[go: up one dir, main page]

WO2008001306A2 - Streptomycète et composé bioactif produit par celui-ci - Google Patents

Streptomycète et composé bioactif produit par celui-ci Download PDF

Info

Publication number
WO2008001306A2
WO2008001306A2 PCT/IB2007/052468 IB2007052468W WO2008001306A2 WO 2008001306 A2 WO2008001306 A2 WO 2008001306A2 IB 2007052468 W IB2007052468 W IB 2007052468W WO 2008001306 A2 WO2008001306 A2 WO 2008001306A2
Authority
WO
WIPO (PCT)
Prior art keywords
strain
diphenyloxazole
derivatives
variants
microorganism
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2007/052468
Other languages
English (en)
Other versions
WO2008001306B1 (fr
WO2008001306A3 (fr
Inventor
Paul Robert Meyers
Marilize Le Roes
David W Gammon
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South African Medical Research Council
University of Cape Town
Original Assignee
South African Medical Research Council
University of Cape Town
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by South African Medical Research Council, University of Cape Town filed Critical South African Medical Research Council
Publication of WO2008001306A2 publication Critical patent/WO2008001306A2/fr
Publication of WO2008001306A3 publication Critical patent/WO2008001306A3/fr
Publication of WO2008001306B1 publication Critical patent/WO2008001306B1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Definitions

