Macrolide antibiotic

When pure CYP107W1 and oligomycin C interact, type I binding occurs, with a Kd value of 14.4 μM [2].

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Oligomycin C (compound II) showed strong antifungal activity against several plant pathogenic fungi as determined by minimum inhibitory concentration (MIC) assays. The MIC values were: against Aspergillus niger ATCC 10335 – 2 μg/mL; against Alternaria alternata ATCC 13963 – 6 μg/mL; against Botrytis cinerea (local isolate) – 6 μg/mL; against Phytophthora capsici (local isolate) – 10 μg/mL. No activity was found against the bacterial strains Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Pseudomonas fluorescens ATCC 13525 [1].
In a separate study, Oligomycin C was used as a substrate for purified CYP107W1 enzyme. The enzyme converted oligomycin C to oligomycin A via regioselective hydroxylation at the C12 position, as confirmed by LC-mass spectrometry (retention time of oligomycin C: 44.4 min; retention time of oligomycin A: 30.3 min; molecular weight increase of 16 from 775 to 791) [2].

Oligomycin C exhibited a good control effect on tobacco brown spot caused by Alternaria alternata in in vivo tests at a concentration of 60 μg/mL [1].

Streptomyces avermitilis contains 33 cytochrome P450 genes in its genome, many of which play important roles in the biosynthesis process of antimicrobial agents. Here, we characterized the biochemical function and structure of CYP107W1 from S. avermitilis, which is responsible for the 12-hydroxylation reaction of oligomycin C. CYP107W1 was expressed and purified from Escherichia coli. Purified proteins exhibited the typical CO-binding spectrum of P450. Interaction of oligomycin C and oligomycin A (12-hydroxylated oligomycin C) with purified CYP107W1 resulted in a type I binding with Kd values of 14.4 ± 0.7 μM and 2.0 ± 0.1 μM, respectively. LC-mass spectrometry analysis showed that CYP107W1 produced oligomycin A by regioselectively hydroxylating C12 of oligomycin C. Steady-state kinetic analysis yielded a kcat value of 0.2 min(-1) and a Km value of 18 μM. The crystal structure of CYP107W1 was determined at 2.1 Å resolution. The overall P450 folding conformations are well conserved, and the open access binding pocket for the large macrolide oligomycin C was observed above the distal side of heme. This study of CYP107W1 can help a better understanding of clinically important P450 enzymes as well as their optimization and engineering for synthesizing novel antibacterial agents and other pharmaceutically important compounds.[2]

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The interaction of Oligomycin C with purified CYP107W1 enzyme was assessed by spectral binding titration. Purified CYP107W1 enzyme was diluted to 3 μM in 100 mM potassium phosphate buffer (pH 7.4) and divided between two glass cuvettes. Spectra between 350-500 nm were recorded while subsequently adding increasing concentrations of oligomycin C. The difference in absorbance between the wavelength maximum and minimum was plotted against ligand concentration. Binding of oligomycin C resulted in a type I binding mode (shift from low-spin to high-spin spectra with an increase at 385 nm and a decrease at 420 nm), indicating displacement of an iron-bound water molecule. The calculated dissociation constant (Kd) for oligomycin C binding to CYP107W1 was 14.4 ± 0.7 μM [2].
The catalytic activity of CYP107W1 toward Oligomycin C was determined using an in vitro hydroxylation assay. The reaction mixture contained 200 pmol purified P450 enzyme, 40 μg/mL spinach ferredoxin, and 0.04 U/mL spinach ferredoxin reductase in 0.50 mL of 100 mM potassium phosphate buffer (pH 7.4) along with specified amounts of oligomycin C. Reactions were initiated by adding 50 μL of an NADPH-generating system (10 mM glucose 6-phosphate, 0.5 mM NADP+, and 1.0 IU/mL glucose 6-phosphate dehydrogenase). After 30 min incubation at 37°C, reactions were terminated by adding 1 mL CH2Cl2, followed by vortex mixing and centrifugation. Reaction products were recovered from the organic phase after drying under N2. LC-mass spectrometry analysis was performed on a Shimadzu LCMS-2010 EV system using a Shimpack VP-ODS column (2.0 mm i.d. × 250 mm) with a mobile phase of CH3CN–MeOH–H2O (60:16:24, v/v/v) at a flow rate of 0.15 mL/min. Mass spectra were recorded by electrospray ionization in negative mode. The product was identified as oligomycin A (12‑hydroxylated oligomycin C) [2].
Steady-state kinetic analysis of Oligomycin C hydroxylation by CYP107W1 was performed. The kinetic parameters were obtained using the Michaelis-Menten equation: the kcat value was approximately 0.21 ± 0.01 min⁻¹, and the calculated Km value was 17.9 ± 1.3 μM [2].

