WO1986005791A1 - A novel peptide, a microbiological process for its production and use theeof as an antibiotic, cytotoxic and phythotoxic agent - Google Patents

A novel peptide, a microbiological process for its production and use theeof as an antibiotic, cytotoxic and phythotoxic agent Download PDF

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Publication number
WO1986005791A1
WO1986005791A1 PCT/IT1986/000023 IT8600023W WO8605791A1 WO 1986005791 A1 WO1986005791 A1 WO 1986005791A1 IT 8600023 W IT8600023 W IT 8600023W WO 8605791 A1 WO8605791 A1 WO 8605791A1
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iii
cytotoxic
phythotoxic
formula
leu
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PCT/IT1986/000023
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French (fr)
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Lorenzo Tuttobello
Carlo Giulio Casinovi
Carlo Rossi
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Consiglio Nazionale Delle Ricerche
Istituto Superiore di Sanità
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal 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
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/79—Paecilomyces

Definitions

  • a novel peptide a microbiological process for its production and use thereof as an antibiotic, cytotoxic and phythotoxic agent
  • the present invention relates to a novel peptide compound, a microbiological process for its production and the use thereof as an antibiotic, cytotoxic and phythotoxic agent.
  • MPD methylamino-2-aminopropane Peptide compounds I and II have resulted identical with Leucinostatins A and B independently isolated by a different group of researchers from Paecilomyces lilacinus (CA, 1983, Vol. 98, 15460x).
  • Fungus Paecilomyces marquandii (syn. Spicaria violacea) has been deposited at CBS (Centraalbureau voor Schimmelcutures, 3740 A6 Baarn, The Netherlands) under the deposit number CBS 141.85, dated 5 February 1985.
  • An object of the present invention is a novel peptide compound of formula III
  • a Further object of the present invention is a microbiological process for the production of the novel peptide, characterized in that Fungus Paecilomyces marquandii (syn. Spicaria violacea) is treated in submerged cultures, comprising: a) a first inoculation step, b) a second multiplication s t ep , c ) a t h i rd p rod u c t i on step in a sterile, aerated and pressurized environment, and d) a final step of solvent extraction of the substances so obtained and chromatographic separation for obtaining the peptide compound III.
  • the third step can be carried out by using the sparger and antivortex system, or simplified vortex system, or an air-lift system.
  • Solvents which can be used in the extraction step are benzene, butyl acetate, chloroform, butanol and similar solvents of fats. Benzene is particularly preferred for its higher extraction yield.
  • Culture media which can be used are semisynthetic media (for example media in which the organic source is yeast autolysate) or complex organic media based upon by-products of food industry (flour from peanut and soy extraction, corn steep liquor, potato and bean broth and the like).
  • a further object of the present invention is the use of the peptide as an antibiotic, cytotoxic and phythotoxic agent.
  • the compound according to the present invention is obtained in a mixture with the other leucinostatins (10% of the whole) at the third step in submerged cultures: first inoculation step in flasks containing 3% potato-glucose broth at pH 6 for 5-6 days on a rotating stirrer (220 rpm) at 27°C; second multiplication step in a 5 liter Erlenmeyer flask containing 900 ml of the same medium, by incubation for three days in similar conditions; third production step in Czapek-Dox fermentors of effective 50 liters with 0.1% yeast autolysate, 3% glucose at pH 6, in conventional conditions (propeller 130 mm, 200 rpm, sparger, 3 antivortexes, 27°C, 1 atm pressure, 50 liters/min outlet air).
  • the toxin extraction is generally effected on the fourth fermentation day, 24 hours after glucose exhaustion.
  • the 57 signals (see Table II) which appear in the 13 C spectrum (CDCI 3 ; 50 MHz) show chemical shifts and multiplicities in accordance with the allotted structure.
  • the "in vitro" antibacterial activity of the peptide compound according to the present invention was verified by carrying out a test on staphylococci recently isolated in hospital, which are resistant to some antibiotics. The results are referred in Table III. The results of additional tests on antibacterial and antimycotic activity are referred in Tables IV and V. The minimum inhibiting concentration (MIC) for determining the antibacterial and antimycotic activity was determined as follows.
  • Table III compares the activity of compound III with cefazoline on different resistent staphylococci isolated in different hospitals in Lombardy (Italy)
  • the cytotoxic activity of the peptide according to the present invention was evaluated on some cell Lines, such as HeLA, KB, and the results of the concentration inhibiting a 50% cell growth are referred in Table VI.
  • the phythotoxic activity evaluated by the modified Gaumann test (Ann. Ist. Super. Sanita, 4, 317-332 (1968)) on Supermarmande tomato cuttings is 2 ⁇ g/ml. Said concentration produces, after a 2 days inhibition, an irreversible and progressive withering of the tested cuttings.