  • This invention relates to an isolated organism and to compounds recovered therefrom.
  • this invention relates to 'Streptomyces polyantibioticus' strain SPR and 21-57 being an antibacterial compound produced by the organism.
  • the invention further relates to the use of 21-57 as an antibacterial agent.
  • Tuberculosis has become the leading cause of natural death in South Africa, killing 1000 people/month, most of whom are in their most economically productive years.
  • South Africa is one of the 22 most burdened countries in the world, with an average incidence rate of 536 TB cases per 100 000 people in the population - the third highest incidence in the world (Global Tuberculosis Control, WHO Report, 2005).
  • WHO Report Global Tuberculosis Control
  • the family Streptomycetaceae contains the genera Streptomyces, Kitasatospora and Streptacidiphilus. All of these genera are phylogenetically related, but differ in chemical composition and in the formation of distinct phylogenetic clades in phylogenetic trees.
  • the genus Kitasatospora (initially Kitasatosporia) was first proposed in 1982 by Omura et al. and was subsequently included in the genus Streptomyces (Groth et al., 2003). It was re-established in 1997 (Zhang et al.) and has subsequently been recognised as a genus separate from the genus Streptomyces.
  • the genus Streptomyces was first described in 1943 by Henrici & Waksman. After a period of classification and re-classification of genera such as Actinopycnidium, Actinosporangium, Chainia, Elytrosporangium, Microellobosporia, Kitasatoa and Streptoverticillium (Anderson & Wellington, 2001), the genus Streptomyces today consists of 519 validly published species (Euzeby, 2005). The systematics of the genus Streptomyces at species level is, however, still in a state of confusion and the genus is believed to be overclassified - by far having the highest amount of validly described species (Groth et al., 1999).
  • the genus Streptomyces has long been known as a rich source of antibiotics.
  • Members of the genus Streptomyces are aerobic, Gram positive and have a DNA G+C mol% of 69 - 78%, the highest reported for actinomycetes (Anderson & Wellington, 2001). They produce extensively branching substrate and aerial mycelia; sporangia are not produced. With age, the aerial mycelium differentiates into chains of spores. Spore chain morphology and spore surface ornamentation are often used as distinguishing features amongst members of this genus.
  • the genus is characterized as having a cell wall chemotype I (Lechevalier & Lechevalier, 1970) - LL-DAP and glycine are present in their peptidoglycan, and no characteristic sugars are present in whole-cell sugar hydrolysates (Anderson & Wellington, 2001; Lechevalier & Lechevalier, 1970).
  • Saturated iso- and ante/so-fatty acids, hexa- or octahydrogenated menaquinones with nine isoprene units, and a phospholipid pattern of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides, are characteristic of this genus (Locci, 1989). Furthermore, streptomycetes have an A3 ⁇ peptidoglycan type and lack mycolic acids (Locci, 1989).
  • antibiotics are secondary metabolites with a typically low molecular weight (ranging from 150 - 5000 Da) and are chemically diverse. They could comprise only carbon and hydrogen atoms, but mostly they contain carbon, hydrogen, oxygen and nitrogen atoms, while some also contain sulphur, phosphorus or halogens. In addition, almost all organic chemical functional groups are represented (Lancini et al., 1995). The role of secondary metabolites in bacteria has been disputed for a long time (Berdy, 2005). In actinomycetes, they are typically produced after vegetative growth and when the producer is entering a phase of dormancy or spore formation (Challis & Hopwood, 2003). Various hypotheses exist to explain the role of antibiotics in the antibiotic producers.
  • the process of isolating, purifying and identifying novel compounds involves a range of professionals: microbiologists, fermentation technologists, bioengineers and chemists, and encompasses all the processes involved from the isolation of the bacterium from a soil sample to a vial containing the purified compound. Due to the high re-isolation incidence of known antibiotics, it is essential to determine as soon as possible whether or not a compound is novel (Lancini et al., 1995). The identification of antibiotics from fermentation media is a labour-intensive and time-consuming process (Berdy, 2005; Sandvoss et al., 2001; Wilson, 2000).
  • the microorganism is an actinomycete strain. More preferably the microorganism is from the genus Streptomyces, The microorganism may be from the family Streptomycetaceae and is most preferably Streptomyces polyantibioticus.
  • the microorganism preferably includes genetic material of GenBank accession number DQ141528 (16S-rDNA-sequence) (http://www.ncbi.nlm.nih.gov).
  • a biologically pure culture having identifying characteristics of GenBank accession number DQ141528 (16S-rDNA-sequence).
  • 2,5- diphenyloxazole or variants or derivatives thereof recovered from a microorganism having identifying characteristics of GenBank accession number DQ141528 (16S- rDNA-sequence).
  • 2,5- diphenyloxazole or variants or derivatives thereof as an antimicrobial agent including use in the treatment of influenza, tuberculosis and/or other bacterial infections.
  • compositions for use as an antimicrobial agent comprising an effective amount of 2,5-diphenyloxazole or variants or derivatives thereof.
  • 2,5- diphenyloxazole or variants or derivatives thereof for use in the manufacture of a medicament for use as an antimicrobial agent including use in the treatment of influenza, tuberculosis and/or other bacterial infections.
  • an isolated polynucleotide sequence comprising a polynecleotide of GenBank accession number DQ141528 (16S-rDNA-sequence).
