Biosynthesis of 2'-deoxycoformycin: evidence for ring expansion of the adenine moiety of adenosine to a tetrahydroimidazo[4,5-d][1,3]diazepine system.

2'-Deoxycoformycin (2'-dCF), a nucleoside antitumor agent produced in trace quantities by Streptomyces antibioticus, has been shown in earlier work to originate from the intact carbon-nitrogen framework of adenosine. Additional experiments using 13C and two-dimensional Fourier transform NMR techniques, together with radiolabeling studies, identify the C-1 of D-ribose, and not the tetrahydrofolate "C-1 pool", as the source of the C-7 carbon in the aglycon of 2'-dCF. These results show that the adenine portion of adenosine (or a nucleotide thereof) undergoes a unique ring expansion, by insertion of a -CH2- unit between the N-1 and C-6 of the adenine ring, to furnish the 1,3-diazepine portion of 2'-dCF.

Bacterial DNA reactivity of the novel antitumor antibiotics PD 114,759 and PD 115,028 (veractamycins A and B).

The novel antitumor antibiotics PD 114,759 and PD 115,028 were evaluated for their ability to cause repairable DNA damage and the induction of SOS functions in bacterial systems. PD 114,759 and PD 115,028 were preferentially toxic to DNA repair-defective Escherichia coli WP100 uvrA recA in comparison to wild-type E. coli WP2 at concentrations of 10 approximately 30 micrograms/ml in agar diffusion assays. Both compounds were inducers of cell filamentation and prophage lambda (two E. coli SOS functions) at concentrations of 0.1 approximately 1 microgram/ml. In addition, the ability of PD 114,759 and PD 115,028 to retain their filamentation-inducing effects under both aerobic conditions and anaerobic conditions suggests that a bioreductive, rather than an oxygen-requiring, mechanism is involved in the DNA-reactive effects of these agents.

Biological effects of acetomycin. I. Activity against tumor cells in vitro and in vivo.

The antibiotic acetomycin was active in vitro against HCT-8 human colon adenocarcinoma cells (IC50, 1.5 microgram/ml) and L1210 murine leukemia cells (IC50, 2.2 micrograms/ml). Acetomycin also had marked activity in the human tumor stem cell assay, with a 33% overall response rate (less than or equal to 30% survival) against 49 primary tumors. However, acetomycin was inactive in four in vivo tumor assay systems (L1210 and P388 leukemias, B16 melanoma and the MX-1 mammary xenograft system). This lack of in vivo activity may result from metabolic inactivation of acetomycin.

Biological effects of acetomycin. II. Inactivation by esterases in vitro.

Acetomycin has antitumor activity in vitro but not in vivo. HCT-8 human colon adenocarcinoma assays in the presence of a drug metabolizing system (rat liver S9 fraction) demonstrated that liver enzymes inactivated acetomycin. The structure of acetomycin suggested that an esterase could be the key inactivating enzyme. Assays with porcine liver esterase (EC 3.1.1.1) showed that this enzyme rapidly abolishes the activity of acetomycin against HCT-8 cells. The potential utility of acetomycin as an antitumor agent thus depends on finding a means of preventing esterase inactivation.

Antimycotic effects of the novel antitumor agents fostriecin (CI-920), PD 113,270 and PD 113,271.

The novel fermentation products fostriecin and analogs PD 113,270 and PD 113,271 are structurally related polyene lactone phosphates that have antitumor activity in vitro and in vivo. They have no antibacterial effects, but they were inhibitory to yeasts (agar diffusion method) with MICs of 3 approximately 300 micrograms/ml. Fostriecin or its analogs were active vs. 29 of 46 yeast species (11 genera). Ten of 12 cultures of Candida sp. were not sensitive to any of the analogs, while 11 of 14 cultures of Saccharomyces sp. were inhibited by one or more of the agents. Sensitivity patterns were of three types: Twelve cultures were sensitive only to PD 113,270; fostriecin and PD 113,271 (but not PD 113,270) were active vs. 7 cultures; and 9 cultures were sensitive to all three compounds. Dephosphorylation of the compounds resulted in the loss of antimycotic effects. Activity vs. the yeasts was related to studies of uptake and activity against cancer cells.

The Escherichia coli K-12 SOS chromotest agar spot test for simple, rapid detection of genotoxic agents.

The Escherichia coli K-12 SOS chromotest is a colorimetric (beta-galactosidase induction) system for detecting genotoxic chemicals as agents which induce filamentation in response to DNA damage. The chromotest was modified from a liquid suspension assay to a simple, convenient agar spot test, which was performed in the manner of a related colorimetric prophage induction assay (BIA). Chromotest agar dishes yielded optimal results after 16-18 h incubation, presumably because of the agar growth characteristics of tester strain PQ37. Of 44 tested chemicals, nitro aromatics, cytotoxic/antitumor agents, polycyclic hydrocarbons and aflatoxins showed good activity. Alkylating agents such as MNNG and MMS were active only at high concentrations. Compounds active in both the chromotest and BIA were active at 10-100-fold lower concentrations in the chromotest. The chromotest appeared to be less effective than the Salmonella Ames mutagenicity test in the detection of diverse classes of chemical carcinogens. The chromotest may be a useful alternative to the BIA in the study of particular classes of genotoxic compounds.