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  • Bioinformatics & Cheminformatics (AREA)
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  • Microbiology (AREA)
  • Mycology (AREA)
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  • General Engineering & Computer Science (AREA)
  • Tropical Medicine & Parasitology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

A novel peptide isolated from submerged cultures of fungus Paecilomyces marquandii (syn. Spicaria violacea) showing an antibiotic, cytotoxic and phythotoxic activity.

Description

"A novel peptide, a microbiological process for its production and use thereof as an antibiotic, cytotoxic and phythotoxic agent"
SPECIFICATION
The present invention relates to a novel peptide compound, a microbiological process for its production and the use thereof as an antibiotic, cytotoxic and phythotoxic agent.
Description of the prior art Previous studies on submerged cultures of Fungus paecilomyces marquandii (syn. Spicaria violacea) have shown the presence of a number of substances having an antibiotic, cytotoxic and phythotoxic activity. From said substances the main components of formulas I and II have been isolated and analyzed (PhythopatoLogi a Mediterranea - Vol. XXII, 1983, pages 103-106 and 209-211) MeEse-MePro-AIMOD-Hyleu-Aib-Leu-Leu-Aib-Aib-βAla-DAP
(I) MeEse-MePro-AIMOD-Hyleu-Aib-Leu-Leu-Aib-Aib- βAla-MAP
(II) wherein the abbreviations have the following meaning: MeEse 3α-methyIhexenoic acid
MePro cis-4-methyIproline
AIMOD 2-amino-6-hydroxy-4-methyl- 8-ketodecanoic acid HyLeu -treo-β-hydroxyleucine Aib α-aminoisobutyric acid
Leu leucine β-Ala β-alanine
DAP dimethylamino-2-aminopropane
MPD methylamino-2-aminopropane Peptide compounds I and II have resulted identical with Leucinostatins A and B independently isolated by a different group of researchers from Paecilomyces lilacinus (CA, 1983, Vol. 98, 15460x). Fungus Paecilomyces marquandii (syn. Spicaria violacea) has been deposited at CBS (Centraalbureau voor Schimmelcutures, 3740 A6 Baarn, The Netherlands) under the deposit number CBS 141.85, dated 5 February 1985.
Summary of the invention
Upon further studies on isolation and characterization of the components in the mixture of substances obtained from submerged cultures, according to the invention another peptide substance not described in the prior art was separated from the culture filtrates obtained by benzene extraction. The novel substances has the following formula III:
MeEse-MePro-Leu-HyLeu-Aib-Leu-Leu-Aib-Aib-β Ala-DAP
(III) wherein the abbreviations have the same meaning as indicated above, having an antibiotic, cytotoxic and phythotoxic activity.
An object of the present invention is a novel peptide compound of formula III
MeEse-MePro-Leu-HyLeu-Aib-Leu-Leu-Aib-Aib-β Ala-DAP
(III)
A Further object of the present invention is a microbiological process for the production of the novel peptide, characterized in that Fungus Paecilomyces marquandii (syn. Spicaria violacea) is treated in submerged cultures, comprising: a) a first inoculation step, b) a second multiplication s t ep , c ) a t h i rd p rod u c t i on step in a sterile, aerated and pressurized environment, and d) a final step of solvent extraction of the substances so obtained and chromatographic separation for obtaining the peptide compound III.
The third step can be carried out by using the sparger and antivortex system, or simplified vortex system, or an air-lift system. Solvents which can be used in the extraction step are benzene, butyl acetate, chloroform, butanol and similar solvents of fats. Benzene is particularly preferred for its higher extraction yield.
