Studies on the mechanism of action of A80915A, a semi-naphthoquinone natural product, as an inhibitor of gastric (H(+)-K+)-ATPase.

A semi-naphthoquinone natural product, A80915A, produced by Streptomyces aculeolatus was found to be a potent inhibitor of gastric (H(+)-K+)-ATPase, the enzyme responsible for acid secretion in the stomach. Enzyme activity was measured by potassium-stimulated hydrolysis of ATP or p-nitrophenolphosphate with enzyme prepared from the stomach fundic mucosa of pigs. Concentration-dependent inhibition was observed with an IC50 of about 2-3 microM for both ATPase and p-nitrophenylphosphatase. A Hill plot indicated that the enzyme has two binding sites for A80915A. Inhibition was not affected by the presence of the reducing agent dithiothreitol, indicating a lack of involvement of enzyme sulfhydryl groups. A 30-min incubation of enzyme with increasing drug concentrations followed by a 10-fold dilution did not alter the IC50, indicating that A80915A does not covalently modify the enzyme. Coincubation of enzyme with 3.8 microM A80915A resulted in time-dependent inhibition. The rate of inhibition was slowed significantly by the presence of 20 mM potassium, rubidium and ammonium but not by 20 mM sodium, lithium and choline, or by 40 mM sucrose. The level of inhibition was influenced by the order of addition of potassium and drug to the enzyme. Taken together, these studies indicate that inhibition by A80915A is dependent on the conformation of gastric (H(+)-K+)-ATPase and that potassium slows the rate of inhibition by converting the enzyme to a conformation where the drug binding site is not as accessible. The mode of action of A80915A is distinct from that of two well characterized proton pump inhibitors, omeprazole and SCH 28080.

A54145, a new lipopeptide antibiotic complex: discovery, taxonomy, fermentation and HPLC.

A54145 is a complex of new lipopeptide antibiotics that inhibits Gram-positive bacteria and acts as a growth promotant for broiler chicks. Eight factors; A, B, C, D, E, F, A1 and B1; have been isolated and characterized. They contain four similar peptide nuclei, each of which is acylated with either an 2-decanoyl, n-decanoyl, or undecanoyl side chain. Taxonomic studies ascertained that the producing microorganism was a strain of Streptomyces fradiae. Fermentation studies determined that superior antibiotic yields were obtained in stirred bioreactors in a soybean flour-molasses medium employing a continuous glucose feed. These findings, interwoven with the selection of hyper-productive mutants, increased fermentation yields from less than 50 micrograms/ml to more than 1 mg/ml. An analytical HPLC system was developed for the identification and subsequent quantitation of each factor of the A54145 complex.

A54145, a new lipopeptide antibiotic complex: isolation and characterization.

A54145 is a complex of acidic lipopeptide antibiotics which are produced by Streptomyces fradiae and are active against Gram-positive bacteria. The A54145 complex was isolated by adsorption on Diaion HP-20 nonfunctionalized macroreticular resin and/or ion exchange on Amberlite IRA-68 anion exchange resin. Antibacterial factors A, A1, B, B1, C, D, E, and F were obtained in purified form by repeated preparative reverse phase HPLC on C8 and/or C18 bonded-phase supports. The molecular formulae of the factors are C72H109N17O27 (factors A and A1), C73H111N17O27 (factors B, B1, C, and D), C74H113N17O27 (factor E), and C71H107N17O27 (factor F). The identities of the acyl side chains were established as 8-methylnonanoyl (factors F, A, and B1), n-decanoyl (factors A1 and B), and 8-methyldecanoyl (factors C, D, and E).

A54145, a new lipopeptide antibiotic complex: microbial and chemical modification.

A54145 is a complex of acidic lipopeptide antibiotics produced by Streptomyces fradiae NRRL 18158, NRRL 18159, and NRRL 18160. Each antibiotic factor consists of a peptide core bearing an N-terminal acyl substituent. N-Lys-tert-BOC-protected A54145 complex was deacylated by Actinoplanes utahensis; three protected core peptides were isolated. A54145 antibiotic analogs were synthesized by acylation of the tryptophan N-terminus with 2,4,5-trichlorophenyl active esters, followed by deblocking with trifluoroacetic acid.

A54145 a new lipopeptide antibiotic complex: microbiological evaluation.

