ATP synthase; Antibiotic
The primary target is the mitochondrial F1F0 ATP synthase (EC 3.6.3.14), specifically the Fo subunit proton channel. Oligomycin B binds to the Fo component and blocks proton translocation through the inner mitochondrial membrane, inhibiting both ATP synthesis and ATP hydrolysis. It is a nonselective inhibitor that affects both synthase and hydrolase activities. Interaction with ATP synthase shows complex, multi-step kinetics. The compound also acts as a mitochondrial respiratory-chain inhibitor.

Four antibiotics (pamamycin, oligomycin A, oligomycin B and echinosporin) were isolated and characterized from the fermentation broth of the marine Streptomyces strains B8496 and B8739. Bioassays revealed that each of these compounds impaired motility and caused subsequent lysis of P. viticola zoospores in a dose- and time-dependent manner. Pamamycin displayed the strongest motility inhibitory and lytic activities (IC50 0.1 μg mL(-1)) followed by oligomycin B (IC50 0.15 and 0.2 μg mL(-1)) and oligomycin F (IC50 0.3 and 0.5 μg mL(-1)). Oligomycin A and echinosporin also showed motility inhibitory activities against the zoospores with IC50 values of 3.0 and 10.0 μg mL(-1), respectively. This is the first report of motility inhibitory and lytic activities of these antibiotics against zoospores of a phytopathogenic peronosporomycete. Structures of all the isolated compounds were determined based on detailed spectroscopic analysis.[2]

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In vitro, Oligomycin B blocks mitochondrial ATP synthesis, proton translocation, and O2 uptake in isolated mitochondria. It elevates mitochondrial membrane potential and induces apoptosis and necrosis in various cell types. The compound is active at nanomolar concentrations and is used as a reference inhibitor in mitochondrial respiration studies. Oligomycin B is also active against bacterial ATP synthases. It suppresses TNF-induced apoptosis by inhibiting the F0 part of H+-ATP synthase.

Production of zoospores and bioassay[2]
For the isolation of sporangia of Plasmopara viticola, infected leaves of grapevine. The strain was maintained by continuous culturing on the lower surface of young grapevine leaves on Petri dishes containing 1.5% water agar at 25°C and 95% relative humidity (Islam and Tiedemann 2011; Islam, Tiedemann and Laatsch 2011). On the sixth day of cultivation, the sporangiophores containing the lemon-shaped sporangia were harvested into an Eppendorf vial by a micro-vacuum cleaner. The freshly harvested sporangia were separated from sporangiophores by filtration through a nylon sieve (50 mesh), washed twice with distilled water and then incubated in sterilized tap water (3 × 104 sporangia per mL) in the dark for 6 h at room temperature (23°C) to release zoospores. The zoospores remained motile up to 12 h in sterilized water, and they were used for the motility bioassay (Islam and Tahara 2001). Stock solutions of the antibiotics were first prepared in dimethyl sulfoxide (DMSO) and then diluted with distilled water. The final concentration of DMSO in the zoospore suspension never exceeded 1% (v/v), a condition that does not affect zoospore motility. The effects of compounds on motility and viability of zoospores were assayed using protocols described earlier (Islam et al.2005; Abdalla et al.2011; Islam, Tiedemann and Laatsch 2011). Motility of the zoospores was observed under a light microscope at 100-fold magnification. Quantification of time-course changes of motility and lysis of the zoospores were carried out as described earlier (Islam, Tiedemann and Laatsch 2011). Each treatment was replicated three times. The mean value % ±SE (standard error) of the affected spores in each treatment was calculated and the statistical significance was determined by one- and two-way Analysis of Varience (ANOVA) and t-test. Two-way ANOVA (see Table S1, Supporting Information) was used to test the significance of differences in motility inhibition and lytic activities of the antibiotics at different times and doses of treatment. The difference is considered statistically significant with a value of P < 0.05.

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In vivo, Oligomycin B reduces the rate of ATP depletion during ischemia in cardiac tissue, consistent with ATP synthase as an early target. As a mitochondrial toxin, it is used in animal studies to investigate the role of ATP synthase in ischemia-reperfusion injury and metabolic disorders. However, systemic toxicity limits its therapeutic application. It remains a research tool for studying mitochondrial bioenergetics in vivo.

