Hiroyuki Suda

An endothelin receptor (ETA) antagonist isolated from Streptomyces misakiensis.

A competitive endothelin (ET) antagonist, BE-18257B, was isolated from the fermentation products of Streptomyces misakiensis. It is a novel cyclic pentapeptide, cyclo(-D-Glu-L-Ala-allo-D-Ile-L-Leu-D-Trp-), and binds to ETA receptors (ET-1 selective) in cardiovascular tissues, but not to ETB receptors (equally sensitive to isopeptides of ET family) in kidney, adrenal gland and cerebellum tissues. BE-18257B also antagonizes ET-1-induced vasoconstriction in rabbit iliac artery and pressor action in rats. Thus it is a selective ETA antagonist and should provide a valuable tool for elucidation of the pharmacological and pathophysiological roles of ET-1.

Inhibition of aminopeptidase B and leucine aminopeptidase by bestatin and its stereoisomer.

Abstract Bestatin, [(2 S ,3 R )-3-amino-2-hydroxy-4-phenylbutanoyl]-( S )-leucine, inhibited aminopeptidase B and leucine aminopeptidase in a competitive manner and their K i values were calculated to be 6 × 10 −8 and 2 × 10 −8 M , respectively. Among all stereoisomers of bestatin synthesized, those which have a (2 S )-configuration in the 3-amino-2-hydroxy-4-phenylbutanoyl moiety showed marked inhibition against aminopeptidase B and leucine aminopeptidase compared with the other isomers which have (2 R )-configuration. One of the isomers, [(2 S ,3 S )-3-amino-2-hydroxy-4-phenylbutanoyl]-( R )-leucine, showed somewhat stronger activity against aminopeptidase B than bestatin. Aminopeptidase B appears to be a metallo-exopeptidase. It is proposed that bestatin and its active isomers are effective due to a mechanism other than a chelating action at the active center.

In vitro biological profile of a highly potent novel endothelin (ET) antagonist BQ-123 selective for the ETA receptor

Summary: The novel endothelin (ET) receptor antagonists BE‐18257A and BE‐18257B were isolated from the fermentation products of Streptomyces misakiensis. The above‐mentioned compounds inhibited [125I]ET‐1 binding to ETA receptors (selective for ET‐1) on porcine aortic vascular smooth muscle cells (VSMCs) with IC50 values of 1.4 and 0.47 μM, respectively. [125I]ET‐1 binding to ETB receptors (nonselective to ET isopeptides) in cerebellar membranes was not inhibited by either of these compounds even at 100 μM. The synthesized analogue BQ‐123 induced extremely potent inhibition of [125I]ET‐1 binding to ETA receptors (IC50 of 7.3 nM), but it barely inhibited [125I]ET‐1 binding to ETB receptors (IC50 of 18 μM) and binding of various other peptides to their receptors. BQ‐123 shifted the concentration‐response curve for ET‐1 toward the right in porcine isolated coronary arteries, indicative of competitive antagonism for the ETA receptor. However, there was a small amount of BQ‐123‐insensitive vasocontraction that paralleled the incomplete inhibition of [125I]ET‐1 binding in the membrane of the vascular smooth muscle layer. These data suggest that the artery contracts via both ETA and ETB receptors and that BQ‐123 selectively inhibits ETA‐mediated contraction. Furthermore, BQ‐123 revealed large tissue and species differences in the distribution of ETA receptors. Thus, the potent ETA antagonist BQ‐123 should be useful in clarifying the (patho)physiological roles of ETA receptors.

Aminopeptidase activities on the surface of mammalian cells.

