EP4577208A1 - Composés antimicrobiens, leurs procédés de production et leurs utilisations - Google Patents

Composés antimicrobiens, leurs procédés de production et leurs utilisations

Info

Publication number
EP4577208A1
EP4577208A1 EP23857830.6A EP23857830A EP4577208A1 EP 4577208 A1 EP4577208 A1 EP 4577208A1 EP 23857830 A EP23857830 A EP 23857830A EP 4577208 A1 EP4577208 A1 EP 4577208A1
Authority
EP
European Patent Office
Prior art keywords
formula
antimicrobial compound
microbial cell
compound
engineered
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.)
Pending
Application number
EP23857830.6A
Other languages
German (de)
English (en)
Inventor
Fong Tian Wong
Kuan Chieh CHING
Siew Bee NG
Yee Hwee LIM
Yoganathan s/o KANAGA SUNDARAM
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.)
Agency for Science Technology and Research Singapore
Original Assignee
Agency for Science Technology and Research Singapore
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 Agency for Science Technology and Research Singapore filed Critical Agency for Science Technology and Research Singapore
Publication of EP4577208A1 publication Critical patent/EP4577208A1/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces

Definitions

  • the present invention relates, in general terms, to antimicrobial compounds, their methods of production and uses thereof.
  • NPs Natural products
  • BGCs biosynthetic gene clusters
  • the microbial cell is a bacterium.
  • the bacterium is Streptomyces sp.
  • Compounds 1 and 2 were tested for their antimicrobial activity against a panel of microorganisms consisting of Gram-positive and Gram-negative bacteria, as well as against one fungal strain. Namely, A. baumannii (ATCC® 19606TM), K. aerogenes (ATCC® 13048TM), P. aeruginosa (ATCC® 9027TM), S. aureus Rosenbach (ATCC® 25923TM) and A. fumigatus (ATCC® 46645TM).
  • Target organism MIC a MBC/MFC b MIC a MBC/MFC b
  • Target cell line ATCC® number IC50 (pM)
  • Figure 1 Previously, other structurally similar tetramic acid analogs (Figure 1) have been reported to exhibit antimicrobial activity, mainly against Gram-positive bacteria. For instance, equisetin had been described for its activity against Staphylococcus erythraea and Staphylococcus aureus; ascosalipyrrolidone A was active against Bacillus megaterium, Mycoptypha microsporosum, and Microbotyryum violaceum; BU-4514N and the fungal metabolite altersetin had inhibitory activity against several Gram-positive bacteria.
  • Overexpression FAS cassette consist of asOp*-FAS (accession code: WP_011030732).
  • Overexpression RedD cassette consists of tesOp*-RedD (accession code: AI.112849.1).
  • fcasOp* is a strong constitutive promoter.
  • the integration plasmid was derived by inserting the overexpression cassette into pSET152. Integration is mediated by atP site of the Streptomyces phage OC31. The completed plasmid was conjugated into Streptomyces sp A58051 from the Natural Organism Library collection (SIFBI, NPL) and genetically integrated mutants were screened and sequenced (Supplementary Figure).
  • FAS mutants are labelled as A100020 and A100023 and RedD integration mutant is A100292 ( Figure 2).
  • Wild type Streptomyces and edited mutants were cultured on ISP2 plates [malt extract broth 10 g/L, Bacto yeast extract 4 g/L, glucose 4 g/L, 20 g/L agar Bacto] at 30 °C for 5 days. Three agar plugs of 5 mm diameter from the culture plate was then used to inoculate into 4 x 250 mL Erlenmeyer flasks each containing 50 mL SV2 seed media [glucose 15 g/L, glycerol 15 g/L, soya peptone 15 g/L, calcium carbonate 1 g/L, pH 7.0] and incubated for 4 days at 30 °C, with shaking at 200 rpm.
  • a volume of 2.5 mL of the homogenized seed cultures were then inoculated into 250 mL Erlenmeyer flasks each containing 50 mL of ferment medium, CA07LB [glycerol 15 g/L, oatmeal 30 g/L, yeast extract 5 g/L, potassium dihydrogen phosphate 5 g/L, disodium hydrogen phosphate dodecahydrate 5 g/L, magnesium chloride hexahydrate 1 g/L] or CA10LB [soluble starch 20 g/L, soybean flour 15 g/L, potassium dihydrogen phosphate 3 g/L, disodium hydrogen phosphate dodecahydrate 2 g/L, magnesium sulphate heptahydrate 0.5 g/L, trace salt solution 1 mL/L (iron(II) heptahydrate 2 g/L, manganese chloride tetrahydrate 2 g/L, zinc sulfate heptahydrate 2 g/L, copper(II) sul
  • cultures were freeze dried.
  • the lyophilized samples were extracted overnight with methanol.