For non-cellular ATP synthase assays, submitochondrial particles or purified F1F0 ATP synthase are incubated with Oligomycin B in assay buffer containing ATP or ADP. ATP synthesis or hydrolysis is measured by monitoring NADH oxidation at 340 nm using a coupled enzyme system or by measuring 32P-ATP formation. IC50 values are determined from dose-response curves. Oxygen consumption is measured using a Clark electrode.

Scabrosin esters (SEs), which have been recently isolated from the lichen Xanthoparmelia scabrosa, belong to the epipolythiodioxopiperazine (ETP) class of secondary metabolites characterized by possession of a reactive disulfide bond. Colony forming assays show that these toxins are active against human tumor cell lines at nanomolar concentrations. Other members of the ETP class of toxins such as gliotoxin have been shown to induce apoptosis in cells, although the cellular target(s) of the ETP toxins is currently unknown. ETP toxins have been shown to inhibit a variety of enzymes via interaction with sensitive cysteine residues. Here we show that the typical scabrosin ester acetate butyrate induces early mitochondrial membrane hyperpolarization assessed by JC-1 staining accompanied by apoptotic cell death. The toxin lowers ATP in intact cells and inhibits the rate of ATP synthesis in permeabilzed cells. Comparison with the effects of the known ATP synthase inhibitor oligomycin B is consistent with ATP synthase as an early target in scabrosin ester-induced cell death.[1]

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For cell-based assays, cells are cultured and treated with Oligomycin B (typically 1-10 µM) for 1-24 hours. Mitochondrial membrane potential is measured using TMRM or JC-1. Cellular ATP levels are measured using luciferase-based assays. Apoptosis is assessed by Annexin V/PI staining and caspase-3/7 activity. Mitochondrial respiration is measured using Seahorse extracellular flux analysis with oligomycin as an inhibitor of ATP-linked respiration.

Production of zoospores and bioassay[2]
For the isolation of sporangia of Plasmopara viticola, infected leaves of grapevine. The strain was maintained by continuous culturing on the lower surface of young grapevine leaves on Petri dishes containing 1.5% water agar at 25°C and 95% relative humidity (Islam and Tiedemann 2011; Islam, Tiedemann and Laatsch 2011). On the sixth day of cultivation, the sporangiophores containing the lemon-shaped sporangia were harvested into an Eppendorf vial by a micro-vacuum cleaner. The freshly harvested sporangia were separated from sporangiophores by filtration through a nylon sieve (50 mesh), washed twice with distilled water and then incubated in sterilized tap water (3 × 104 sporangia per mL) in the dark for 6 h at room temperature (23°C) to release zoospores. The zoospores remained motile up to 12 h in sterilized water, and they were used for the motility bioassay (Islam and Tahara 2001). Stock solutions of the antibiotics were first prepared in dimethyl sulfoxide (DMSO) and then diluted with distilled water. The final concentration of DMSO in the zoospore suspension never exceeded 1% (v/v), a condition that does not affect zoospore motility. The effects of compounds on motility and viability of zoospores were assayed using protocols described earlier (Islam et al.2005; Abdalla et al.2011; Islam, Tiedemann and Laatsch 2011). Motility of the zoospores was observed under a light microscope at 100-fold magnification. Quantification of time-course changes of motility and lysis of the zoospores were carried out as described earlier (Islam, Tiedemann and Laatsch 2011). Each treatment was replicated three times. The mean value % ±SE (standard error) of the affected spores in each treatment was calculated and the statistical significance was determined by one- and two-way Analysis of Varience (ANOVA) and t-test. Two-way ANOVA (see Table S1, Supporting Information) was used to test the significance of differences in motility inhibition and lytic activities of the antibiotics at different times and doses of treatment. The difference is considered statistically significant with a value of P < 0.05.

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For in vivo experiments, Oligomycin B is administered to rodents via intraperitoneal or intravenous injection at doses of 0.1-1 mg/kg. For ischemia studies, animals undergo coronary artery ligation followed by reperfusion, and ATP depletion rates are measured in cardiac tissue by HPLC. Mitochondrial function is assessed by high-resolution respirometry. However, systemic toxicity may limit chronic administration.

