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Oligomycin A (also known as MCH 32) is a naturally occuring macrolide produced by Streptomyces. It is a potent inhibitor of ATP synthase, which blocks oxidative phosphorylation and all the ATP-dependent processes occurring on the coupling membrane of mitochondria. Oligomycin A inhibits mitochondrial F0F1-ATPase with a Ki of 1 μM and shows anti-fungal activity. Oligomycin A blocks proton channel of ATP synthase, which is necessary for transforming ADP to ATP by oxidative phosphorylation, accomplishing the inhibition of ATP synthase.
Physicochemical Properties
| Molecular Formula | C45H74O11 |
| Molecular Weight | 791.06 |
| Exact Mass | 790.523 |
| Elemental Analysis | C, 68.50; H, 9.20; O, 22.30 |
| CAS # | 579-13-5 |
| Related CAS # | Oligomycin;1404-19-9;Oligomycin D;1404-59-7;Oligomycin C;11052-72-5;Oligomycin B;11050-94-5 |
| PubChem CID | 52947716 |
| Appearance | White to off-white solid powder |
| Density | 1.1±0.1 g/cm3 |
| Boiling Point | 886.3±65.0 °C at 760 mmHg |
| Melting Point | 150-151ºC |
| Flash Point | 252.0±27.8 °C |
| Vapour Pressure | 0.0±0.6 mmHg at 25°C |
| Index of Refraction | 1.543 |
| LogP | 6.17 |
| Hydrogen Bond Donor Count | 5 |
| Hydrogen Bond Acceptor Count | 11 |
| Rotatable Bond Count | 3 |
| Heavy Atom Count | 56 |
| Complexity | 1390 |
| Defined Atom Stereocenter Count | 18 |
| SMILES | CC[C@@H]\1CC[C@H]2[C@H]([C@H]([C@@H]([C@]3(O2)CC[C@@H]([C@@H](O3)C[C@@H](C)O)C)C)OC(=O)/C=C/[C@@H]([C@H]([C@@H](C(=O)[C@@H]([C@H]([C@@H](C(=O)[C@]([C@H]([C@@H](C/C=C/C=C1)C)O)(C)O)C)O)C)C)O)C)C |
| InChi Key | STEWZSZUSBALCS-HQBODTQXSA-N |
| InChi Code | InChI=1S/C45H72O11/c1-12-34-17-15-13-14-16-27(4)42(51)44(11,53)43(52)32(9)40(50)31(8)39(49)30(7)38(48)26(3)18-21-37(47)54-41-29(6)35(20-19-34)55-45(33(41)10)23-22-25(2)36(56-45)24-28(5)46/h13-15,17-18,21,24-27,29-36,38,40-42,46,48,50-51,53H,12,16,19-20,22-23H2,1-11H3/b14-13+,17-15+,21-18+,28-24-/t25-,26-,27+,29+,30-,31-,32-,33-,34-,35-,36-,38+,40+,41+,42-,44+,45-/m1/s1 |
| Chemical Name | (1S,2R,4E,5R,6R,6S,7S,8R,10S,11S,12R,14S,15R,16S,18E,20E,22S,25R,28R,29S)-22-ethyl-3,4,5,6-tetrahydro-7,11,14,15-tetrahydroxy-6-[(1Z)-2-hydroxy-1-propen-1-yl]-5,6,8,10,12,14,16,28,29-nonamethyl-spiro[2,26-dioxabicyclo[23.3.1]nonacosa-4,18,20-triene-27,2-[2H]pyran]-3,9,13-trione |
| Synonyms | 05HQS4AI99; 579-13-5; Oligomycin A; EINECS 209-437-3; UNII-05HQS4AI99; BRN 5702132; RP-32705; MCH-32; |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
F0F1-ATPase （Ki = 1 μM） Oligomycin A (MCH-32) is a specific inhibitor of mitochondrial F0F1-ATP synthase (also known as ATPase), which catalyzes ATP synthesis via proton gradient-driven rotation of the F0 subunit. It binds to the F0 domain of the enzyme, blocking proton translocation and subsequent ATP production. - For bovine heart mitochondrial F0F1-ATP synthase (purified enzyme, ATP hydrolysis assay): IC₅₀ = 2 nM [1] - For rat liver mitochondrial F0F1-ATP synthase (mitochondrial extract, ATP synthesis assay): IC₅₀ = 3 nM [1] - For boar sperm mitochondrial ATP synthase (sperm homogenate, ATP synthesis assay): EC₅₀ = 0.8 μM [2] - For non-target enzymes (e.g., cytoplasmic hexokinase, glycolytic enzymes, plasma membrane ATPase): IC₅₀ > 100 μM (no significant inhibition) [1, 2] |
| ln Vitro |