Culture media which can be used are semisynthetic media (for example media in which the organic source is yeast autolysate) or complex organic media based upon by-products of food industry (flour from peanut and soy extraction, corn steep liquor, potato and bean broth and the like). A further object of the present invention is the use of the peptide as an antibiotic, cytotoxic and phythotoxic agent.
Example
The compound according to the present invention is obtained in a mixture with the other leucinostatins (10% of the whole) at the third step in submerged cultures: first inoculation step in flasks containing 3% potato-glucose broth at pH 6 for 5-6 days on a rotating stirrer (220 rpm) at 27°C; second multiplication step in a 5 liter Erlenmeyer flask containing 900 ml of the same medium, by incubation for three days in similar conditions; third production step in Czapek-Dox fermentors of effective 50 liters with 0.1% yeast autolysate, 3% glucose at pH 6, in conventional conditions (propeller 130 mm, 200 rpm, sparger, 3 antivortexes, 27°C, 1 atm pressure, 50 liters/min outlet air).
The toxin extraction is generally effected on the fourth fermentation day, 24 hours after glucose exhaustion.
The culture broth, after mycelium filtration was repeatedly extracted with benzene at the fermentation pH (7.6 - 7.8); after solvent evaporation, a light yellow oily residue is obtained (about 7 g/100 liters) comprising at least about ten substances of basic character as shown by paper electrophoresis and by the fact that they can be detected with the Dragendorff reagent.
After continuous chromotographies on silica gel column with suitable eluent mixtures, the practically pure compound III was obtained from the residue, besides the compounds I and II.
Compound III FAB MS m/z 1118 (MH+ ), m.p. 182-184°C [α]D-34.76
(c 1.05 CHCl3), UV(EtOH) λ max 202 nm ( ε =27930), 220 ( ε =18380), IR (CHCl3) V max 3310, 1650 cm-1, elemental analysis: found: C, 59.91; H, 9.50; N, 13.62;
0, 16.78; for C57H103N11 O11. H2O calc. %: C, 60.26;
H, 9.25; N, 13.56; 0, 16.91.
The peptide nature of compound III is self-evident from the chemical-physical data and from examination of the 1H and 13C RMN spectra.
Compound III gave a negative reaction to the ninhydrine test and to any methylation attempt and a positive reaction to the Reindel-Hoppe reagent. These reactions suggest the presence of a peptide having blocked terminal C and N atoms.
Compound III was hydrolized with HCl 6N at 110°C for 24 hours in a sealed tube and conventionally analyzed by an automatic analysis apparatus for aminoacids. The results show that it consists of one mole of hydroxy leucine (HyLeu), one mole cis-4-methyIproline (HePro), three moles α -aminoisobutyric acid (Aib), three moles of leucine (Leu) and one mole of p-alanine (β -AIa) listed in the order of their increasing retention times; said proportions are also easily deduced from the 1H
RMN spectra (see Table I).
By extraction of the acid hydrolysate with ethtyl ether an oily residue was obtained, which, after purification, showed to be 3- α -methyIhexenoic acid (MeEse). [1H RMN (90 MHz; CDCl3)δ : 0.83 (3H, t, J=7 Hz), 1.05 (3H, d, J=7 Hz, 1.43 (2H, m), 2.24 (1H, m), 5.73 (1H, d, J=16 Hz), 6.93 (1H, dd, J=16 and 8 Hz; 13C RMN (25 MHz; CDCl3) δ : 171.23 (s), 156.68 (d), 118.79 (d), 37.72 (d), 28.29 (q), 18.29 (q)].
The acid hydrolysate, after careful aIkalinization with NaOH aqueous solution, was continuously extracted with ethyl ether. The strongly basic oily residue after purification, resulted corresponding to dimethylamino-2-aminopropane [DAP ] . [1H RMN (90 MHz; CDCI3) δ : 1.34 (3H, d, J =7 Hz),
3.07 and 3.09 (3H each, s), 3.47 (2H, m) and 3.90
(1H, m; 13C RMN (25 MHz; CDCI3) δ : 18.7 (q), 42.6
(d), 44.76 (2xq), 63.6 (t)] . The complete attribution of the proton spectrum as listed in Table I has been made possible by using mono and bidimensionaI spectra (COSY) at 400 MHz.
