Macrolide antibiotic; K+-dependent ATPas; vacuolar H+-ATPase (V-ATPase)
Bafilomycin B1 targets vacuolar H+-ATPases (V-ATPases), which are proton pumps responsible for acidifying intracellular compartments such as lysosomes, endosomes, and vacuoles. It also inhibits K+-dependent ATPase from E. coli. V-ATPases are involved in multiple cellular processes, including the maintenance of intracellular ion homeostasis by maintaining acidic pH within the cell. The compound acts as a specific inhibitor of V-ATPases, distinguishing them from other types of ATPases such as F1F0 ATPases and E1E2 ATPases.

Various membrane ATPases have been tested for their sensitivity to bafilomycin A1, a macrolide antibiotic. F1F0 ATPases from bacteria and mitochondria are not affected by this antibiotic. In contrast, E1E2 ATPases--e.g., the K+-dependent (Kdp) ATPase from Escherichia coli, the Na+,K+-ATPase from ox brain, and the Ca2+-ATPase from sarcoplasmic reticulum--are moderately sensitive to this inhibitor. Finally, membrane ATPases from Neurospora vacuoles, chromaffin granules, and plant vacuoles are extremely sensitive. From this we conclude that bafilomycin A1 is a valuable tool for distinguishing among the three different types of ATPases and represents the first relatively specific potent inhibitor of vacuolar ATPases[1].

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Vacuolar proton-translocating ATPase (V-ATPase) is located in fungal vacuolar membranes. It is involved in multiple cellular processes, including the maintenance of intracellular ion homeostasis by maintaining acidic pH within the cell. The importance of V-ATPase in virulence has been demonstrated in several pathogenic fungi, including Candida albicans. However, it remains to be determined in the clinically important fungal pathogen Candida glabrata. Increasing multidrug resistance of C. glabrata is becoming a critical issue in the clinical setting. In the current study, we demonstrated that the plecomacrolide V-ATPase inhibitor bafilomycin B1 exerts a synergistic effect with azole antifungal agents, including fluconazole and voriconazole, against a C. glabrata wild-type strain. Furthermore, the deletion of the VPH2 gene encoding an assembly factor of V-ATPase was sufficient to interfere with V-ATPase function in C. glabrata, resulting in impaired pH homeostasis in the vacuole and increased sensitivity to a variety of environmental stresses, such as alkaline conditions (pH 7.4), ion stress (Na+, Ca2+, Mn2+, and Zn2+ stress), exposure to the calcineurin inhibitor FK506 and antifungal agents (azoles and amphotericin B), and iron limitation. In addition, virulence of C. glabrata Δvph2 mutant in a mouse model of disseminated candidiasis was reduced in comparison with that of the wild-type and VPH2-reconstituted strains. These findings support the notion that V-ATPase is a potential attractive target for the development of effective antifungal strategies[2].

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In vitro studies have shown that Bafilomycin B1 is a potent inhibitor of V-ATPase activity. Various membrane ATPases have been tested for their sensitivity to bafilomycin A1 (an analog): F1F0 ATPases from bacteria and mitochondria are not affected; E1E2 ATPases (e.g., K+-dependent ATPase, Na+,K+-ATPase, Ca2+-ATPase) are moderately sensitive; while V-ATPases from fungal vacuoles, chromaffin granules, and plant vacuoles are extremely sensitive. Bafilomycin A1 completely inhibits H+ translocation at 10 nM. The compound is a valuable tool for distinguishing among the three different types of ATPases.

Vacuolar H+-adenosine triphosphatase (V-ATPase) stimulates vesicular acidification that may activate cytoplasmic enzymes, hormone secretion and membrane recycling of transporters. We investigated the effect of blockade of V-ATPase by bafilomycin B1 on renal gluconeogenesis, mitochondrial enzymes, and insulin secretion in type 2 diabetic rats. Spontaneous type 2 diabetic Torii rats were treated with intraperitoneal injection of bafilomycin B1 for 1 week, and the kidneys were examined after 24 h of starvation in metabolic cages. The renal expression and activity of V-ATPase were increased in the brush border membrane of the proximal tubules in diabetic rats. The blockade of V-ATPase by bafilomycin B1 reduced renal V-ATPase activity and urinary ammonium in diabetic rats. Treatment with bafilomycin suppressed the enhanced renal gluconeogenesis enzymes and mitochondrial electron transport enzymes in type 2 diabetic rats and reduced the renal cytoplasmic glucose levels. The insulin index and pancreatic insulin granules were decreased in diabetic rats with increased V-ATPase expression in islet cells, and treatment with bafilomycin B1 reversed these changes and increased the insulin secretion index. Hepatosteatosis in type 2 diabetic rats was ameliorated by bafilomycin treatment. As a consequence, treatment with bafilomycin B1 significantly decreased the plasma glucose level after 24 h of starvation in diabetic rats. In conclusion, a V-ATPase inhibitor improved plasma glucose levels in type 2 diabetes by inhibiting renal mitochondrial gluconeogenesis and improving insulin secretion.[3]