A54145 complex is made up of eight factors; A, A1, B, B1, C, D, E, and F which were active in vitro (MIC 0.25 approximately greater than 32 micrograms/ml) against Gram-positive aerobic organisms. The complex, factor B and B1 were found to be active against two strains of Clostridium perfringens. A calcium dependence study on some of the factors showed that their in vitro antibacterial activity was greatly enhanced by the presence of calcium (50 mg/liter) in the media. Resistance build-up was seen when Staphylococcus sp. and Streptococcus sp. were passed seven times in the presence of sublethal concentrations of A54145 antibiotics. This resistance disappeared immediately when the resistant organisms were passed in the absence of the antibiotics. Factor A was very effective against Staphylococcus aureus and Streptococcus pyogenes infections in mice (sc ED50s of 3.3 approximately 2.4 mg/kg x 2, respectively). Factor B was more active against S. pyogenes in vivo (sc ED50, 0.9 mg/kg x 2). Acute mouse toxicities were determined with these antibiotics. Semisynthetic derivatives were evaluated.

A80915, a new antibiotic complex produced by Streptomyces aculeolatus. Discovery, taxonomy, fermentation, isolation, characterization, and antibacterial evaluation.

New semi-naphthaquinone antibiotics A80915A, B, C, and D were isolated from the fermented broth of Streptomyces aculeolatus A80915 (NRRL 18422). Factors A and C, present in both the broth filtrate and mycelial methanol extract, and factors B and D, found predominantly in the broth filtrate, were recovered by extraction with ethyl acetate. Purification of the individual factors was accomplished by preparative reverse phase high performance liquid chromatograph on C18 bonded silica supports. Factors A through D show antimicrobial activity against Gram-positive aerobic and anaerobic organisms in vitro. Mechanism of action studies demonstrated nearly complete inhibition of macromolecular biosynthesis (protein, RNA, DNA, and cell wall) by A80915 factors A through D. A less highly cyclized semi-naphthaquinone, A80915 factor G, was isolated from the broth of the strain fermented in an alternate medium.

Deacylation of echinocandin B by Actinoplanes utahensis.

Echinocandin B (ECB) is a lipopeptide antifungal agent produced by several species of Aspergillus. The lipid side chain of cyclic lipopeptides is known to be an important determinant of their antibiotic activity and toxicity. Deacylation of another lipopeptide antibiotic, A21978C, had formerly been accomplished with Actinoplanes utahensis. In spite of the structural dissimilarities between the peptide cores and acyl side chains of A21978C and ECB, A. utahensis also removed the linoleoyl acyl unit from the amino terminus of ECB to yield the bioinactive cyclic peptide core, or "nucleus". The ECB nucleus, which contained a new titratable group at the N-terminus, was subsequently employed for chemical reacylation with other side chains to yield a variety of novel ECB analogs. One of these, cilofungin (LY121019), containing an N-(4-n-octyloxybenzoyl)acyl unit, is currently undergoing clinical evaluation.

Synthesis of new analogs of echinocandin B by enzymatic deacylation and chemical reacylation of the echinocandin B peptide: synthesis of the antifungal agent cilofungin (LY121019).

The antifungal antibiotic, echinocandin B (ECB), was modified by a sequential procedure in which the initial step involved enzymatic removal of the native N-linoleoyl group from the N-terminus using an Actinoplanes utahensis culture. The resulting product, ECB nucleus, was reacylated using active esters or acid halides of various substituted acids to give a series of ECB analogs. These analogs possessed anti-Candida activity both in vitro and in vivo (mice). Other studies have shown that one of these, cilofungin, the 4-n-octyloxybenzoyl-ECB analog (LY121019), has excellent anti-Candida activity, low toxicity and is superior to other available antifungal antibiotics.

Enzymatic and chemical modifications of lipopeptide antibiotic A21978C: the synthesis and evaluation of daptomycin (LY146032).

The novel lipopeptide antibiotic A21978C complex is active against Gram-positive organisms. This complex consists of a common peptide nucleus with various lipid acyl groups at the N-terminus characteristic of each individual factor. The fatty acid acyl group is removed by incubation of the A21978C complex with Actinoplanes utahensis to give the peptide nucleus. This peptide nucleus has the same amino acid sequence as A21978C. New analogs of A21978C were synthesized by acylation of the N-terminus of a tert-butoxycarbonyl (tert-BOC)-protected nucleus and subsequent deprotection. 1H NMR showed that the newly introduced acyl group was at the desired N-terminus. Three major groups of analogs were synthesized bearing fatty acid acyl, amino-aroyl and extended peptide side chains. Each analog was evaluated for antimicrobial activity and acute toxicity. Of these analogs, the n-decanoyl analog of A21978C (LY146032) gave the best survival in the mouse acute toxicity test at a high dose of 1,000 mg/kg, iv and was chosen for further study. This analog has been named daptomycin.

