WF14865A and B, novel cathepsins B and L inhibitors, were produced and isolated separately from the culture mycelium of a fungal strain Aphanoascus fulvescens No. 14865. Spectroscopic analysis revealed that both WF 14865A and B were composed of trans-epoxysuccinyl moieties, 1-H-imidazole-2-ylamine, and isoleucine or leucine. These compounds inhibited human cathepsins B and L with IC50 values in the range of 8.4-72 nM in vitro. Though their in vitro properties were typical as trans-epoxysuccmyl type inhibitors, they exerted strong bone resorption inhibitory effects in low-calcium-diet-fed mouse model at 3.2-10mg/kg.
In the course of our screening for inhibitors of sphingosine kinase, we found an active compound from a culture broth of a discomycete, Trichopezizella barbata SANK 25395. The structure of the compound, named F-12509A, was elucidated by a combination of spectroscopic analyses, to be a new sesquiterpene quinone consisting of a drimane moiety and a dihydroxybenzoquinone. Enzyme kinetic analyses showed that F-12509A inhibits sphingosine kinase activity in a competitive manner with respect to sphingosine, with a Ki value of 18 μM.
A potent sigma (σ) receptor ligand was isolated from the culture broth of Streptomyces longispororuber #525. The active compound was identified to be (2R-trans)-2-butyl-5-heptylpyrrolidine by spectroscopic and chemical studies. The compound exhibited high affinity and selectivity for σ receptors. The IC50 values toward σ1, σ2 and dopamine D2 receptors were 2.0, 22.7 and more than 40, 000 nM, respectively. Its (2S-trans)- and (±)-cis-isomers, both synthesized, were also found to be high affinity σ ligands.
A novel bioactive macrolide, IB-96212 has been isolated from the fermentation broth of a marine actinomycete, L-25-ES25-008. The strain belongs to the genus Micromonospora. The macrolide showed a very strong cytotoxic activity against P-388, and lower but significant activity against A-549, HT-29, and MEL-28 cell lines. We describe the isolation, taxonomy and fermentation of the producing strain as well as the isolation of IB-96212.
IB-96212, is a new member of spiroketal containing macrolide class of fermentationderived natural products isolated from mycelial extracts of Micromonospora sp. The structure consists of a new aglycone which possesses a 26-membered macrolide ring system and of one deoxy sugar identified as L-rhodinose, this structure represents the first reported spiroketal macrolide natural product related to other macrolides, such as oligomycins, dunaimycins, citovaricin, rutamycin and ossamycin.
The role of two thioesterase genes in the premature release of polyketide synthase intermediates during rifamycin biosynthesis in the Amycolatopsis mediterranei S699 strain was investigated. Creation of an in-frame deletion in the rifR gene led to a 30-60% decrease in the production of both rifamycin B by the S699 strain or a series of tetra- to decaketide shunt products of polyketide chain assembly by the rifF strain. Since a similar percentage decrease was seen in both genetic backgrounds, we conclude that the RifR thioesterase 2 is not involved in premature release of the carbon chain assembly intermediates. Similarly, fusion of the Saccharopolyspora erythraea DEB S3 thioesterase 1 domain to the C-terminus of the RifE PKS subunit did not result in a noticeable increase in the amount of the undecaketide intermediate formed nor in the amounts of the tetra- to decaketide shunt products. Hence, premature release of the carbon chain assembly intermediates is an unusual property of the Rif PKS itself.
A new member of the angucycline family, vineomycin C (3), together with four known metabolites saquayamycin Al (1), A-7884 (2), rabelomycin (5) and xanthomegnin (6) were isolated from microbial extracts. The structures were determined by ID and 2D NMR techniques and chemical degradation. Compounds 1-3 and 5 were isolated from a fermentation of Streptomyces sp., while 6 was isolated from a fungal fermentation extract. All five compounds have shown potent inhibitory activity in the inducible nitric oxide synthase (iNOS) assay.
The gene cluster encoding the deoxyoleandolide polyketide synthase (OlePKS) was isolated from the oleandomycin producing strain Streptomyces antibioticus. Sequencing of the first two genes encoding OlePKS, together with the previously identified third gene revealed an overall genetic and protein architecture similar to that of the erythromycin gene cluster encoding the 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea. When the entire OlePKS (10, 487 amino acids) was expressed in the heterologous host Streptomyces lividans, it produced 8, 8a-deoxyoleandolide, an aglycone precursor of oleandomycin. The role of the P-450 monooxygenase, OleP, in oleandomycin biosynthesis was also examined in vivo by co-expression with DEBS in S. lividans. The production of 8, 8adihydroxy-6-deoxyerythronolide B and other derivatives indicates that OleP is involved in the epoxidation pathway of oleandomycin biosynthesis. Since there are currently no genetic systems available for manipulation of the natural oleandomycin producing strain, the heterologous expression system reported here provides a useful tool for studying this important macrolide antibiotic.