Activities of hydrolytic enzymes on the surface of monkey kidney, canine kidney, L. FM3A and various tumor cells were determined and compared with those in the cell homogenate. Although aminopeptidase (EC 3.4.11.-) activities were always detected on the surface membrane in mammalian cells, trypsin, chymotrypsin and elastase activities were not detected while slight glycosidase activity was detected in a suspension of cultured cells. The activities of alanine-, leucine-, methionine- and phenylalanine-aminopeptidases were rather high but aminopeptidase A, proline-, valine-, glycyl propline dipeptidyl-and glycyl propyl leucine-tripeptidyl-aminopeptidases showed relatively low activities. Aminopeptidase activity was also demonstrated in the isolated membrane fractions. The specific activities of enzymes in these membrane fractions were not significantly greater than in cell homogenate so it was concluded that these enzyme activities were rather loosely bound to the cell membrane. Further evidence for the localization of the aminopeptidase activities on the cell surface was obtained by using glass-bead-bound substrate and detecting the release of the terminal residues. When bestatin, a specific inhibitor against aminopeptidase B and leucine aminopeptidase, was included in the assay system for the enzyme activities on the cell surface, the enzymes were commonly inhibited in all types of cells.

Studies on inhibitory effect of phosphoramidon and its analogs on thermolysin.

Abstract Phosphoramidon, N -(α- l -rhamnopyranosyloxyhydroxyphosphinyl)- l -leucyl- l -tryptophan, and its analog, N -phosphoryl- l -leucyl- l -tryptophan, inhibited thermolysin in a competitive manner and K i values were calculated to be 2.8 × 10 −8 and 2.0 × 10 −9 m , respectively. The l -rhamnose moiety in phosphoramidon was suggested to be not involved in inhibition of thermolysin. A phosphoramidon analog containing histidine instead of tryptophan showed weaker inhibition. Spectrophotometric titration based on difference ultraviolet absorption spectra of the enzyme-inhibitor complex showed equimolar binding of the inhibitor to the enzyme.

BE-31405, a new antifungal antibiotic produced by Penicillium minioluteum. I. Description of producing organism, fermentation, isolation, physico-chemical and biological properties.

A new antifungal antibiotic, BE-31405, was isolated from the culture broth of a fungal strain, Penicillium minioluteum F31405. BE-31405 was isolated by adsorption on high porous polymer resin (Diaion HP-20), followed by solvent extraction, precipitation and crystallization. BE-31405 showed potent growth inhibitory activity against pathogenic fungal strains such as Candida albicans, Candida glabrata and Cryptococcus neoformans, but did not show cytotoxic activity against mammalian cells such as P388 mouse leukemia. The mechanism studies indicated that BE-31405 inhibited the protein synthesis of C. albicans but not of mammalian cells.

Purification and properties of N-formylmethionine aminopeptidase from rat liver

A specific enzyme for the liberation of N-terminal N-formylmethionine from N-formylmethionyl peptides was purified 4750-fold from rat liver by successive applications of (NH4)SO4 precipitation, DEAE-cellulose, N-formylbestatin-AH-Sepharose 4B and AH-Sepharose 4B chromatography followed by Sepharose CL-6B gel filtration. The molecular weight was determined by gel filtration on Sepharose CL-6B as 290 000 +/- 5000. This was suggested to be a tetramer consisting of a subunit which was shown by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis to have a 72 000 +/- 2000 molecular weight. The optimum pH of the enzyme was 7.8. Cd2+ and Hg2+ were highly toxic to the enzyme. Michaelis constants of N-formylmethionyl leucine and N-formylmethionine beta-naphthylamide were 0.03 and 0.2 mM, respectively.

Release of a plasma membrane-bound triaminopeptidase activity from mammalian cells by thermolysin.

Abstract Aminopeptidase and other enzyme activities on cellular surface were determined in the presence and absence of endopeptidases. Gly-Pro-Leu-AP activity was specifically released into the medium by thermolysin treatment, while the other activities retained on the cellular surface were markedly decreased. A similar phenomenon was also observed in rat liver membrane, mouse FM3A, spleen lymphocyte and other cells. Structural rearrangement of some protein components in the cell plasma membrane was suggested.

Total synthesis of a new topoisomerase II inhibitor BE 10988

Abstract The total synthesis of a new topoisomerase II inhibitor BE 10988 is described. And the structure was unambiguously established by this synthesis.

New cytotoxic agents, BE-52440A and B, produced by a streptomycete

New cytotoxic substances, designated BE-52440A and B, were isolated from the mycelium of Streptomyces sp. A52440. The active principles were extracted from the mycelium by methanol and purified by silica gel column chromatography. BE-52440A and B exhibited cytotoxic activity against murine and human tumor cell lines.