is correlated with earliest changes in cell hypotrophy, and precedes or occurs in tandem with evidence of significant cell loss. They also demonstrate that, while some cell loss occurs earlier than previously believed, the majority of cells remains morphologically intact well beyond what is typically thought to be the window of opportunity for intervention. The results thus raise the question of how long after the ischemic event pharmaceutic intervention might be employed to salvage substantial numbers of neurons.