Intraperitoneal LD50 in mice: 2900 ug/kg

[1]. Evidence that the lichen-derived scabrosin esters target mitochondrial ATP synthase in P388D1 cells. Toxicol Appl Pharmacol. 2003 Aug 1;190(3):232-40.

[2]. Oligomycins and pamamycin homologs impair motility and induce lysis of zoospores of the grapevine downy mildew pathogen, Plasmopara viticola. FEMS Microbiol Lett. 2016 Aug;363(16).

Oligomycin B is an oligomycin with the molecular formula C45H72O12. It is a derivative of oligomycin A, in which the spirocyclic ring with a 2-hydroxypropyl substituent is replaced by a carbonyl group directly attached to the carbon atom at the center of the spirocyclic ring. It is a non-selective inhibitor of mitochondrial F1F0 ATP synthase and functions as an inhibitor of EC 3.6.3.14 (H(+)-transporter dual-zone ATPase). It is an oligomycin, triketone, and pentanol compound. 28-oxooligomycin A has been reported in the Chinese honeybee (Apis cerana), and relevant data are available.

C45H72O12

805.04598

804.502

11050-94-5

Oligomycin A;579-13-5;Oligomycin;1404-19-9;Oligomycin D;1404-59-7;Oligomycin C;11052-72-5

76958645

Typically exists as White to off-white solid at room temperature

1.2±0.1 g/cm3

911.4±65.0 °C at 760 mmHg

160-161℃

260.3±27.8 °C

0.0±0.6 mmHg at 25°C

1.547

Originated from microorganisms: the marine Streptomyces strains B8496 and B8739

5.5

5

12

3

57

1470

18

CC[C@@H]1C=CC=CC[C@H](C)[C@H]([C@@](C)(C(=O)[C@H](C)[C@H]([C@H](C)C(=O)[C@H](C)[C@H]([C@H](C)C=CC(=O)O[C@H]2[C@@H](C)[C@@H](CC1)O[C@]3([C@@H]2C)C(=O)C[C@@H](C)[C@@H](C[C@H](C)O)O3)O)O)O)O

QPRQJOHKNJIMGN-WVUAJZTGSA-N

InChI=1S/C45H72O12/c1-12-33-17-15-13-14-16-25(3)42(52)44(11,54)43(53)31(9)40(51)30(8)39(50)29(7)38(49)24(2)18-21-37(48)55-41-28(6)34(20-19-33)56-45(32(41)10)36(47)22-26(4)35(57-45)23-27(5)46/h13-15,17-18,21,24-35,38,40-42,46,49,51-52,54H,12,16,19-20,22-23H2,1-11H3/b14-13+,17-15+,21-18+/t24-,25+,26-,27+,28+,29-,30-,31-,32-,33-,34-,35-,38+,40+,41+,42-,44+,45+/m0/s1

(1R,4E,5'S,6S,6'S,7R,8S,10R,11R,12S,14R,15S,16R,18E,20E,22R,25S,27S,28S,29R)-22-ethyl-7,11,14,15-tetrahydroxy-6'-[(2R)-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]-3,3',9,13-tetrone

Oligomycin B; 11050-94-5; Oligomycin A, 28-oxo-; 387OE420H7; EINECS 234-275-5; BRN 5705502; (1R,4E,5'S,6S,6'S,7R,8S,10R,11R,12S,14R,15S,16R,18E,20E,22R,25S,27S,28S,29R)-22-ethyl-7,11,14,15-tetrahydroxy-6'-[(2R)-2-hydroxypropyl]-5',6,8,10,12,14,16,28,29-nonamethyl-5',6'-dihydro-3H,9H,13H-spiro[2,26-dioxabicyclo[23.3.1]nonacosa-4,18,20-triene-27,2'-pyran]-3,3',9,13(4'H)-tetrone; 28-OXOOLIGOMYCIN A;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

DMSO: ~50 mg/mL (~62.1 mM; with ultrasonication)

Solubility in Formulation 1: ≥ 1.25 mg/mL (1.55 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), Clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of DMSO stock solution (12.5 mg/mL) to 400 μL of PEG300 and mix well; then add 50 μL of Tween-80 and mix well; finally add 450 μL of physiological saline and adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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