Deacylation of A21978C, an acidic lipopeptide antibiotic complex, by Actinoplanes utahensis.

A21978C, produced by Streptomyces roseosporus NRRL 11379, is an acidic lipopeptide antibiotic complex that inhibits Gram-positive bacteria. Individual factors of the complex possess an identical peptide core or "nucleus", and are differentiated by the distinctive fatty acid acyl group attached to the N-terminus of the nucleus. Certain members of the family Actinoplanaceae deacylated A21978C to yield the unaltered nucleus, which was then reacylated to form new analogs. Actinoplanes utahensis NRRL 12052 was the most efficient of these cultures, producing up to 500 micrograms of nucleus per ml of culture broth per hour. Eacylation was also accomplished with semi-pure and tert-butoxycarbonyl (tert-BOC)-A21978C. In the latter, the ornithine amino group was blocked to prevent formation of diacyl analogs during reacylation. The acylase was an endoenzyme present in submerged cultures of A. utahensis from less than 18 to greater than 168 hours of incubation. Whole cells suspended in phosphate buffer or entrapped in polyacrylamide gel also deacylated A21978C efficiently.

Structure elucidation of A58365A and A58365B, angiotensin converting enzyme inhibitors produced by Streptomyces chromofuscus.

A58365A and A58365B, angiotensin converting enzyme inhibitors isolated from the culture filtrate of Streptomyces chromofuscus NRRL 15098, are homologous compounds of molecular formulas C12H13NO6 and C13H15NO6. The molecular similarities of the two inhibitors were established by comparison of their 1H NMR, 13C NMR, and UV spectra. Catalytic hydrogenation of A58365A led to a tetrahydro-deoxy derivative, C12H17NO5; extensive 1H NMR decoupling studies at 360 MHz allowed all the non-exchangeable protons of the derivative to be connected in a continuous substructure. This fragment was combined with information from other spectroscopic methods to suggest the structures for A58365A (1) and A58365B (2); the conclusions were confirmed by an X-ray crystallographic analysis of A58365A-dimethyl ester.

Method for the detection and quantitation of chitinase inhibitors in fermentation broths; isolation and insect life cycle effect of A82516.

A new method of screening for chitinase inhibitors in crude fermentation broths as a means of discovering new insecticidal leads has been developed. In this procedure soluble Remazol brilliant violet 5R dye-coupled chitin degradation products released from insoluble chitin azure substrate by hydrolysis with Streptomyces griseus chitinase are filtered in 0.45 micron Millititer HA 96 well filtration plates and collected in 96 well microtiter plates. Inhibitors of this reaction are detected by a decrease in absorbance (570 nm) of the filtrate. A chitinase inhibitor, designated A82516, produced by culture A82516 was discovered using this screen. Purified A82516 was found to have an IC50 of 3.7 X 10(-6) M for S. griseus chitinase. At a test concentration of 0.27 mg/ml, A82516 was 100% effective in preventing development of house fly larvae to pupae. Allosamidin, a recently reported chitinase inhibitor in vitro, has spectral properties identical to A82516.

A21978C, a complex of new acidic peptide antibiotics: isolation, chemistry, and mass spectral structure elucidation.

A21978C, produced by Streptomyces roseosporus, NRRL 11379, is a complex of new acidic lipopeptolide antibiotics which inhibits Gram-positive bacteria. HPLC separation of the various components from the purified complex resulted in the isolation of A21978C1, -C2 and -C3 (major components) and -C4, -C5, and -C0 (minor components). Each of these components was fermented with cultures of Actinoplanes utahensis (NRRL 12052) to give the identical inactive peptide ("A21978C nucleus") by removal of the fatty acid acyl groups from the N-terminus. This peptide was composed of 13 amino acids: L-kynurenine, L-threo-3-methylglutamic acid, L-asparagine, L-aspartic acid (3 residues), glycine (2 residues), L-tryptophan, L-ornithine, D-alanine, D-serine and L-threonine. The amino acid sequence was determined using a combination of the Edman degradation and gas chromatography mass spectrum (GC-MS) analysis of appropriately derivatized peptides obtained from partial hydrolysis. Each major component was shown to be acylated with a branched chain fatty acid at the N-terminus and the structure of this fatty acid was determined by 1H NMR and mass spectral methods. A structure for A21978C was assigned on the basis of this degradative and physico-chemical information.

Preparation and evaluation of 3,4''-ester derivatives of 16-membered macrolide antibiotics related to tylosin.