  • the extract mixture was passed through cellulose filter paper (Whatman Grade 4, 1004-185) and the filtrate was then dried using rotary evaporator.
  • A100020 mutant was grown on Bennet's agar (Himedia, M694) plates at 28°C for 5 days. Three agar plugs of 5mm diameter from the culture plate was then used to inoculate into 4 x 250 mL Erlenmeyer flasks each containing 50 mL of SV2 seed media and incubated for 4 days at 28°C, with shaking at 200 rpm.
  • a volume of 2.5 mL of the homogenized seed cultures were then inoculated into 250 mL Erlenmeyer flasks each containing 50 mL of ferment medium, CA07LB. All the cultures were fermented at 28°C for 10 days shaking at 200 rpm with 50 mm throw.
  • cultures were harvested and freeze dried.
  • the lyophilized cultures were extracted overnight with methanol.
  • the extract mixture was passed through cellulose filter paper (Whatman Grade 4, 1004-185) and the filtrate was then dried using rotary evaporator.
  • the extracts were analysed on an Agilent 1290 Infinity LC System coupled to an Agilent 6540 accurate-mass quadrupole time-of-flight (QTOF) mass spectrometer. 5 pL of extract was injected onto a Waters Acquity UPLC BEH Cis column, 2.1 x 50 mm, 1.7 pm. Mobile phases were water (A) and acetonitrile (B), both with 0.1 % formic acid. The analysis was performed at flow rate of 0.5 mL/min, under gradient elution of 2% B to 100% B in 8 min. Both MS and MS/MS data were acquired in positive and/or negative electrospray ionization (ESI) mode.
  • ESI electrospray ionization
  • the typical QTOF operating parameters were as follows: sheath gas nitrogen, 12 L/min at 325 °C; drying gas nitrogen flow, 12 L/min at 350 °C; nebulizer pressure, 50 psi; nozzle voltage, 1.5 kV; capillary voltage, 4 kV. Lock masses in positive ion mode: purine ion at m/z 121.0509 and HP-0921 ion at m/z 922.0098.
  • the dried extracts obtained from 1 L of fermentation were combined and partitioned with 240 mL CH2Cl2/MeOH/H2O in a ratio of 1: 1 : 1.
  • the aqueous MeOH layer was washed with 80 mL CH2CI2 (x2) and dried under reduce pressure via Buchi rotary evaporator.
  • the dried crude extract (6 g) was soaked and resuspended with 20 mL MeOH, sonicated for 5 min and centrifuged to separate the insoluble from the soluble.
  • the supernatants were transferred to 50 mL round bottom flaks and dried using Buchi rotary evaporator.
  • JASCO P-2000 digital polarimeter was used for specific rotations measurement.
  • NMR spectra were collected using Bruker DRX-400 NMR spectrometer with Cryoprobe. 5-mm BBI H, G-COSY, multiplicity-edited G-HSQC, and G-HMBC spectra) or BBO ( 13 C spectra) probe heads equipped with z-gradients. The T H and 13 C NMR chemical shifts were referenced to the residual solvent peaks for MeOH-cfo at 6H 3.31 and 6c 49.0 ppm.
  • QTOF time-of-flight
  • the minimum inhibition concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC) of the isolated compounds against a panel of microbial pathogens were determined using the microbroth dilution method. This is done according to the Clinical Laboratory Standards Institute (CLSI) guidelines, with slight modifications. Antibacterial assays were carried out with Acinetobacter baumannii (ATCC® 19606TM), Klebsiella aerogenes (ATCC® 13048TM), Pseudomonas aeruginosa (ATCC® 9027TM) and Staphylococcus aureus Rosenbach (ATCC® 25923TM) at 5 x 10 5 cells/mL.
  • Acinetobacter baumannii ATCC® 19606TM
  • Klebsiella aerogenes ATCC® 13048TM
  • Pseudomonas aeruginosa ATCC® 9027TM
  • Staphylococcus aureus Rosenbach ATCC® 25923TM
  • MBC/MFC was then evaluated by transferring 5 pL of the treated culture into fresh media in 384-well microtitre plates. The plates were incubated under the same conditions, and MBC/MFC was determined by measuring the optical density at 600 nm. All assays were performed in triplicates to ensure reproducibility.
  • A549 human lung carcinoma cells (ATCC® CCL- 185TM) were seeded at 3.3 x 10 4 cells/mL in a 384-well microplate. The cells were treated with the compounds for 72 hours and incubated at 37 °C in the presence of 5% CO2. Standard inhibitor Puromycin (Sigma-Aldrich) was used as the assay control for cytotoxicity testing.
  • the microplates were incubated with PrestoBlueTM cell viability reagent (ThermoFisher Scientific, USA) for 2 hours, followed by fluorescence reading at excitation 560 nm and emission 590 nm.
  • PrestoBlueTM cell viability reagent ThermoFisher Scientific, USA