PD 116,152, a novel phenazine antitumor antibiotic. Discovery, fermentation, culture characterization and biological activity.

A novel phenazine antitumor antibiotic is described, produced by Streptomyces lomondensis subsp. galanosa NRRL 15738. The antibiotic is selectively active versus the bacterium Streptococcus pneumoniae (MIC less than 0.46 microgram/ml); the antitumor activity versus murine P388 leukemia is T/C 149.

Evaluation of galactosyltransferase isoenzyme II in a human colon carcinoma-derived cell line, HCT-8.

Polyacrylamide gel electrophoresis of galactosyltransferase (GT) extracted from a human colon adenocarcinoma cell line, HCT-8, demonstrated the presence of two peaks of activity: a slow-moving peak, referred to as GT-II, and a more anodally migrating peak, designated as GT-I, which was also found for normal human serum. However, if GT solubilized from HCT-8 cells was separated by isoelectric focusing, no unique isoenzymes could be detected. Total GT activity from HCT-8 cells was purified by alpha-lactalbumin-Sepharose affinity chromatography followed by ion exchange chromatography on either DEAE-cellulose or FPLC using a Mono Q anion exchange resin. Three major peaks of activity were resolved from anion exchange chromatography. Electrophoresis of each peak revealed a GT pattern identical with that originally observed for the crude (detergent) solubilized homogenate. No enrichment of either GT-I or GT-II was observed in the three enzyme fractions. The data suggest that GT-II may be an artifactual activity of cancer cells composed of GT-I associated with some contaminating protein.

Effect of phosphate and copper on the fermentation of hydroheptin.

The organism Streptomyces chartreusis IMRU 3962 produced a mixture of the antibiotics hydroheptin and chartreusin in fermentation broth. The addition of increasing levels of phosphate resulted in a corresponding increase in the production of both antibiotics, with maximum yields of 400 to 450 micrograms of chartreusin per ml and 80 to 100 micrograms of hydroheptin per ml at 0.45 to 0.55 M phosphate. Chartreusin was invariably produced at a higher ratio; however, a reversal in ratio to favor hydroheptin was attained when 0.03% copper sulfate was added to the medium, particularly at a 0.2 M KH2PO4 level, with antibiotic yields of 125 micrograms of hydroheptin per ml and 40 micrograms of chartreusin per ml.

Potent antitumor antibiotic complex: PD 114,759, PD 115,028, PD 119,707, and PD 119,193.

Four novel antitumor antibiotics (PD 114,759, PD 115,028, PD 119,707 and PD 119,193) are produced as a complex by a new species of Actinomadura. The proposed name for the culture is Actinomadura verrucosospora subsp. veractimyces ATCC 39363. The antibiotics are extremely bioactive, with MIC values of less than 0.006 ng/ml against several bacteria and ID50 values of 0.003 approximately 0.107 ng/ml against L1210 leukemia cells in vitro. Antitumor activities vs. P388 leukemia in vivo were observed at doses of 0.313, 0.40, and 0.5 micrograms/kg (daily X 5) for PD 119,707, PD 115,028, and PD 114,759, respectively.

2'-Chloropentostatin: discovery, fermentation and biological activity.

2'-Chloropentostatin (2-CP) is a new nucleoside antibiotic produced by Actinomadura sp. ATCC 39365. A selectively sensitive assay organism, Enterococcus faecalis PD 05045 (MIC 0.005 micrograms/ml) was instrumental in the discovery of this compound. 2-CP is a tight-binding inhibitor of adenosine deaminase (Ki = 1.1 X 10(-10) M).

Collagen-production inhibitors evaluated as antitumor agents.

Proline analogues such as cis-4-hydroxy-L-proline (CHP) and L-azetidine-2-carboxylic acid (A2C) were tested for their antitumor activity in tissue culture and in vivo. In culture, CHP specifically inhibited those tumor cells that synthesized basement-membrane collagen. CHP appeared to selectively inhibit collagen biosynthesis with only a slight effect on protein synthesis. Culturing cells on type IV collagen matrix did not alter the antiproliferative effect of CHP. The inhibition of 450.1 mouse mammary tumor cells was fully reversible when cultures were incubated for 6 or 12 hours with 25 micrograms CHP/ml but was irreversible after 24 hours of exposure. Of the proline analogues tested against 450.1 tumor cells, A2C and CHP were the most potent inhibitors of cell growth. These two compounds were therefore tested in vivo using 3 transplantable tumors, all of which synthesized basement-membrane collagen. CHP and A2C were given twice daily to mice for 7 to 10 days at doses ranging from 50 mg/kg (body wt) to 600 mg/kg (body wt) per injection. Both CHP and A2C were completely inactive against the 450.1 mammary tumor and the EHS sarcoma. Both compounds also caused considerable liver toxicity. Against CD8F1 mammary tumors, treatment with maximum tolerated doses of CHP and A2C resulted in a slight but insignificant inhibition of tumor growth. While our studies confirmed previous findings that CHP specifically inhibited those tumor cells that synthesized basement-membrane collagen, CHP and A2C did not appear to be efficacious antitumor agents.