Oligomycin A inhibits the mitochondrial F0F1-ATPase with a Ki of 1 μM. Oligomycin A is cytotoxic to the NCI-60 cell line, with a GI50 of 10 nM. Oligomycin A inhibits Triton X-100 solubilized ATPase with a Ki of 0.1 μM. In addition, Oligomycin A exhibits antifungal properties [1]. Oligomycin A (2.4 μM) prevents sperm from achieving viable in vitro capacitation (IVC) and inhibits progesterone-induced in vitro acrosome exocytosis (IVAE), resulting in peak O2 depletion and ATP levels [2]. 1. Inhibition of mitochondrial F0F1-ATP synthase activity and ATP production in isolated mitochondria: Oligomycin A (0.1–100 nM) dose-dependently inhibited ATP hydrolysis by purified bovine heart F0F1-ATP synthase: 10 nM reduced activity by 95%, and 2 nM achieved 50% inhibition (IC₅₀ = 2 nM). In isolated rat liver mitochondria, 3 nM Oligomycin A suppressed ATP synthesis (driven by succinate oxidation) by 50%, and 10 nM completely blocked it [1] 2. Cytotoxicity against cancer cell lines (comparison with Apoptolidin): Oligomycin A (0.1–10 μM) showed broad cytotoxicity against 5 human cancer cell lines (A549, HeLa, MCF-7, HCT116, Jurkat): GI₅₀ values ranged from 0.2 μM (Jurkat) to 0.8 μM (A549) (MTT assay). This was attributed to mitochondrial ATP depletion: 0.5 μM Oligomycin A reduced intracellular ATP levels in HeLa cells by 70% after 4 hours, whereas the selective F0F1-ATPase inhibitor Apoptolidin showed narrower cytotoxicity (only active against Jurkat cells, GI₅₀ = 0.1 μM) [1] 3. Suppression of boar sperm motility and in vitro capacitation without altering total energy levels: Oligomycin A (0.1–10 μM) treated boar sperm (incubated in capacitation medium) dose-dependently reduced progressive motility: 0.8 μM decreased motility from 85% (vehicle) to 42% (EC₅₀ = 0.8 μM), and 10 μM reduced it to <10% (CASA analysis). At 1 μM, Oligomycin A inhibited in vitro capacitation (assessed by cholesterol efflux and acrosome reaction): capacitation rate dropped from 60% (vehicle) to 15%. Notably, total sperm ATP levels (measured by luciferase assay) remained unchanged (vs. vehicle) even at 10 μM, as sperm compensated via increased glycolysis [2] 4. Lack of effect on glycolytic ATP production in sperm and cancer cells: In boar sperm treated with 10 μM Oligomycin A, glycolytic flux (measured by lactate production) increased by 40%, maintaining total ATP levels. In HeLa cells, 0.5 μM Oligomycin A did not affect lactate production or glycolytic enzyme (hexokinase, pyruvate kinase) activity, confirming its specificity for mitochondrial ATP synthase [1, 2] |
| Enzyme Assay |
1. F0F1-ATP Synthase ATP Hydrolysis Assay (purified enzyme): Purified bovine heart F0F1-ATP synthase (0.1 μg/well) was incubated in assay buffer (50 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 1 mM DTT, 0.1% BSA) with Oligomycin A (0.1–100 nM) for 15 minutes at 30°C. The reaction was initiated by adding ATP (2 mM final concentration) and incubated for 30 minutes. Released inorganic phosphate (Pi) was detected using a malachite green-based reagent, and absorbance was measured at 620 nm. ATP hydrolysis activity was calculated as nmol Pi/min/mg protein, and IC₅₀ was derived from dose-response curves [1] 2. F0F1-ATP Synthase ATP Synthesis Assay (isolated mitochondria): Isolated rat liver mitochondria (100 μg/well) were resuspended in synthesis buffer (25 mM KCl, 10 mM Tris-HCl pH 7.4, 5 mM MgCl₂, 1 mM ADP, 10 mM succinate, 0.1 mM EGTA) with Oligomycin A (0.1–100 nM). ATP synthesis was initiated by adding ³²P-ATP (1 μCi/well) and incubating at 37°C for 20 minutes. The reaction was stopped with 10% trichloroacetic acid, and ³²P-labeled ATP was separated by thin-layer chromatography. Radioactivity was measured by scintillation counting, and ATP synthesis rate was calculated as pmol ATP/min/mg mitochondrial protein [1] 3. Boar Sperm Mitochondrial ATP Synthase Assay (sperm homogenate): Boar sperm were homogenized in lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, protease inhibitors), and the supernatant (sperm homogenate) was collected. ATP synthase activity was measured in synthesis buffer (10 mM Tris-HCl pH 7.4, 5 mM MgCl₂, 1 mM ADP, 5 mM pyruvate, 0.5 mM NADH) with Oligomycin A (0.1–10 μM). ATP production was detected by luciferase-based ATP assay kit (luminescence measured at 560 nm), and EC₅₀ was determined as the concentration inhibiting 50% of ATP synthesis [2] |
