Naoki Higuchi

Synthesis of a new cell penetrating calpain inhibitor (calpeptin)

N-terminal of Leu-norleucinal or Leu-methioninal was modified to obtain a cell penetrative peptide inhibitor against calpain. Benzyloxycarbonyl (Z) derivatives had less active against papain than phenylbutyryl derivatives and leupeptin. Z-Leu-nLeu-H (calpeptin) was more sensitive to calpain I than Z-Leu-Met-H and leupeptin. Calpeptin was most potent among synthesized inhibitors in terms of preventing the Ca2+-ionophore induced degradation of actin binding protein and P235 in intact platelets. After 30 min incubation with intact platelets, calpeptin completely abolished calpain activity in platelets but no effect was observed in case of leupeptin. Calpeptin also inhibited 20K phosphorylation in platelets stimulated by thrombin, ionomycin or collagen. Thus calpeptin was found to be a useful cell-penetrative calpain inhibitor.

Synthesis and Inhibitory Activity of Acyl-Peptidyl-Pyrrolidine Derivatives Toward Post-Proline Cleaving Enzyme; A Study of Subsite Specificity

Several pyrrolidine derivatives have been synthesized and examined for their inhibitory activity on post-proline cleaving enzymes from Flavobacterium meningosepticum and bovine brain. Almost all the compounds tested in this study inhibited the activity of both enzymes at low IC50 values (from nM to microM) but a specificity difference was observed with alkylacyl-peptidyl-pyrrolidine derivatives which strongly inhibited only the bacterial enzyme. The most effective inhibitors have a proline residue on their P2 sites and a substituted or unsubstituted phenoxybutyryl moiety on their P3 sites. Thus phenoxybutyryl-prolyl-pyrrolidine is the most effective partial structure of the inhibitors. The best inhibitors found were: 4-(4-benzylphenoxy)butyryl-prolyl-pyrrolidine for bacterial enzyme (IC50 1.4 nM) and 4-phenylbutyryl-thioprolyl-pyrrolidine for bovine brain enzyme (IC50 67 nM). In the passive avoidance test, using amnesic rats experimentally induced with scopolamine, the pyrrolidine derivatives which had potent inhibitory activity toward post-proline cleaving enzymes also showed strong anti-amnesic activities at doses of 1-5 mg/kg, i.p.

Synthesis and Inhibitory Activity of Acylpeptidyl-Prolinal Derivatives Toward Post-Proline Cleaving Enzyme as Nootropic Agents

Several prolinal derivatives were synthesized and examined for their inhibitory activity on post-proline cleaving enzymes from Flavobacterium meningosepticum and bovine brain and their possible properties as nootropic agents. Almost all the compounds tested inhibited the activity of both enzymes at low IC50 values of the order of nM, but a specificity difference was observed with alkylacyl-prolinal derivatives which strongly inhibited only the bacterial enzyme. Prolyl-prolinal derivatives were the most effective inhibitors for both enzymes. In the passive avoidance test using amnesic rats experimentally induced with scopolamine, the prolinal derivatives that have potent inhibitory activity toward post-proline cleaving enzymes showed also strong anti-amnesic activities at dose of 10-1000 micrograms/kg, i.p. Some of the compounds showed a bell-shape dose dependency. These results suggest that the post-proline cleaving enzymes play an important role in the regulation of learning and memory consolidation in the brain and inhibitors of these enzymes are suggested as possible candidates for nootropic agents, particularly for an anti-amnesic drug.

Purification and Structure of Novel Cysteine Proteinase Inhibitors, Staccopins P1 and P2, from Staphylococcus tanabeensis

A new type of cysteine proteinase inhibitors, staccopin PI and P2, are low molecular weight (lower than 1,000) peptide derivatives isolated first from Staphylococcus sp. Staccopins strongly inhibited only cysteine proteinases like calpain and papain, but were not active against serine proteinases like trypsin and chymotrypsin. The purification procedures included HP-20 adsorption chromatography, DEAE-cellulose, CM-cellulose, Sephadex LH-20 column chromatography, and high-performance liquid chromatography. The yields were 49 mg and 18 mg from 20 liters each of culture fluid. These proteinase inhibitors are pentapeptides containing valine and phenylalaninal for staccopin PI or tyrosinal for staccopin P2, and the N-terminals of both peptides are free. Thus the structures are H-valyl-valyl-valyl-valyl-phenylalaninal for staccopin PI and H-valyl-valyl-valyl- valyl-tyrosinal for staccopin P2. The amino acid sequence-inhibitory activity relationships of staccopins and other low molecular weight peptide cysteine ...