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15-Deoxyspergualin (DSG) inhibited growth of mouse EL-4 lymphoma cells with an IC50 0.02 microg/ml. Even though the cells were treated with DSG for only 4 hours and then washed, the antiproliferative effect lasted long with an IC50 0.4 microg/ml. DSG has spermidine and guanidine moieties in its structure. One decomposed element containing guanidine moiety inhibited the growth at higher doses than DSG, but the effect did not last long unlike DSG. While another element containing spermidine moiety did not affect the growth, it diminished the long-lasting antiproliferative effect of DSG by pretreatment of the cells. Pretreatment with polyamines such as putrescine, spermidine, and spermine also diminished the effect of DSG. Furthermore, N-alkylation of spermidine moiety in DSG abolished the antiproliferative effect. These results suggested that DSG binds to the cells through its spermidine moiety and exerts its long-lasting antiproliferative effect.

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IC202A, a new immunosuppressive compound, was isolated from the culture filtrate of Streptoalloteichus sp. 1454-19. It showed a suppressive effect on mixed lymphocyte culture reaction with an IC50 value of 3.6 microg/ml and mitogen induced lymphocyte blastogenesis in vitro.

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IC202A (1) was isolated from the culture filtrate of Streptoalloteichus sp. 1454-19. The structure of 1 was determined by spectral analysis including a variety of two-dimentional NMR and FAB-MS experiments. IC202A is a ferrioxamine-related compound containing a butylidene N-oxide function.

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IC202B (1) and C (2) were isolated from the culture filtrate of Streptoalloteichus sp. 1454-19. The structures were elucidated by various NMR spectral analyses including 1H-15N HMBC and FAB-MS experiments. IC202B and C showed immunosuppressive activity on a mixed lymphocyte culture reaction at the same IC50 value of 1.6 microg/ml.

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In order to investigate systematically the substrate or subsite specificity of two sperm proteases, acrosin and spermosin (a novel trypsin-like protease) of the ascidian, Halocynthia roretzi, the effects of peptidyl-argininals on the purified enzymes as well as on fertilization were examined. Among four benzyloxycarbonyl (Z)-Leu-X-argininals (X = Pro, Leu, Ser, and Gly), Z-Leu-Pro-argininal showed the strongest inhibition toward the spermosin activity. On the P3 site specificity, Val-Pro-argininal derivatives showed a stronger inhibition than a Leu-Pro-argininal derivative, suggesting the preference of Val rather than Leu residue at the P3 position. Similar results were obtained by analyzing the hydrolyzing activity of the fluorogenic peptide substrates: it hydrolyzed Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide (MCA) most efficiently, and Boc-Asp(O-benzyl)-Pro-Arg-MCA was the next best substrate, but Gly-Pro-Arg (or Lys)-MCAs were hardly hydrolyzed. On the other hand, acrosin was found to prefer Leu or Pro residue rather than Gly or Ser residue at the P2 position as revealed by comparing the Ki values of peptidyl-argininals. Detailed kinetic analysis on the inhibitory abilities of peptidyl-argininals toward the purified enzymes and the ascidian fertilization suggested that both acrosin and spermosin are involved in ascidian fertilization.

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Several pyrrolidine-containing analogues of poststatin were synthesized and examined for their inhibitory activity against prolyl endopeptidase and cathepsin B in vitro. Replacement of the postine residue with 2-oxo-2-(2-pyrrolidinyl)acetic acid increased the selectivity and inhibitory activity against prolyl endopeptidase. Benzyloxycarbonyl-L-phenylalanyl-(S)-2-oxo-2- (2-pyrrolidinyl)acetyl-D-phenylalanine was about 46 times as active to propyl endopeptidase as natural poststatin.

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The structure of cytostatin was determined to be 5,6-dihydro-5-methyl-6-(6-hydroxy,1,5-dimethyl-4-phosphonooxy-7,9, 11-tridecatrienyl)-2H-pyran-2-one sodium salt on the basis of physico-chemical properties and NMR studies.

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In our search for inhibitors of cell adhesion to components of extracellular matrix (ECM), fibronectin, laminin and collagen type IV, we succeeded in finding a novel cyclic hexadepsipeptide antibiotic, named IC101, which was isolated from cultured mycelium of Streptomyces albulus MJ202-72F3. It was purified by centrifugal partition chromatography, preparative reverse phase HPLC and Sephadex LH-20 and was obtained as a white powder. IC101 strongly inhibited cell adhesion to ECM components, suppressed immune responses in vitro and in vivo, and exhibited antimicrobial activity on Gram-positive bacteria.

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The biosynthesis of delaminomycin A, produced by Streptomyces albulus MJ202-72F3, was investigated by feeding 13C-labeled compounds followed by 13C NMR analyses. The results indicate that delaminomycin A is derived from six acetate units, five propionate units and one glycine unit.