Novel antitumor antibiotics, CI-940 (PD 114,720) and PD 114,721. Taxonomy, fermentation and biological activity.

The novel, broad-spectrum antitumor antibiotics, CI-940 (PD 114,720) and PD 114,721 are components of bioactive compounds produced by a novel actinomycete (ATCC 39366). The organism is unique in that whole cell analysis revealed LL-diaminopimelic acid and major amounts of arabinose. These new antibiotics exhibit potent antitumor activity in vitro and in vivo versus an array of tumors and possess strong antibacterial and antifungal activity. The above antibiotics are produced in 7,570-liter fermentors at yields of 5 approximately 8 micrograms/ml.

Fermentation by a new daunomycin-producing organism, Streptomyces insignis ATCC 31913.

A new organism belonging to the grey series of streptomycetes is described which produces 55 to 75 micrograms of daunomycin per ml in a sparged fermentor. This organism is not taxonomically related to other known daunomycin producers. Its proposed name in Streptomyces insignis ATCC 31913.

Novel antitumor agents CI-920, PD 113,270 and PD 113,271. I. Taxonomy, fermentation and biological properties.

CI-920 (PD 110,161) and two analogues (PD 113,270 and PD 113,271) are novel antitumor compounds produced by a new actinomycete characterized as Streptomyces pulveraceus subsp. fostreus ATCC 31906. The antitumor compounds are predominantly produced during the stationary (idiophase) growth phase of the organism. CI-920 is active versus the murine P388 lymphocytic and L1210 lymphoid leukemia with T/C values of 246 and 207, respectively. This compound has no significant antimicrobial activity.

Epithienamycins-novel beta-lactams related to thienamycin. I. Production and antibacterial activity.

The epithienamycins are cell wall active antibiotics structurally related to N-acetylthienamycin. We have found forty-three isolated of Streptomyces flavogriseus which are capable of producing members of the epithienamycin family. Six major epithienamycin components, and xanthomycin, have been isolated from fermentation broth. Fermentation conditions can be varied to enrich for certain members of the epithienamycin family. All six components show activity in vitro versus a broad spectrum of bacterial species. The weight potencies vary 27 fold from the most active to least active.

Hydroheptin: a water-soluble polyene macrolide. I. Taxonomy, fermentation and isolation.

A soil isolate of Streptomyces, which has been deposited in the culture collection of the Waksman Institute of Microbiology, Rutgers University as IMRU 3962, produces a new heptaene macrolide antifungal antibiotic, hydroheptin. The producing microorganism, which co-produces the antibiotic, chartreusin, has been identified as a strain of Streptomyces chartreusis. Fermentation and bioassay procedures were developed for the production and analysis of hydroheptin and chartreusin. Isolation and purification procedures based on solvent extraction and precipitation of an organic acid yielded a relatively pure product of hydroheptin.

Hydroheptin: a water-soluble polyene macrolide. II. Chemical and biological properties.

Hydroheptin, a new polyene macrolide antifungal antibiotic, is co-produced with the antibiotic, chartreusin, by a strain of Streptomyces chartreusis designated as IMRU 3962 isolated in our laboratory. The unique water-solubility of this antibiotic at neutrality, revealing in aqueous solution molecular dispersion and an ultraviolet-visible absorption spectrum characteristic of an all-trans heptaene chromophore, clearly distinguishes it from all previously-described and naturally-occurring heptaene macrolides. The isolation and identification of the amino sugar, mycosamine (3-amino-3,6-dideoxy-D-mannose), in acid hydrolysates of hydroheptin and the absence of an aromatic amine upon retrograde alkaline dealdolization of the molecule certainly characterize the antibiotic as a member of the non-aromatic heptaene macrolide group. Chromatographic and countercurrent distribution studies likewise support its novelty. With little or no demonstrable activity against bacteria, hydroheptin as compared to other non-aromatic heptaene macrolides exhibits excellent but somewhat less activity against a wide variety of yeasts and fungi. Likewise, its parenteral toxicity appears to be less than that of other heptaene macrolides.

Avermectins, new family of potent anthelmintic agents: producing organism and fermentation.

The avermectins are a complex of chemically related agents which exhibit extraordinarily potent anthelmintic activity. They are produced by a novel species of actinomycete, NRRL 8165, which we have named Streptomyces avermitilis. The morphological and cultural characteristics which differentiate the producing organism from other species are described. The avermectins have been identified as a series of macrocyclic lactone derivatives which, in contrast to the macrolide or polyene antibiotics, lack significant antibacterial or antifungal activity. The avermectin complex is fully active against the gastrointestinal nematode Nematospiroides dubius when fed to infected mice for 6 days at 0.0002% of the diet. Fermentation development, including medium modification and strain selection, resulted in increasing the broth yields from 9 to 500 mug/ml.