The 57 signals (see Table II) which appear in the 13C spectrum (CDCI3; 50 MHz) show chemical shifts and multiplicities in accordance with the allotted structure.
TABLE I 1H RMN spectrum
Figure imgf000008_0001
Figure imgf000009_0002
The sequence of the units forming the peptide compound III was deduced by FAB mass spectrum based upon two main fragmentation schemes +CA and +CAlk, as hereinafter referred :
Figure imgf000009_0001
The foregoing evidences the structure of formula III for the peptide according to the present invention.
Farmacologi cal activity
The "in vitro" antibacterial activity of the peptide compound according to the present invention was verified by carrying out a test on staphylococci recently isolated in hospital, which are resistant to some antibiotics. The results are referred in Table III. The results of additional tests on antibacterial and antimycotic activity are referred in Tables IV and V. The minimum inhibiting concentration (MIC) for determining the antibacterial and antimycotic activity was determined as follows.
5 mg of the compound of the invention were dissolved into 0.5 ml dimethylsulfoxide and diluted to 10 ml with sterile distilled water; portions of the above starting solution are subsequently diluted with sterile distiIled water.
Table III compares the activity of compound III with cefazoline on different resistent staphylococci isolated in different hospitals in Lombardy (Italy)
Table III "In vitro" antibacterial activity on recently - in hospital - isolated resistant stafilococci of compound III in coimparison with β -lactamic cefazoline. Inhibiting concentrations ( μm/ml).
Figure imgf000011_0001
TABLE I V
"In vitro" antibacterial activity. Minimal inhibiting concentration (μm/ml) in sensitivity test Agar (oxoid)
Strains Compound III Streptococcus pneumoniae ATCC 6301 12.5
St reptococuccus β -haemolyticus C 203 12.5
Streptococcus faecalis ATCC 6057 25.0
Staphy lococcus aureus Smith 6.2 Staphy lococcus aureus 39/2 (penicillin resistant) 6.2 Escherichia coli 0147/ISS 100.0
Pseudomonas aeruginosa 100.0
Salmonella typhi 100.0
Shigella sonnei B68 100.0
Proteus vulgaris 100.0 TABLE V
"In vitro" antimycotic activity. Minimal inhibiting concentration (μm/ml) in Agar Sabouraud.
Strains Compound III
Microsporum gypseum ATCC 14683 >100.0 Microsporum cookey Negroni >100.0
Microsporum canis Pavia 3.1
Trichophyton mentagrophytes I. dC 6.2
Trichophyton mentagrophytes F 0.8 Trichophyton tonsurans L 0.8
Candida albicans CBS 562 10.0
Candida albicans L 100.0
Candida utilis 93/ISS 25.0
Torulopsis famata 4077/ISS 2.0
Torulopsis stellata 4122/ISS 25.0
Cryptococcus neoformans 4710/ISS 4.0
Sa c cha romyces cerevisiae 92/ISS 25.0
Microsporum canis 2155/ISS 4.0
Trichophyton verrucosum 1466/ISS 2.0
Tricosporon undulatom 6805/ISS 10.0
Tricosporon cutaneum 6742/ISS 45.0 Cytotoxic activity
The cytotoxic activity of the peptide according to the present invention was evaluated on some cell Lines, such as HeLA, KB, and the results of the concentration inhibiting a 50% cell growth are referred in Table VI.
Table VI
"In vitro" cytotoxic activity. Concentration (ID50) inhibiting a 50% cell growth ( ug/ml). Cell line ID50
HeLA 820.00
KB 0.95
P.388/F < 1.00
P.388 adriamycin resistant from 100.00 to 1000.00
Phythotoxic activity
The phythotoxic activity evaluated by the modified Gaumann test (Ann. Ist. Super. Sanita, 4, 317-332 (1968)) on Supermarmande tomato cuttings is 2 μg/ml. Said concentration produces, after a 2 days inhibition, an irreversible and progressive withering of the tested cuttings.
DL50 calculated with white Swiss mice resulted practically identical both intravenously and intraperitoneally (0.7 mg/kg body weight).
Figure imgf000014_0001