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Vacuolar H+-adenosine triphosphatase (ATPase) plays important roles in urinary acid excretion, vesicular acidification to activate enzymes, and the membrane recycling of transporters in the kidney. As acidosis stimulates renal gluconeogenesis, we investigated the effect of blockade of H+-ATPase on renal gluconeogenesis in diabetic rats. Diabetes mellitus was induced by a single injection of streptozotocin, and a group of DM rats was treated with bafilomycin B1 intraperitoneally for 8 days. In diabetic rats, the renal expression and activity of H+-ATPase were increased with elevated urinary ammonium excretion. The blockade of H+-ATPase by bafilomycin B1 reduced the renal H+-ATPase activity and urinary ammonium excretion in diabetic rats. Treatment with bafilomycin suppressed the enhancement of the renal gluconeogenesis enzymes phosphoenol pyruvate carboxykinase and glucose-6-phosphatase in diabetic rats and reduced the renal cytoplasmic glucose levels, whereas hepatic gluconeogenesis did not change significantly. After a 24-h starvation period, bafilomycin decreased the plasma glucose level to a normal level in diabetic rats. The suppression of renal gluconeogenesis by an H+-ATPase inhibitor may therefore be a new therapeutic target for the treatment of diabetes mellitus[4].

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In vivo, Bafilomycin B1 has been shown to inhibit the pit-forming activity of osteoclasts, which is dependent on V-ATPase function. Bafilomycin A1 triggers the proliferative potential of senescent cancer cells in vitro and in NOD/SCID mice. It also induces caspase-independent cell death in hepatocellular carcinoma cells via targeting of autophagy and MAPK pathways. The compound's effects on V-ATPase inhibition have been studied in various animal models to understand the role of V-ATPases in physiology and disease.

Drug susceptibility assays[2]
Susceptibility to fluconazole, voriconazole, and the V-ATPase inhibitor bafilomycin B1, alone or in combination, was examined by using broth microdilution test, essentially according to the Clinical and Laboratory Standards Institute (CLSI) M27-S4 protoco and the previous report with minor modifications. Briefly, C. glabrata cells were incubated in SC at 35°C for 48 h. The minimum drug concentration that inhibited cell growth by more than 80% relative to drug-free control was defined as the minimum inhibitory concentration (MIC). Fractional inhibitory concentration (FIC) was calculated by using the following formula: FIC for drug A = (MIC of drug A in combination with drug B)/(MIC of drug A alone). The sum of FIC for drug A and FIC for drug B was defined as the FIC index (FICI). Drug interaction was classified as synergistic if FICI was ≤0.5.

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The in vitro enzyme assay for Bafilomycin B1 typically involves measuring V-ATPase activity in isolated membrane vesicles or purified enzyme preparations. The enzyme is incubated with ATP and a proton gradient is generated. The proton pumping activity is measured using fluorescence-based assays with pH-sensitive dyes such as acridine orange or by monitoring the quenching of fluorescence. Varying concentrations of bafilomycin B1 are added to determine the IC50 for inhibition. The inhibition of V-ATPase activity is expressed as a percentage of the control activity. Data are analyzed using nonlinear regression.

In vitro cellular assays for Bafilomycin B1 are conducted using various cell types, such as osteoclasts, cancer cells, or primary cells. Cells are treated with varying concentrations of bafilomycin B1 (typically in the nanomolar to micromolar range). V-ATPase activity is assessed by measuring the acidification of intracellular compartments using fluorescent dyes such as LysoTracker or acridine orange. Autophagy is evaluated by monitoring the accumulation of autophagosomes (e.g., by LC3-II detection via Western blot). Cell viability and apoptosis are assessed using standard assays such as MTT, LDH release, or caspase activity assays.