  • the microorganism has been deposited in two international culture collections, namely, the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, the German Culture Collection, http://www.dsmz.de) and the Actinobacterial Culture Collection of the Agricultural Research Service of the US Department of Agriculture (http://nrrl.ncaur.usda.gov).
  • Figure 1 is a Cryo-scanning electron micrograph of strain SPR T grown on Middlebrook 7H9-glucose agar at 30 0 C for 90 days. Clustered sporangiophores bearing mature sporangia are clearly visible, with smooth, rod-shaped spores contained within the sporangia. The bar represents 1 ⁇ m.
  • Figure 2 is an unrooted 16S-rDNA phylogenetic tree obtained by the neighbour- joining method, showing the position of strain SPR T among its phylogenetic neighbours and representatives of sporangium-producing genera.
  • the 16S-rDNA sequence of Arthrobacter globiformis JCM 1332 T was used as an out-group. All sequences were edited to the longest common region (1409 bp).
  • GenBank sequence accession numbers are given in parentheses. Numbers at the nodes show the % bootstrap values. Asterisks indicate the clades that were conserved when the neighbour-joining, minimum evolution and maximum parsimony methods were used in constructing the phylogenetic tree.
  • Figure 3 is an rRNA operon copy number of strain SPR T .
  • Genomic DNA fragments were fractionated on a 0.7% agarose gel for 24 h, transferred to a nylon membrane and hybridized with the rDNA probes.
  • Lanes 1 and 2 represent digested genomic DNA hybridized with the 16S-rDNA probe
  • lanes 3 to 6 represent digested genomic DNA hybridized with the 23S- rDNA probe.
  • Figure 4 is schematic diagram of the isolation and purification of the bioactive compound, 21-57.
  • Figure 5 is bioautography against M. tuberculosis H37Rv. Kanamycin was used as a positive control. Amounts spotted are indicated. Areas of white indicate areas of inhibition of growth; darker areas represent areas where bacteria are alive.
  • Figure 6 is a) the molecular structure of 21-57 as determined by X-ray crystallography (structure provided by Associate Professor Susan Bourne, X-ray Crystallography Unit, Department of Chemistry, University of Cape Town) and b) the molecular structure of 21-57 with IUPAC numbering which was followed for the assignment of NMR spectra data (Table 3).
  • Figure 7 is a schematic representation of the biosynthetic process involved in oxazole and thiazole production (Roy et al., 1999).
  • Figure 8 illustrates chemical structures of natural products which contain one or more oxazole moieties.
  • Figure 9 is a) the biosynthetic gene cluster and structure of yersiniabactin produced by Yersinia pestis; and b) the function of a truncated HMWP2 with the final products (hydroxyphenyl) thiazoline-cysteine adduct and (hydroxyphenyl) thiazoline carboxylate (Roy et al. 1999).
  • Figure 10 shows the gene sequence of "streptomyces polyantibioticus” strain SPR T (SEQ ID NO:1).
  • an actinomycete with unusual morphology was isolated from soil collected from the banks of the Umgeni River, Kwa-Zulu Natal, South Africa.
  • Strain SPR T was isolated on Middlebrook 7H9 agar [Difco Laboratories; supplemented with 10mM glucose; albumin-dextrose (AD) supplement omitted] after incubation at 28°C for 7 days. Following isolation, strain SPR T was maintained on Middlebrook 7H9-glucose agar.
  • Antimicrobial activity was determined by the sloppy-agar overlay technique.
  • the isolate was stab-inoculated into Middlebrook 7H9-glucose agar, Czapek Solution agar (CZ) (Atlas, 1993) and yeast extract-malt extract (ISP 2 - Shirling & Gottlieb, 1966) agar plates, in duplicate. Plates were incubated for 10 days at 30 0 C (the duplicate set at 37°C), and overlaid with 6 ml Luria sloppy agar (Sambrook et al., 1989) containing the test bacterium.
  • Bacillus coagulans ATCC 7050 T Enterococcus faecium vanA (vancomycin resistant), Enterococcus phoeniculicola JLB-1 T , a Micrococcus sp. (clinical isolate), Mycobacterium aurum A+ and a Streptococcus sp. (clinical isolate); weak activity was exhibited against Citrobacter braaki strain 90 (clinical isolate), Enterobacter cloacae strain 67 (clinical isolate), Escherichia coli ATCC 25922 and Klebsiella oxytoca strain K52 (clinical isolate; resistant to augmentin and cefuroxime).
  • Strain SPR T was cultured in 500 ml Hacene's Medium (HM; Hacene & Lefebvre, 1995) for 10 days at 30 0 C on a rocking shaker. The culture was filtered through a coffee filter (House of Coffees, 1X4 sized filters). The mycelial mass of the isolate was extracted with methanol and activity determined by bioautography.
  • strain SPR T The morphological characteristics of strain SPR T were determined using standard methods (Locci, 1989). The isolate was grown on Middlebrook 7H9-glucose agar for 90 days at 30 0 C and the morphology observed under a light microscope and by cryo- scanning electron microscopy.
  • Standard techniques were used for the determination of catalase and oxidase activity. Staining was performed using the standard Gram staining technique. Acid- fast staining (1% sulphuric acid used in decolourisation step) and acid-alcohol-fast staining (Ziehl-Neelsen) were performed using standard methods.
  • Freeze-dried cells used in chemotaxonomic tests were obtained from a 500 ml ISP 2 culture of strain SPR T , which was cultivated on a rocking shaker at 30 0 C for 5 days.
  • the DAP isomer and whole-cell sugar pattern were determined by the method of Hasegawa et al. (1983), with the exception that freeze-dried cells were used instead of colonies from agar plates.
  • the fatty acid methyl esters were prepared according to Chou et al. (1998) and were analysed by gas chromatography on a Zebron GC column, ZB-1 (0.25 mm by 30 m). The temperature of the column was fixed at 210 0 C and the temperature of the injector was programmed to increase from 150°C to 22O 0 C at a rate of 4°C per minute. The peaks were identified by comparison with fatty acid methyl ester standards (FAM Standard: MIX GLC-80; SEPULCO). Mycolic acids were isolated and analysed as described by Minnikin et al. (1975). Phospholipids were isolated as described by Minnikin et al. (1984).