By sequestering cytosolic calcineurin into a molecular complex with cyclophilin and its consequent T-cell dysfunction, some cyclosporins, such as CsA and FR901459 ([Thr2-Leu5-Leu10]-CsA), display potent immunosuppressive activity. Independently on this property, cyclosporins may display one or more other biological activities mediated by interaction with cell surface glycoproteins. Several cyclosporins inhibit the function of human MDR1-encoded P-glycoprotein (Pgp), a flippase known to cause cancer multidrug resistance, but also expressed by some normal immunocompetent cells and by normal epithelial cells which control drug bioavailability in vivo. CsA is known to be a potent Pgp inhibitor with a 3.2 μM IC50 in an assay where the most potent derivative SDZ PSC 833 gives a 0.49 μM IC50. FR901459 is now shown to be a good Pgp inhibitor, being 2-fold weaker only (IC50 of 6μM) than CsA. Some cyclosporins may also inhibit the function of the human FPR 1-encoded formyl peptide receptor (FPR), a chemotactic receptor whose absence is known to impair antibacterial immunity. Yet this inhibition is very weak for all, but one of them, CsH, whose 0.15μM IC50 makes it a much more potent FPR inhibitor than CsA (IC50 >10 μM in the same assay). FR901459 is now shown to be a very potent inhibitor of FPR function (IC50 of 0.6 μM). Since CsH shows little Pgpinhibitory activity and has no known immunosuppressive activity, FR901459 displays a unique pharmacological profile: like CsA, it inhibits T-cell function; less than CsA, it can inhibit Pgp function on selected leukocyte subsets and on epithelial barriers known to control drug bioavailability; however, much more efficiently than CsA, it can inhibit the FPR function, a receptor involved in some leukocytic inflammatory responses to chemotactic pep tides.
Current Japanese clinical practice involves the usage of large amounts of new macrolides such as clarithromycin and roxithromycin for the treatment of diffuse panbronchiolitis, Helicobacter pylori and Mycobacterium avium complex infections. In this study, the phenotypes, genotypes, and macrolide resistance mechanisms of macrolide-inactivating Escherichia coli recovered in Japan from 1996 to 1997, were investigated. The isolation rate of erythromycin A highly-resistant E. coli (MIC ≥ 1600μg/ml) in Japan slightly increased from 0.5% in 1986 to 1.2% in 1997. In six macrolide-resistant strains, recovered from the strains collected for this study during 1996 to 1997, the inactivation of macrolide could be detected with or without added ATP in the assay system. The appearance of erythromycin A-inactivating enzyme independent of ATP was novel from Japanese isolates, and the 1H NMR spectra of oleandomycin hydrolyzed by the three ATP-independent isolates were examined. It was clearly shown that the lactone ring at the position of C-13 was cleaved as 13-H signal in aglycon of oleandomycin upper shifted. These results suggested the first detection of macrolide-lactone ring-hydrolase from clinical isolates in Japan. These results suggested the first detection of an ATP-independent macrolide-hydrolyzing enzyme from Japanese clinical isolates. Substrate specificity of the macrolide-hydrolyzing enzyme was determined with twelve macrolides including the newer members of this group and it was found that not only erythromycin A but also the new macrolides, such as clarithromycin, roxithromycin, and azithromycin were inactivated. The NMR data, broad spectrum of activity, and independence of co-enzyme supported our naming of the enzyme as a macrolide esterase. PCR methodology was employed to detect an ereB-like gene from the 3 isolates producing macrolide esterase, and one of these was subsequently shown to contain both ereB-like, and emB-like genes. It was also clearly shown that the other three isolates, which inactivated macrolide in the presence of ATP, had an mphA-like gene.
The structure of glomosporin, an antifungal antibiotic, was elucidated by NMR and MS spectroscopic studies. Glomosporin is a novel cyclic depsipeptide with an amino acid sequence Ser-Ala-Asp-Asn-Asn-Ser-Thr, and a 3, 4-dihydroxy-4-methylhexadecanoic acid side chain.
Micacocidin (3), a Zn-free derivative of micacocidin A (1), was prepared to evaluate its antimicrobial activity in comparison with 1 and to obtain a starting material for chemical modification of 1. The structure of 3, quite unlike those of any previously known antimicrobial agents, was elucidated by 1-D and 2-D homonuclear and heteronuclear NMR and mass spectroscopy. Micacocidin (3) thus prepared exhibited weak or no antibacterial activity except against Mycoplasma species, i.e.3 showed stronger activity than 1. It is noteworthy that 3 displayed high activity against fungi such as Candida, Aspergillus and Trichophyton species.
Pironetin (1) and demethylpironetin (2) are potent inhibitors of tubulin assembly. They arrested the mammalian cell cycle in M-phase and showed antitumor activity against a murine tumor cell line, P388 leukemia, transplanted in mice. To investigate the chemical and biological properties of 1, we synthesized several derivatives and investigated the structure-activity relationships. All synthesized derivatives decreased biological activities, such as inhibition of cell cycle progression, and disruption of the microtubule network in situ. The most drastic decrease was observed in 6, 8 and 10. These results suggested that α, β-unsaturated lactone, chirality at the 7-position bearing a hydroxyl group and the terminal portion of the alkyl chain are important for microtubule inhibitory activity of pironetins.