A large group of ester derivatives of tylosin-related macrolides was prepared in which the hydroxyl groups at C-3 and C-4'' were acylated by either chemical or biochemical methods. Most of the derivatives exhibited excellent in vitro antimicrobial activity. However, only the 3,4''-diacyl derivatives of tylosin and macrocin showed any significant improvements of in vivo efficacy against experimental infections in rodents.

Isolation of A58365A and A58365B, angiotensin converting enzyme inhibitors produced by Streptomyces chromofuscus.

A58365A and A58365B, angiotensin converting enzyme inhibitors, were isolated from the culture filtrate of Streptomyces chromofuscus NRRL 15098. A58365A and A58365B are homologous nitrogen-containing bicyclic structures of molecular formulae C12H13NO6 and C13H15NO6.

Microbial transformation of A23187, a divalent cation ionophore antibiotic.

A23187 is an ionophore antibiotic that forms dimeric complexes with divalent cations such an Mn2+ and Ca2+. Over 200 randomly selected soil microorganisms were incubated with A23187. None of these cultures was capable of transforming this compound. In contrast, many microorganisms were able to modify the methyl ester of A23187. The transformation products produced by one culture, Streptomyces chartreusis, were isolated and identified as 16-hydroxy-N-demethyl A23187 methyl ester, 16-hydroxy-A23187 methyl ester, and N-demethyl A23187 methyl ester. These ester derivatives lack most of the ionophore properties of the acids and cannot readily form dimeric complexes with divalent cations. However, they could be hydrolyzed by a mild treatment with ethanolic KOH to free acids that possess good ionophore activity. The use of the ester substrate in conjunction with the hydrolysis procedure is, at the present time, the only known method for microbiologically producing A23187 derivatives.

Microbiological transformations of nabilone, a synthetic cannabinoid.

A screening program was conducted to find microorganisms that modify the synthetic cannabinoid nabilone. After purification, the products from three cultures were analyzed by spectral methods to determine their chemical structures. An optically active 9S-hydroxy-6aR,10aR-trans cannabinoid was isolated from a culture of an unidentified soil bacterium designated A24007. From Bacillus cereus cultures were isolated a 9S,6'-dihydroxy-6aR,10aR-trans cannabinoid, a 9S-hydroxy-6'-keto-6aR,10aR-trans cannabinoid, a 9-keto-6'-hydroxy-6aS,10aS-trans cannabinoid, and a 6',9-diketo-6aS,10aS-trans cannabinoid. All of these products were optically active, as was a 9S-hydroxy-6aS,10AS-trans cannabinoid also isolated from B. cereus cultures. A series of acidic products were isolated from cultures of Nocardia salmonicolor. All of these products contained a carboxylic acid group at the terminal end of three-position alkyl side chains having varying numbers of carbon atoms. Two of the acidic products contained a 9-keto group, whereas all other carboxylic acid products were 9-hydroxy cannabinoids. The array of products obtained from incubation of nabilone indicates the usefulness of microbial transformations in the preparation of new cannabinoids.

Microbiological transformation of cannabinoids.

Microorganisms were screened for their ability to modify 2 synthetic cannabinoid substrates (I and II). Structure analyses revealed that microorganisms transformed the substrates by (a) primary oxidation of the side chain, beta-oxidation of the side chain, ketone formation on the side chain or cyclohexene ring, (b) secondary hydroxylation on the side chain, (c) aromatization of the cyclohexene ring, and (d) tertiary hydroxylation at the b/c ring juncture.

Immobilization of a cephalosporin acetylesterase by containment within an ultrafiltration device.

A cephalosporin acetylesterase produced by Bacillus subtilis catalyzes the deacetylation of 7-aminocephalosporanic acid (7-ACA). Previous reports from our laboratory described the kinetic constants that characterize the reaction: Km = 2.8 X 10(-3)M, Kia acetate = 5 X 10(-2)M, and Kid deacetyl-7-ACA = 3.6 X 10(-2)M. These constants were used to predict the time course of the reaction using the following equation for dual competitive product inhibition. (see article) where St =mg/ml 7-ACA, At =mg/ml acetate, Dt =mg/ml deacetyl-7-ACA. The predicted time course closely matched the time course measured experimentally. The equation also was solved without the inhibition terms and the solution indicated that product inhibition caused about a 30% increase in the time required for complete (greater than 97%) hydrolysis of a 24 mg/ml 7-ACA solution. The esterase was immobilized by containment within an ultrafiltration device. With this technique the enzyme was reused 20 times over an 11 day span to deacetylate 7-ACA solutions containing 4 to 24 mg/ml 7-ACA. The specific activity after the 20th use was the same as the activity prior to the first use, indicating little enzyme inactiviation occurred.