  • Menaquinones were analysed by the DSMZ Identification Service (Braunschweig, Germany) using an HPLC separation method. Phospholipids were analysed by two-dimensional thin-layer chromatography (TLC) on silica gel 60 F 254 (Merck) plates as described by Komagata & Suzuki (1987). The peptidoglycan type was determined as described by Komagata & Suzuki (1987) - analysis was performed by TLC. The base composition (G+C mol%) was determined in 1.0 X standard saline citrate (SSC) using the thermal denaturation method described by Mandel & Marmur (1968).
  • SSC standard saline citrate
  • Genomic DNA was digested singly with restriction endonucleases: Sa/I and Seal for the determination of 16S-rRNA operon multiplicity; and Sa/I, EcoRV, SspBI and SnaBI for the determination of 23S-rRNA operon multiplicity.
  • the digested genomic DNA was electrophoresed on a 0.7% agarose gel for 24 h. Copy numbers for both the 16S-rRNA gene and 23S-rRNA gene were determined by Southern hybridization analysis (Sambrook et a/., 1989). Digoxigenin-dUTP (Roche) labelled DNA probes were used.
  • the probe for the determination of 16S-rRNA copy number was amplified from strain SPR T using the universal bacterial primers F1 and R5 (Cook & Meyers, 2003), and the probe for the determination of the 23S-rRNA copy number was amplified using the primers described by Wang & Zhang (2000): forward primer 5'- CCGATGAAGGACGTGGGA-3' (positions 46 to 63*) and reverse primer 5'- ACCAGTGAGCTATTACGC-3' (positions 1212 to 1195 * ). Amplified, unlabeled 16S- rDNA and 23S-rDNA were used as positive controls. Hybridization and visualization were performed as recommended in the Roche DIG manual (http://www.roche.com). * Streptomyces ambofaciens ATCC 23877 T 23S-rRNA gene numbering
  • Mycolic acids were not detected and strain SPR T had an A3 ⁇ peptidoglycan type [presence of LL-DAP and glycine as determined by TLC - Schleifer & Kandler (1972)].
  • the predominant fatty acids were determined to be of the /so and ante/so-branched type (28% /-C16:0; 18% a/-C15:0; 15% C16:0; 14% ai- C17:0; 10% /-C15:0; 8% /-C17:0), similar to the pattern observed in the genera Oerskovia, Micropolyspora, Saccharopolyspora, Streptomyces, Glycomyces and Kitasatospora.
  • Phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and two unknown phospholipids were detected by 2D TLC analysis.
  • the predominant menaquinones were determined to be MK-9 (H 4 ) (60%) and MK-9 (H 6 ) (40%), with traces of MK-9 (H 2 ) ( ⁇ 3%), the same pattern observed for the genera Actinomadura and Spi ⁇ llospora.
  • the G+C mol% was 74.4% ( ⁇ 0.2%) when determined in 1.0 X SSC (in duplicate).
  • Table 1 A comparison of the chemotaxonomic characteristics of strain SPR , sporangiate genera and the genus Streptomyces
  • DAP 2,6-diaminopimelic acid
  • PG phosphatidylglycerol
  • PE phosphatidylethanolamine
  • Pl phosphatidylinositol
  • PIM phosphatidylinositol mannosides
  • DPG diphosphatidylglycerol
  • a 1459 bp 16S-rDNA sequence was obtained for strain SPR T .
  • a BLAST (Basic Local Alignment Search Tool) search showed 97% homology to members of the genus Streptomyces.
  • a phylogenetic tree of streptomycete strains, representatives of the sporangiate genera, the type species of the genera Kitasatospora and Streptacidiphilus, and strain SPR T ( Figure 2) showed that strain SPR T clustered with members of the genus Streptomyces.
  • strain SPR T contains seven copies of the 16S-rRNA and 23S-rRNA genes.
  • the only enzyme which did not give a clear result was EcoRV (lane 4).
  • Most members of the genus Streptomyces have 6 copies of the rRNA operon ⁇ rrndb; Klappenbach et al., 2001), but Streptomyces venezuelae has seven copies (Klappenbach et al., 2000; La Farina et al., 1996).
  • Some members of the Gamma proteobacteria such as Escherichia coli, Shigella flexneri, Pseudomonas putida, Samonella spp.
  • Yersinia pestis are known to have seven copies of the rRNA operon (Acinas et al., 2004), with some strains of E. coli having up to 36 copies (Klappenbach et al., 2001).
  • Other bacterial strains with seven copies of the rRNA operon include Oceanobacillus iheyensis HTE831 T , Streptococcus agalactiae NEM316 and Streptococcus agalactiae 2603 V/R (Acinas et al., 2004). Klappenbach et al. (2001) showed that there is no phylogenetic relatedness in species with the same number of rRNA operons and that the copy number is dependent on the natural environment of the bacteria.
  • strain SPR T is clearly related to the genus Streptomyces, with homology values of 97% to validly described members of the genus.
  • the group formed a distinct phylogenetic clade and the genus Kitasatospora was resurrected (Zhang ef a/., 1997).
  • Strain SPR T clearly differs from members of the genus Streptomyces: morphologically in the production of sporangia, which resemble those produced by members of the genus Streptosporangium, and chemically in containing characteristic sugars in its whole-cell hydrolysate that are similar to the sugars observed for members of the genera Actinoalloteichus and Actinomadura.
  • the rest of the chemical characteristics of strain SPR T are similar to those of the genus Streptomyces, with the exception of the menaquinone pattern.
  • MK-9 H 41 H 6
  • MK-9 H 61 H 8
  • the tetra-hydrogenated MK-9 menaquinone has been detected in some streptomycetes, including S. hebeiensis DSM 41837 T (Xu ef a/., 2004), S. scabrisporus NRRL B- 24202 T (Ping et a/., 2004), S. scopiformis A25 T (Li et a/., 2002), S.
  • sodiiphilus CIP 107975 T Li ef a/., 2005
  • S. thermocoprophilus B19 T Kim ef a/., 2000
  • S. yeochonensis NRRL B-24245 1* Kim ef a/., 2004