During the screening program for fungicides, one actinomycete strain ECO 00047 was isolated with the potential activity against fungus. According to the morphology and analysis of the nucleotide sequence of the 16S rRNA gene (1500 bp) this isolate was identified as Streptomyces diastaticus. The active compounds were separated by silica gel column chromatography, Sephadex LH-20 gel filtration and then purified by flash chromatography on C18 (20-45 microm). The chemical structure of the bioactive compounds I and II were elucidated, based on the spectroscopic data of MS, IR, UV, 1H-NMR, 13C-NMR and X-ray single crystal diffraction analysis. Compounds I and II were identical with oligomycins A and C, the macrolide antibiotics which have been known to be produced by Streptomyces diastatochromogenes, S. libani and S. avermitilis. The two compounds exhibited a strong activity against Aspergillus niger, Alternaria alternata, Botrytis cinerea and Phytophthora capsici but no activity toward bacteria. Although the two above antibiotics were known, their isolation has so far not been reported from S. diastaticus.[1]

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The antifungal activity of Oligomycin C was determined by a 2-fold dilution method using potato dextrose agar (PDA) as assay medium for fungi. The test organisms included Aspergillus niger ATCC 10335, Alternaria alternata ATCC 13963, Botrytis cinerea (local isolate), and Phytophthora capsici (local isolate). MIC values (in μg/mL) were read after a 48‑h incubation at 30°C [1].

Oligomycin C was tested in vivo for control of tobacco brown spot caused by Alternaria alternata at a concentration of 60 μg/mL. No further details on animal model, dosing regimen, formulation, or route of administration are provided in the literature [1].

Intraperitoneal LD50 in mice: 8300 ug/kg

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[1]. Oligomycins A and C, major secondary metabolites isolated from the newly isolated strain Streptomyces diastaticus. Folia Microbiol (Praha). 2010;55(1):10-16.

[2]. Functional characterization of CYP107W1 from Streptomyces avermitilis and biosynthesis of macrolide oligomycin A. Arch Biochem Biophys. 2015;575:1-7.

See also: Oligomycin C (note moved to).

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Oligomycin C is a macrolide antibiotic known to be produced by various Streptomyces species including S. diastatochromogenes, S. libani, and S. avermitilis. Its isolation from S. diastaticus had not been reported prior to this study [1]. Oligomycins are known to inhibit ATP synthase by blocking its proton channel, which is necessary for the oxidative phosphorylation of ADP [2]. In this work, oligomycin C was converted to oligomycin A by the cytochrome P450 enzyme CYP107W1 from S. avermitilis, which catalyzes regioselective hydroxylation at the C12 position [2]. The crystal structure of CYP107W1 (determined at 2.1 Å resolution, PDB ID: 4WPVZ) revealed a large, open substrate-binding pocket with hydrophobic residues (e.g., Ala239, Thr243, Met85, Ser88, Leu89, Val90, Leu285, Gly289, Gly290, Ile291, Ile292, Ser392, Ile393, Ile394) that may accommodate the bulky hydrophobic chain of oligomycin C [2].

C45H74O10

775.06306

774.528

11052-72-5

Oligomycin A;579-13-5;Oligomycin;1404-19-9;Oligomycin B;11050-94-5

5472287

Typically exists as solid at room temperature

1.12g/cm3

876.2ºC at 760mmHg

249.4ºC

0mmHg at 25°C

1.535

6.767

4

10

3

55

1340

17

CC[C@H]1C=CC=CC[C@@H](C)[C@H](O)[C@H](C)C(=O)[C@@H](C)[C@H](O)[C@@H](C)C(=O)[C@@H](C)[C@H](O)[C@@H](C)C=CC(=O)OC2[C@@H](C)C(OC3([C@@H]2C)CC[C@H](C)[C@H](C[C@H](O)C)O3)CC1 |t:3,5,30,&1:2,8,10,12,16,18,20,24,26,28,36,41,45,47,49|

CMMLZMMKTYEOKV-HQCSJJBPSA-N

InChI=1S/C45H74O10/c1-12-35-17-15-13-14-16-26(3)39(48)30(7)41(50)32(9)43(52)33(10)42(51)31(8)40(49)27(4)18-21-38(47)53-44-29(6)36(20-19-35)54-45(34(44)11)23-22-25(2)37(55-45)24-28(5)46/h13-15,17-18,21,25-37,39-40,43-44,46,48-49,52H,12,16,19-20,22-24H2,1-11H3/b14-13+,17-15+,21-18+/t25-,26+,27-,28?,29+,30-,31-,32+,33-,34-,35-,36-,37-,39-,40+,43-,44+,45-/m1/s1

(1S,4E,5'R,6R,6'R,7S,8R,10S,11R,12R,14R,15R,16S,18E,20E,22S,25R,27S,28R,29S)-22-ethyl-7,11,15-trihydroxy-6'-(2-hydroxypropyl)-5',6,8,10,12,14,16,28,29-nonamethylspiro[2,26-dioxabicyclo[23.3.1]nonacosa-4,18,20-triene-27,2'-oxane]-3,9,13-trione

Oligomycin C; 12-deoxyoligomycin A; 11052-72-5; Oligomycin A, 12-deoxy-; 4F7MA81184; (-)-Oligomycin C; (1R,4E,5'S,6S,6'S,7R,8S,10R,11S,12S,14S,15S,16R,18E,20E,22R,25S,27R,28S,29R)-22-ethyl-7,11,15-trihydroxy-6'-[(2R)-2-hydroxypropyl]-5',6,8,10,12,14,16,28,29-nonamethyl-3',4',5',6'-tetrahydro-3H,9H,13H-spiro[2,26-dioxabicyclo[23.3.1]nonacosa-4,18,20-triene-27,2'-pyran]-3,9,13-trione; UNII-4F7MA81184;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)