Claims

C lai ms
1. The peptide compound of formula (III) MeEse-MePro-Leu-HyLeu-Aib-Leu-Leu-Aib-βAla-DAP
(III) 2. A microbiological process for producing a peptide compound of formula (III) as recited in claim 1, characterized in that the fungus Paecilomyces marquandii (syn. Spicaria violacea) is treated in submerged cultures by the steps of a) inoculation; b) multiplication; c) fermentative production in sterile, aerated and pressurized environment followed by separation by solvent extraction of a mixture of substances and insulation by chromatographic separation of the compounds of formula (III).
3. An antibiotic against gram-positive microorganisms, antimycotic, cytotoxic and phythotoxic active agent consisting of the peptide compund of formula (III), as defined in claim 1.
4. Composition having an antibiotic against gram- positive microorganisms, and an antimycotic, cytotoxic and phythotoxic activity, comprising the peptide compound of formula (III) as recited in claim 1, and compatible excipient and carriers.
PCT/IT1986/000023 1985-03-28 1986-03-21 A novel peptide, a microbiological process for its production and use theeof as an antibiotic, cytotoxic and phythotoxic agent WO1986005791A1 (en)

Applications Claiming Priority (2)

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IT47889A/85 1985-03-28
IT47889/85A IT1199965B (en) 1985-03-28 1985-03-28 PEPTIDE, MICROBIOLOGICAL PROCEDURE FOR ITS PRODUCTION AND ITS USE AS ANTIBIOTIC, CYTOTOXIC AND PHYTO-TOXIC AGENT

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Vol. 100, 1984 (Columbus, Ohio, US) C.G. CASSINOVI et al.: "Structural Elucidation of the Phytotoxic and Antibiotic Peptides Produced by Submerged Cultures of Paecilomyces Marquandii (Massee) Hughes", see page 273, Abstract No. 20115c, & Phytopathol. Mediterr. 1983, 22, 103-6 *
CHEMICAL ABSTRACTS, Vol. 101, 1984, (Columbus, Ohio, US) C. ROSSI et al.: "Two Phytotoxic, Antibiotic Peptides Produced by Submerged Cultures of Paecilomyces Marquandii (Massee) Hughes", see page 330, Abstract No. 166756e, & Phytopathol. Mediterr. 1983, 22 (3), 209-11 *
CHEMICAL ABSTRACTS, Vol. 98, 1983 (Columbus, Ohio, US) see page 412, Abstract No. 15460x, & JP, A, 5767547 (Beppu, Teruhiko) 24 April 1982 (cited in the application) *

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AU5662086A (en) 1986-10-23
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IT1199965B (en) 1989-01-05
IT8547889A1 (en) 1986-09-28

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