  • S. scabrisporus NRRL B-24202 T is the only streptomycete that has MK-9 (H 4 ) as one of its predominant menaquinones.
  • strain SPR T is proposed to be a member of the genus Streptomyces.
  • Streptomyces polyantibioticus (po.ly.an.ti.bi.o'ti.cus. N. L. fern. adj. polyantibioticus - referring to the ability to produce numerous antibiotics).
  • Strain SPR T forms brown substrate mycelium and fluffy, white aerial mycelium on inorganic salts-starch agar (ISP 4).
  • a leathery, dark brown substrate mycelium forms on ISP 2, but no sporulation occurs.
  • Brown substrate mycelium with white aerial mycelium forms on oatmeal agar (ISP 3).
  • the colour of the substrate mycelium is not pH sensitive.
  • Good growth is observed on Middlebrook 7H9-glucose agar.
  • a dark brown diffusible pigment is produced on glycerol-asparagine agar (ISP 5) and melanin is produced on peptone-yeast extract-iron agar (ISP 6) and tyrosine agar (ISP 7).
  • the methanol extract from mycelial mass was analysed by thin layer chromatography on silica gel 60 F 2 5 4 (Merck) plates using ethyl acetate:methanol (100:15, v/v) as the mobile phase.
  • Five anti-M. aurum A+ activity spots were detected by bioautography.
  • Strain SPR T grows in the presence of 0.1% 2-phenylethanol (but not 0.3%), 0.0001% crystal violet, 7% NaCI (but not 10%) and 0.1% phenol, but not in the presence of sodium azide (0.01%). Growth was also observed in the presence of lysozyme, capreomycin (20 ⁇ g/ml), cefotaxime (100 ⁇ g/ml), cephaloridine (100 ⁇ g/ml), D- cycloserine (50 ⁇ g/ml), penicillin G (10 i.u.) and viomycin (8 ⁇ g/ml), but not in the presence of gentamicin (100 ⁇ g/ml), kanamycin (10 ⁇ g/ml), lincomycin (100 ⁇ g/ml), neomycin (50 ⁇ g/ml), oleandomycin (100 ⁇ g/ml), rifampicin (50 ⁇ g/ml), streptomycin (100 ⁇ g/ml),
  • Strain SPR T uses DL- ⁇ -amino-n-butyric acid, L-arginine, L-cysteine, L-histidine, L-hydroxyproline, L- phenylalanine, L-serine, L-threonine, L-valine and potassium nitrate as sole nitrogen sources; L-methionine is weakly utilized.
  • H 2 S production occurs and nitrate is reduced (weakly).
  • Lecithinase and lipase activity are observed on egg-yolk agar, but not protease activity.
  • Pectin is hydrolysed, but hippurate is not.
  • Strain SPR T degrades adenine, aesculin, arbutin, casein, hypoxanthine, L-tyrosine, Tween 80, xanthine and DNA, but not allantoin, cellulose, gelatin, guanine, starch, urea and xylan.
  • Strain SPR T is able to utilize the organic acids, sodium acetate, sodium citrate, sodium formate, sodium gluconate, sodium lactate, sodium malate, sodium succinate and sodium tartrate.
  • Sodium butyrate is utilized weakly and sodium benzoate, sodium maleate, sodium mucate, sodium oxalate, sodium salicylate and sodium sorbate are not utilized.
  • the G+C content of the DNA is 74.4% ( ⁇ 0.2%) (1.0 X SSC).
  • an antibacterial agent was isolated, purified and designated 21-57.
  • the activity of 21-57 was determined and its anti-tubercular activity prompted the use of classical structure determination methods, including nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), infra-red spectroscopy (IR) and X-ray crystallography to elucidate the structure of the compound.
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • IR infra-red spectroscopy
  • X-ray crystallography X-ray crystallography
  • the seed cultures were used to inoculate nine 5 litre Erlenmeyer flasks each containing 500 ml of HM.
  • the flasks were incubated on rocking shakers at 30 0 C for a further 192 hours (8 days) for a total fermentation time of 10 days.
  • Isolation and purification of 21-57 is outlined in Figure 4 (method adapted from Oki et al., 1979).
  • the cultures were pooled and filtered through coffee filters (House of Coffees, 1x4 sized filters).
  • the antibacterial compounds were extracted from the mycelial mass with methanol and from the culture filtrate with ethyl acetate by stirring overnight at room temperature on magnetic stirrers.
  • the activity of the fractions was detected through bioautography with Mycobacterium aurum A+ as the test organism.
  • the active fractions were pooled, dried down and re- dissolved in methanol. White crystals precipitated out, leaving a yellow compound in solution. The methanol was removed and the crystals were allowed to dry. The crystals were re-dissolved in 2 ml toluene and subjected to another 40 cm x 4 cm silica gel G (Merck) column with toluene as the elution solvent for further purification. Once again, 5-ml fractions were collected (150 in total). Bioactivity was determined as before. Fractions 21 to 57 were combined, containing 110 mg of the purified bioactive compound, designated 21-57. The yield of 21-57 was 24.44 mg per litre of culture. Reagent grade organic solvents were used in all extractions.
  • All test bacteria were grown in LB (Luria-Bertani) broth except for Mycobacterium bovis BCG (Tokyo), Mycobacterium smegmatis LR222 and Mycobacterium tuberculosis H37Rv.
  • the mycobacteria were grown in Middlebrook 7H9 broth supplemented with 0.05% (v/v) Tween 80 and one tenth volume AD supplement (5% BSA, 2% glucose solution, filter sterilized).
  • test bacteria used in this study were: the Gram positives - Bacillus coagulans ATCC 7050 T , Bacillus megaterium NCIB 2602, Enterococcus faecium VanA (vancomycin resistant), Enterococcus phoeniculicola JLB-1 T , a Micrococcus sp., Mycobacterium aurum A+ (fast-growing, non-pathogenic), Mycobacterium bovis BCG (Tokyo) (fast-growing, non-pathogenic vaccine strain), Mycobacterium smegmatis LR222 (fast-growing, non-pathogenic), Mycobacterium tuberculosis H37Rv (ATCC 27294; slow-growing, pathogenic), Staphylococcus aureus ATCC 25923 (quality control strain for antimicrobial testing), and a Streptococcus sp.
  • M. bovis BCG Tokyo
  • M. smegmatis LR222 were incubated standing at 37°C for 7 days with occasional manual shaking