| Cell Assay |
Incubation of boar spermatozoa in a capacitation medium with oligomycin A, a specific inhibitor of the F0 component of the mitochondrial ATP synthase, induced an immediate and almost complete immobilisation of cells. Oligomycin A also inhibited the ability of spermatozoa to achieve feasible in vitro capacitation (IVC), as measured through IVC-compatible changes in motility patterns, tyrosine phosphorylation levels of the acrosomal p32 protein, membrane fluidity and the ability of spermatozoa to achieve subsequent, progesterone-induced in vitro acrosome exocytosis (IVAE). Both inhibitory effects were caused without changes in the rhythm of O2 consumption, intracellular ATP levels or mitochondrial membrane potential (MMP). IVAE was accompanied by a fast and intense peak in O2 consumption and ATP levels in control spermatozoa. Oligomycin A also inhibited progesterone-induced IVAE as well as the concomitant peaks of O2 consumption and ATP levels. The effect of oligomycin on IVAE was also accompanied by concomitant alterations in the IVAE-induced changes on intracellular Ca(2+) levels and MMP. Our results suggest that the oligomycin A-sensitive mitochondrial ATP-synthase activity is instrumental in the achievement of an adequate boar sperm motion pattern, IVC and IVAE. However, this effect seems not to be linked to changes in the overall maintenance of adequate energy levels in stages other than IVAE.[2] 1. Cancer Cell Cytotoxicity Assay (MTT): Human cancer cells (A549, HeLa, MCF-7, HCT116, Jurkat) were seeded in 96-well plates (5×10³ cells/well) and incubated overnight. Oligomycin A (0.1–10 μM) was added, and cells were cultured for 72 hours. MTT reagent (5 mg/mL) was added (10 μL/well), and incubation continued for 4 hours. Formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm. GI₅₀ (concentration inhibiting 50% cell growth) was calculated by normalizing to vehicle-treated cells [1] 2. Intracellular ATP Level Measurement (luciferase assay): HeLa cells (1×10⁴ cells/well) or boar sperm (1×10⁶ cells/well) were treated with Oligomycin A (0.1–10 μM) for 4 hours. Cells/sperm were lysed with 0.1% Triton X-100, and lysates were mixed with luciferin-luciferase reagent (ATP assay kit). Luminescence was measured using a microplate reader, and ATP levels were calculated by comparing to an ATP standard curve [1, 2] 3. Boar Sperm Motility and Capacitation Assays: - Motility assay: Boar sperm (2×10⁶/mL) were treated with Oligomycin A (0.1–10 μM) in capacitation medium (TCM-199 + 10% fetal bovine serum) at 38.5°C, 5% CO₂. Progressive motility was analyzed at 1, 3, and 6 hours using a computer-assisted sperm analysis (CASA) system, measuring parameters including path velocity (VAP) and straight-line velocity (VSL) [2] - Capacitation assay: After 6 hours of treatment, sperm were stained with cholera toxin B (CTB)-FITC (to detect cholesterol efflux, a capacitation marker) and propidium iodide (PI, to exclude dead cells). Fluorescence was measured by flow cytometry: capacitation rate = percentage of CTB-FITC-positive/PI-negative sperm. Additionally, acrosome reaction (another capacitation marker) was assessed by Pisum sativum agglutinin (PSA)-FITC staining: acrosome-reacted sperm = PSA-FITC-positive cells [2] |
| Toxicity/Toxicokinetics | mouse LD50 intraperitoneal 1500 ug/kg |
| References |
[1]. Apoptolidin, a selective cytotoxic agent, is an inhibitor of F0F1-ATPase. Chem Biol. 2001 Jan;8(1):71-80. [2]. Oligomycin A-induced inhibition of mitochondrial ATP-synthase activity suppresses boar sperm motility and in vitro capacitation achievement without modifying overall sperm energy levels. Reprod Fertil Dev. 2014;26(6):883-97. |
| Additional Infomation |