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Among various protease inhibitors, chymostatin (an inhibitor of sperm chymotrypsin-like protease) strongly inhibited the binding of sperm to the vitelline coat of glycerinated eggs of the ascidian Halocynthia roretzi, whereas leupeptin (an inhibitor for sperm acrosin), antipain, and soybean trypsin inhibitor had no significant inhibitory effects. Dansyl-Val-Pro-argininal (an inhibitor of the sperm trypsin-like protease, spermosin) had an inhibitory effect on the binding of sperm that was much smaller than its effects on fertilization. Since the sperm chymotrypsin-like protease that is involved in ascidian fertilization has been identified as a proteasome (multicatalytic proteinase complex), we tested the effects of several peptidyl argininals, inhibitors of the activities of proteasomes, on this binding process. The ranking of the inhibitory effects of these compounds on the binding of sperm was the same as that of their effects on the chymotrypsin-like activity of the proteasome, reported previously. The potent inhibitors of binding used in these studies had no or minimal effects on sperm motility. These results suggest that a sperm chymotrypsin-like protease (most probably the chymotrypsin-like protease in the proteasome) plays a key role in binding of sperm to the vitelline coat of the ascidian egg.

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Delaminomycins A, B, C and their derivatives were prepared and investigated biological activities of them. Among these compounds, spiro compounds (A2, B2 and C2) showed stronger inhibitory activity than natural products (A1, B1 and C1) on B16 melanoma cells adhesion assay and Con A-induced proliferation of murine splenic lymphocytes assay. In MLCR and antimicrobial assay, however, A1, B1 and C1 showed more potent inhibitory activity than spiro compounds (A2, B2 and C2). On the other hand, as to C-5' substituents of pyrrolidine ring, the order of inhibitory activity was R = OH > R = OCH3 > R = H on Con A-induced proliferation of murine splenic lymphocytes assay. In MLCR and antimicrobial assay, however, the order of inhibitory activities were R = H > R = OCH3 > R = OH. Inhibitory activities of A4 which was lacked pyrrolidine ring were reduced on B16 melanoma cells adhesion assay and on cytotoxicity against tumor cells in vitro in comparison with those of A1.

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Delaminomycins, novel extracellular matrix receptor antagonists, have been isolated from a culture broth of Streptomyces albulus MJ202-72F3. The structure of delaminomycin A was determined to be 3-[[2-[(3E,5E)-2,8-dihydroxy-7-methyl-3,5-decadienyl]- 1,6,8- trimethyl-1,2,4a,5,6,7,8,8a-octahydro-1-naphthyl]carbonyl]-5- hydroxypyrrolidine-2,4-dione by analyses of spectral properties and chemical studies.

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A protease involved in oocyte maturation of a starfish, Asterina pectinifera, was explored. Trypsin-like and chymotrypsin-like activities of the 650-kDa protease in oocyte extract were revealed to increase more than twice under the influence of 1-methyladenine before germinal vesicle breakdown (GVBD) during maturation. The inhibitory potencies of leupeptin and its five analogs against the chymotrypsin-like activity, but not the trypsin-like activity, of this protease was well in accord with those against GVBD (Takagi Sawada et al. (1989). Dev. Biol. 133, 609-612). These results indicate that the chymotrypsin-like activity of the 650-kDa protease (most probably 20 S proteasome) plays a key role in starfish oocyte maturation.

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Inhibition of subsite-substituted leupeptin analogs, potent trypsin inhibitors, on 1-methyladenine-induced germinal vesicle breakdown was investigated in a starfish, Asterina pectinifera. Of benzyloxycarbonyl(Z)-Leu-P2-argininals, the analog with Ser at P2 residue was the strongest inhibitor, and those with Pro, Leu greater than Thr greater than Gly were followed in this order. In Z-P3-Ser-argininals, ranking of the inhibitory ability was as follows: Phe greater than Leu much greater than Pro greater than Ala at P3 residue. Among 11 analogs synthesized, Z-Phe-Ser-argininal showed the strongest inhibition. The inhibitory potency of the analog was 100-fold stronger than that of leupeptin (acetyl-Leu-Leu-argininal). Thus, trypsin-like enzyme possessing a narrow subsite specificity participates in oocyte maturation in the starfish.

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Thirty analogues of leupeptin were synthesized and examined for their inhibitory activities against trypsin, papain, plasmin, kallikrein, thrombin and urokinase in vitro. Benzoyl- and alpha-naphthalenesulfonyl-L-leucyl-L-argininal were 8 times more inhibitory to papain, benzyloxycarbonyl-L-pyroglutamyl-L-leucyl-L-argininal 10 times more to trypsin and plasmin, and DL-2-pipecolyl-L-leucyl-L-argininal 25 times more to kallikrein than leupeptin. Against urokinase, only L-pyroglutamyl-L-leucyl-L-argininal exhibited a potent inhibitory activity. alpha-Naphthalensulfonyl-, dansyl- and benzyloxycarbonyl-(2S,3R)-3-amino-2-hydroxy-4-phenylbutyryl-L-leucyl-L- argininal were inhibitory to thrombin.

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