  • M. tuberculosis H37Rv was incubated at 37°C for 9 days, standing with intermittent agitation in an incubator in a Biosafety Level 3 laboratory, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town (all tests involving M. tuberculosis H37Rv were performed in this Biosafety Level 3 laboratory).
  • the optical density of the test bacterial cultures were determined at 600 nm on a Beckman DU-64 spectrophotometer and adjusted to 0.5 with LB or Middlebrook 7H9 broth depending on the culture (except for M. tuberculosis H37Rv which was not diluted; OD ⁇ 0.6 by comparison with M. smegmatis LR222 cultures of known OD).
  • the antimicrobial spectrum of the purified compound was determined through bioautography.
  • 21-57 is highly non-polar which made the determination of minimal inhibitory concentrations in liquid difficult and standard methods, such as the agar disk diffusion method (Kirby-Bauer method), could not be used in the determination of the antibacterial activity of 21-57.
  • a range of microgram amounts (5 - 100 ⁇ g) of the antibiotic were spotted onto silica gel 60 F 254 (Merck) plates in 5 ⁇ l volumes using Gilson pipettes and allowed to dry for 20 minutes in a fumehood. The same volume (5 ⁇ l) of organic solvent was spotted as a negative control to ensure that it was not the organic solvent in which 21-57 was dissolved that was killing the bacteria.
  • the melting point of 21-57 was determined with a Reichart-Jung Thermovar melting point apparatus fitted with a digital thermometer.
  • the infra-red absorption spectrum was recorded as a chloroform solution with a Perkin-Elmer FT-IR spectrometer and the mass spectrum was determined using MALDI-TOF spectrometry (provided as a service by the Department of Molecular and Cell Biology, University of Cape Town) and was confirmed by high resolution mass spectrometry - service provided by the School of Chemistry, University of Witwatersrand (Johannesburg, South Africa).
  • 1 H and 13 C NMR spectra were recorded on a 600 MHz Bruker NMR spectrometer - service provided by the Department of Chemistry, University of Whybosch (South Africa).
  • Crystals for X-ray crystallography were prepared by dissolving 10 mg of 21-57 in 400 ⁇ l of methanol. The container was covered with Parafilm and the Parafilm was perforated with a sterile needle to allow for the slow evaporation of the solvent. The sample was left at 4°C for 5 days.
  • TLC thin-layer chromatography
  • silica gel 60 F 254 plates (Merck) were used.
  • 21-57 was chromatographed on TLC plates with 100% toluene as the mobile phase and sprayed with 50% sulphuric acid or cerium (IV) ammonium sulphate [30 ml sulphuric acid added to 500 ml distilled water, 63.0 g of cerium (IV) ammonium sulphate added, and diluted to 1 litre with distilled water] and heated at 100 0 C for colour visualization.
  • the compound was also detected with iodine vapours and UV irradiation at 254 nm.
  • the minimum inhibitory amounts of the bioactive compound, 21-57, against various test bacteria are shown in Table 2.
  • the compound seems to have a broad spectrum of activity, but no specific pattern of activity, as seen in its ability to inhibit the growth of some Gram positives and some Gram negatives. No activity was observed against the following bacteria: the Gram positives - Bacillus coagulans ATCC 7050 T , Enterococcus phoeniculicola JLB-1 T , Staphylococcus aureus ATCC 25923 and a Streptococcus sp.
  • M. bovis BCG Tokyo
  • M. tuberculosis H37Rv bioautography results shown in Figure 5
  • M. aurum A+ M. bovis BCG
  • the physico-chemical properties of 21-57 are listed in Table 3.
  • the compound was purified as a white crystalline solid and was more readily soluble in non-polar organic solvents (e.g. hexane, toluene, ethyl acetate and chloroform) than in polar solvents (e.g. ethanol and methanol) and insoluble in water.
  • non-polar organic solvents e.g. hexane, toluene, ethyl acetate and chloroform
  • polar solvents e.g. ethanol and methanol
  • the melting point of the compound was 70 - 72 0 C and the UV/VIS spectrum of this compound showed strong absorbance at 303 nm and 216 nm.
  • the molar extinction coefficients are indicated in Table 3 as determined in methanol.
  • the molecular formula of 21-57, C 15 H 11 NO, was determined from the microanalysis and high-resolution MS and MALDI-TOF MS results.
  • the structure of 21-57 was elucidated from X-ray crystallography data and confirmed by 1 H NMR, 13 C NMR, a DEPT experiment, and 2D NMR spectroscopy ( 1 H- 1 H COSY, HMQC and HSQC).
  • the 1 H and 13 C spectra and the DEPT experiment confirmed the presence of fifteen carbon atoms and eleven protons in 21-57, of which all the carbons are aromatic and four are quaternary carbons at ⁇ 161.15 (C5), 151.25 (C2), 128.02 (C11) and 127.46 (C21). Assignment of the 1 H NMR and 13 C NMR spectra is indicated in Table 4. The NMR data corresponded to the data obtained by Nicolaou et al. (2004) for 2,5- diphenyloxazole.
  • PPO 2,5-diphenyloxazole
  • the carbon at position C2 would be affected the most by the deshielding effect of the adjacent oxygen and nitrogen and would therefore have a chemical shift furthermost downfield of all the carbons.
  • the carbon resonating at ⁇ 161.15 was therefore assigned to C-2.
  • the other quaternary carbon is at position C-5. This carbon is expected to have the second highest chemical shift downfield due to the deshielding effect of the oxygen in the oxazole ring.
  • the carbon with chemical shift ⁇ 151.25 was therefore assigned to this position.
  • the proton at ⁇ 7.62 (m) is not coupled to any of the other protons in 21-57.
  • This proton is bonded to the carbon at ⁇ 123.44 as determined from HSQC data. From the HMQC data, it was determined that this carbon has no long-range association with any other protons, but that the proton at ⁇ 7.62 has long-range association with C-2 ( ⁇ 161.15) and C-5 ( ⁇ 151.25) the two quaternary carbons in the oxazole ring, thereby affirming the assignment of this carbon and proton to position 4.