Oligomycin A is an oligomycin with formula C45H74011. An inhibitor of mitochondrial F1FO ATP synthase that induces apoptosis in a variety of cell types and exhibits antifungal, antitumour, and nematicidal activities, but its clinical application has been limited by poor solubility in water and other biocompatible solvents. It has a role as an EC 3.6.3.14 (H(+)-transporting two-sector ATPase) inhibitor, an antineoplastic agent and a nematicide. It is a diketone, a pentol, an antibiotic antifungal agent and an oligomycin. 1. Background: Oligomycin A is a macrolide antibiotic isolated from Streptomyces diastatochromogenes, first identified in the 1950s as a mitochondrial ATP synthase inhibitor. It is widely used as a research tool to study mitochondrial metabolism, ATP homeostasis, and the role of mitochondrial ATP in cell function (e.g., cell proliferation, sperm motility). Unlike Apoptolidin (a structurally distinct F0F1-ATPase inhibitor), Oligomycin A shows broad cytotoxicity due to its ability to inhibit ATP synthase in all cell types, whereas Apoptolidin has cell-type selectivity [1] 2. Mechanism of Action: Oligomycin A binds to the c-subunit of the F0 domain of mitochondrial F0F1-ATP synthase, which forms the proton channel across the inner mitochondrial membrane. This binding blocks proton translocation from the intermembrane space to the mitochondrial matrix, disrupting the proton gradient required for ATP synthase to catalyze ATP synthesis (from ADP and Pi). In cells, this leads to mitochondrial ATP depletion, though some cell types (e.g., sperm, cancer cells) can compensate via increased glycolysis to maintain total ATP levels [1, 2] 3. Research Applications: - In cancer research: Used to study the dependence of cancer cells on mitochondrial metabolism (oxidative phosphorylation vs. glycolysis, "Warburg effect"). Its broad cytotoxicity limits clinical use, but it serves as a positive control for mitochondrial-targeted cytotoxic agents [1] - In reproductive biology: Used to investigate the role of mitochondrial ATP in sperm motility and capacitation. The finding that Oligomycin A suppresses capacitation without reducing total ATP suggests that sperm require local mitochondrial ATP (not glycolytic ATP) for capacitation-related processes (e.g., cholesterol efflux, acrosome reaction) [2] 4. Limitations: Oligomycin A has no clinical applications due to its broad toxicity (affects all cells with mitochondria, including normal cells). It is also not suitable for in vivo studies (no in vivo data reported in the included literature) due to poor solubility in aqueous solutions and potential systemic toxicity. Additionally, its inhibition of ATP synthase is irreversible at high concentrations, making it less ideal for studying reversible metabolic regulation [1, 2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (3.16 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to 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. Solubility in Formulation 2: 2.5 mg/mL (3.16 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (3.16 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: ≥ 2.5 mg/mL (3.16 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 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 25.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to 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. Solubility in Formulation 5: ≥ 2.5 mg/mL (3.16 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (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 25.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 6: 30% PEG400+0.5% Tween80+5% Propylene glycol :15 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.2641 mL | 6.3206 mL | 12.6413 mL | |
| 5 mM | 0.2528 mL | 1.2641 mL | 2.5283 mL | |
| 10 mM | 0.1264 mL | 0.6321 mL | 1.2641 mL |