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Microbiology (AREA)
  • Communicable Diseases (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Plant Pathology (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente divulgation concerne un procédé de biosynthèse d'un composé antimicrobien de formule (I), le procédé comprenant la culture d'une cellule microbienne qui est modifiée pour surexprimer la synthase d'acyle gras (FAS) ou RedD ; et l'isolement du composé antimicrobien de formule (I) qui est produit par la cellule microbienne ; la biosynthèse du composé antimicrobien de formule (I) étant régulée à la hausse d'au moins environ 2 fois par rapport à un analogue d'acide tétramique dérivé d'une cellule microbienne de type sauvage. La présente divulgation concerne également un composé de formule (I) et leur utilisation dans le traitement d'une maladie ou d'un état microbien.
EP23857830.6A 2022-08-23 2023-06-26 Composés antimicrobiens, leurs procédés de production et leurs utilisations Pending EP4577208A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SG10202250805D 2022-08-23
PCT/SG2023/050447 WO2024043829A1 (fr) 2022-08-23 2023-06-26 Composés antimicrobiens, leurs procédés de production et leurs utilisations

Publications (1)

Publication Number Publication Date
EP4577208A1 true EP4577208A1 (fr) 2025-07-02

Family

ID=90014177

Family Applications (1)

Application Number Title Priority Date Filing Date
EP23857830.6A Pending EP4577208A1 (fr) 2022-08-23 2023-06-26 Composés antimicrobiens, leurs procédés de production et leurs utilisations

Country Status (3)

Country Link
EP (1) EP4577208A1 (fr)
JP (1) JP2025528388A (fr)
WO (1) WO2024043829A1 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000086627A (ja) * 1998-09-10 2000-03-28 Banyu Pharmaceut Co Ltd 抗菌性物質ｂｅ−５４４７６及びその製造法
US9944925B2 (en) * 2013-08-02 2018-04-17 Enevolv, Inc. Processes and host cells for genome, pathway, and biomolecular engineering

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JP2025528388A (ja) 2025-08-28
WO2024043829A1 (fr) 2024-02-29

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