  • the carbon at ⁇ 128.02 is further downfield than the carbon at ⁇ 127.46, suggesting that it is also influenced by the deshielding effect of the oxygen and nitrogen on the oxazole ring and can be assigned as the carbon within ring A at position C-1 '.
  • the carbon at ⁇ 127.46 can therefore be assigned as the carbon within ring B that is bonded to the oxazole ring (C-1").
  • the two protons resonating at ⁇ 8.18 (dd) are shifted the furthest downfield of all the protons, suggesting that their chemical shift is influenced by the electron-withdrawing effect of the oxygen and nitrogen in the oxazole ring.
  • the carbon at ⁇ 127.46 was assigned to C-1" in ring B as mentioned above. From the HMQC spectrum, this carbon has long-range association with four protons, two with a chemical shift of ⁇ 5 7.86 (dd) and two others with a chemical shift of ⁇ 7.56 (m). As argued above for the signal at J 8.18, the signal at ⁇ 7.86 can be assigned to H-2" and H-6" in ring B. From the HSQC spectrum, this signal is correlated with a carbon signal at ⁇ 124.19, which is therefore assigned as C-2" and C-6" in ring B. The two protons at ⁇ 7.56 are bonded to two carbons with chemical shift ⁇ 128.92 (as seen from the HSQC spectrum).
  • the core of 21-57 contains an oxazole moiety.
  • Oxazoles are 5-membered aromatic heterocycles which contain both oxygen and nitrogen (Yeh, 2004).
  • Oxazoles are produced by enzymatic post-translational modifications of peptide-based natural products. Non- ribosomal peptide synthetases are often involved (Walsh, 2004; Keating et al., 2000).
  • the cyclodehydration of serine or threonine yields a dihydroheteroaromatic oxazoline.
  • a two-electron oxidation of the oxazoline in turn, yields an oxazole as a core structure.
  • Certain synthetic antibiotics also contain oxazole functional groups. Some belong to the well-known antibiotic class, ⁇ -lactams, e.g. the isoxazolylpenicillin oxacillin. Its side-chain containing the oxazole functional group provides steric hindrance to the binding of penicillinases. These penicillins have a stronger activity in vitro than that of methicillin, but due to their short half-lives in serum, their activity in vivo is similar to that of methicillin (Lancini et al., 1995).
  • Two sulphonamide drugs are derivatives of sulfanilamide, where one hydrogen of sulfanilamide is replaced by a methyloxazole and a dimethyloxazole, respectively (Prescott et al., 1996).
  • Oxazolines and oxazolidinones are also represented in antibacterials.
  • Phenyloxazolines have the ability to inhibit the growth of certain Gram negatives by inhibiting lipid A production, a molecule that is found in the outer membrane of most Gram negatives (Jackman et al., 2000).
  • the oxazolidinones were first identified in the 1980s (Tanitame et al., 2004). This new class of antibacterials is synthetic and acts during an early stage in protein synthesis - a novel mechanism of action.
  • Figure 8 contains some of the chemical structures of the compounds mentioned in Table 5. References for the chemical structures in Figure 8 are the same as the references indicated in Table 5.
  • step 4 is often accomplished first before the acquisition of biochemical evidence (Lancini et al., 1995).
  • HMWP2 high molecular weight protein 2 and 1
  • HMWP1 comprises the initiation subunit with the aryl carrier protein (ArCP) that via the catalytic action of salicoyl-AMP ligase (YbtE) is acylated with salicylate.
  • ArCP aryl carrier protein
  • YbtE salicoyl-AMP ligase
  • 21-57 could inhibit bacterial growth via the intercalation of DNA, especially when one considers the fact that the molecule has the ability to be planar, similar to that of acridine orange (three fused heterocyclic rings that allow it to act as a DNA intercalating agent), but this will remain purely speculative until experimentally proven.
  • AHH aryl hydrocarbon hydroxylase
  • PPO is oxidatively metabolised by the mono-oxygenase enzyme cytochrome P-450 in mice and several cytochrome P- 450 isozymes in human liver microsomes. PPO is obviously recognised as a potentially toxic agent and is therefore targeted by the mono-oxygenases. This could preclude the use of PPO as an anti-tubercular agent.
  • 21-57 or 2,5-diphenyloxazole could potentially be used as an anti- tubercular agent: it has a low cytotoxicity, it is active against M. tuberculosis H37Rv in vitro, and is available in large amounts from a variety of chemical suppliers.
  • the compound can also be used as a starting point for the chemical synthesis of other oxazole-containing compounds which can be developed with the aim of generating other anti-tuberculosis drugs.
  • the in vivo activity of PPO against M. tuberculosis needs to be determined as recommended by Sood et al. (2005) and the possibility of degradation in the human body by AHH needs to be considered/tested before any further development of this compound as a potential anti-tubercular drug is considered.
  • Thiazole and oxazole peptides biosynthesis and molecular machinery. Natural Product Reports 16, 249 - 263.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un microorganisme isolé du type de souche SPRT (=DSM 44925T = NRRL B-24448T). Le microorganisme comprend, de préférence, du matériel génétique du numéro matricule de GenBank DQ141528 (séquence d'ADNr 16S). L'invention s'étend à un procédé de production de 2,5-diphényloxazole ou de variantes ou dérivés de celui-ci, le procédé comprenant la récupération du 2,5-diphényloxazole ou de variantes ou dérivés de celui-ci à partir d'un microorganisme du type de souche SPRT (=DSM44925T = NRRL B-24448T).
PCT/IB2007/052468 2006-06-26 2007-06-26 Streptomycète et composé bioactif produit par celui-ci Ceased WO2008001306A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ZA2006/05246 2006-06-26
ZA200605246 2006-06-26

Publications (3)

Publication Number Publication Date
WO2008001306A2 true WO2008001306A2 (fr) 2008-01-03
WO2008001306A3 WO2008001306A3 (fr) 2008-08-14
WO2008001306B1 WO2008001306B1 (fr) 2008-10-09

Family

ID=38846067

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2007/052468 Ceased WO2008001306A2 (fr) 2006-06-26 2007-06-26 Streptomycète et composé bioactif produit par celui-ci

Country Status (3)

Country Link
CN (1) CN101535491A (fr)
WO (1) WO2008001306A2 (fr)
ZA (1) ZA200900598B (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109593676B (zh) * 2018-12-21 2023-04-07 江苏大学 一种用于酸菜发酵液中微生物分离的培养基及其制备方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BERDY JANOS: "Bioactive microbial metabolites - A personal view" JOURNAL OF ANTIBIOTICS (TOKYO), vol. 58, no. 1, January 2005 (2005-01), pages 1-26, XP002471302 ISSN: 0021-8820 cited in the application *
DATABASE EMBL [Online] 19 August 2005 (2005-08-19), "Streptoallomorpha polyantibiotica strain SPR 16S ribosomal RNA gene, partial sequence." XP002471303 retrieved from EBI accession no. EMBL:DQ141528 Database accession no. DQ141528 cited in the application *
NICOLAOU K C ET AL: "Chemistry and biology of diazonamide A: Second total synthesis and biological investigations" JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 126, no. 40, 13 October 2004 (2004-10-13), pages 12897-12906, XP002471301 ISSN: 0002-7863 cited in the application *

Also Published As

Publication number Publication date
WO2008001306B1 (fr) 2008-10-09
WO2008001306A3 (fr) 2008-08-14
ZA200900598B (en) 2010-05-26
CN101535491A (zh) 2009-09-16

Similar Documents

Publication Publication Date Title
Singh et al. Optimisation of process parameters for growth and bioactive metabolite produced by a salt-tolerant and alkaliphilic actinomycete, Streptomyces tanashiensis strain A2D
KR930010705B1 (ko) Fr-900520 물질의 제조방법
Iwatsuki et al. Lariatins, novel anti-mycobacterial peptides with a lasso structure, produced by Rhodococcus jostii K01-B0171
EP0627009B1 (fr) Lactones macrocycliques et une des souches productives s'y rapportant
EP0029329B1 (fr) Inhibiteur d'enzyme produit par culture d'un microorganisme de streptomyces, procédé de préparation de celui-ci et une culture du microorganisme
Driche et al. A new Streptomyces strain isolated from Saharan soil produces di-(2-ethylhexyl) phthalate, a metabolite active against methicillin-resistant Staphylococcus aureus
EI-Naggar Kosinostatin, a major secondary metabolite isolated from the culture filtrate of Streptomyces violaceusniger strain HAL64
JPH0415236B2 (fr)
JP4521145B2 (ja) 抗生物質カプラザマイシン類およびその製造法
JP4054576B2 (ja) 抗生物質トリプロペプチン類およびその製造法
CA2061239C (fr) Derives polyhydroxycyclopentane, leur preparation et leur utilisation therapeutique
EP1319666B1 (fr) Composés antibacteriens
Said et al. Enhancement of clavulanic acid production by Streptomyces sp MU-NRC77 via mutation and medium optimization
WO2008001306A2 (fr) Streptomycète et composé bioactif produit par celui-ci
JP3715651B2 (ja) 抗腫瘍イソクマリン類
JPH07102128B2 (ja) ストレプトミセス・サンダエンシスの生物学的純粋培養物
Bundale et al. Isolation of aromatic polyketide producing soil Streptomyces using combinatorial screening strategies
US4764602A (en) Antibiotics, and their production
JPH0676425B2 (ja) Ｗｓ―９３２６ａ、ｗｓ―９３２６ｂおよびそれらの誘導体
JP3830964B2 (ja) 海洋放線菌から単離した新規なチオデプシペプチド
JP4531167B2 (ja) 新規抗細菌化合物
JP2004137175A (ja) 新規抗生物質キガマイシン類とその用途
WO2004057011A1 (fr) Antibiotiques caprazamycines d, g, d1 et g1 et leur procede de production
JP3209791B2 (ja) オキサゾリン誘導体及びその製造法
WO2005111055A1 (fr) Nouvelles kigamicines antibiotiques et utilisation de celles-ci

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200780031851.0

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07805039

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

NENP Non-entry into the national phase

Ref country code: RU

WWE Wipo information: entry into national phase

Ref document number: 626/DELNP/2009

Country of ref document: IN

122 Ep: pct application non-entry in european phase

Ref document number: 07805039

Country of ref document: EP

Kind code of ref document: A2