Sprague–Dawley rats weighing 180–200 g had ad libitum access to tap water and standard rat chow. Diabetes was induced by a single tail vein injection of streptozotocin (STZ, 60 mg/kg body weight) [diabetes mellitus (DM) rats]; the control rats were injected with an equal volume of citrate buffer. Three weeks after STZ injection, a group of DM rats was treated with bafilomycin B1(50, 100, 200 nmol/kg/day intraperitoneally). Twenty-four-hour urine and blood samples were collected using a metabolic cage until day 7 morning under feeding condition with free access to water and food, and then under 24-h starvation conditions without food [16]. On day 8, the rats were anesthetized with pentobarbital (50 mg/kg body weight), and then their kidneys and liver were removed and used for western blotting or immunohistochemistry. Intravenous insulin tolerance tests (ITTs) were performed to assess the degree of insulin resistance, and the extent of insulin resistance was evaluated according to the K index of ITT (KITT) as described previously. [4]

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In vivo animal studies for Bafilomycin B1 are performed in mouse models, such as NOD/SCID mice implanted with cancer cells. The compound is administered via intraperitoneal or intravenous injection. Tumor growth is monitored, and tissues are collected for histopathological analysis. The effects on V-ATPase activity and autophagy are assessed in tumor tissues by measuring lysosomal pH or autophagosome accumulation. Osteoclast function can be evaluated in bone resorption models. Standard study designs with appropriate control groups are employed, with 5-10 animals per group.

Pharmacokinetic data for Bafilomycin B1 are not extensively reported in the available literature. The compound has a molecular weight of 815.99 g/mol and a molecular formula of C44H65NO13. It is a lipophilic macrolide antibiotic with poor aqueous solubility. For in vitro studies, it is typically dissolved in DMSO. For in vivo studies, it is formulated in suitable vehicles (e.g., DMSO/PBS mixtures). The compound's pharmacokinetic properties, including absorption, distribution, metabolism, and excretion, are not well characterized. Its tissue distribution would be influenced by its high lipophilicity.

Toxicology data for Bafilomycin B1 are not extensively reported. As a potent V-ATPase inhibitor, the compound's toxicity is related to its effects on cellular pH homeostasis and autophagy. V-ATPases are essential for normal cellular function, so inhibition can lead to cellular dysfunction and cell death. The compound has been used in studies of cancer and bone resorption, suggesting some therapeutic potential, but its narrow therapeutic window limits its use as a drug. Comprehensive toxicology studies would be required for therapeutic development. The compound is for research use only.

[1]. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7972-6.

[2]. Vacuolar proton-translocating ATPase is required for antifungal resistance and virulence of Candida glabrata. PLoS One. 2019 Jan 23;14(1):e0210883.

[3]. V-ATPase blockade reduces renal gluconeogenesis and improves insulin secretion in type 2 diabetic rats. Hypertens Res. 2020 Oct;43(10):1079-1088.

[4]. H+-ATPase blockade reduced renal gluconeogenesis and plasma glucose in a diabetic rat model. Med Mol Morphol. 2018 Jun;51(2):89-95.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by extracellular β-amyloid (Aβ) deposition and intracellular neurofibrillary tangles (NFTs) in multiple brain regions. NFTs are primarily composed of hyperphosphorylated tau protein (p-Tau). Aβ and p-Tau are two major pathogenic molecules, with tau protein located downstream of Aβ and inducing neuronal degeneration. This study aimed to investigate whether Ginkgo biloba extract EGb 761 could reduce p-Tau levels in the brain and prevent the development and progression of AD. We fed transgenic mice carrying the human P301S tau mutant gene with EGb 761 for 2 months and 5 months, respectively. The results showed that EGb 761 treatment for 5 months significantly improved cognitive function in mice, reduced synaptophysin loss, and restored CREB phosphorylation levels in the mouse brain. EGb 761 treatment for 5 months (instead of 2 months) reduced p-Tau protein levels in the brain and converted pro-inflammatory activation of microglia to anti-inflammatory activation. As a potential therapeutic mechanism, we demonstrated that treatment with EGb 761, particularly its components ginkgolide A, ginkgolides, and flavonoids (rather than purified ginkgolide B or C), enhanced autophagy activity and p-Tau degradation in neuronal lysosomes. Inhibition of ATG5 function or treatment with bafloxacin B1 eliminated the EGb 761-enhanced p-Tau degradation in cultured neurons. Furthermore, we observed that EGb 761 treatment for 5 months (rather than 2 months) inhibited the activity of p38-MAPK and GSK-3β. Therefore, long-term use of the clinically available and well-tolerated herbal extract Ginkgo biloba extract EGb 761 may improve AD pathology by counteracting multiple AD pathogenic processes. [https://pubmed.ncbi.nlm.nih.gov/30010136/](https://pubmed.ncbi.nlm.nih.gov/30010136/) Nonsteroidal anti-inflammatory drugs (NSAIDs) can inhibit tumorigenesis in gastrointestinal tissues and have been proposed as adjuvant therapy to chemotherapy. The ability of cancer epithelial cells to adapt to the tumor microenvironment and resist cytotoxic drugs appears to depend on rescue mechanisms such as autophagy. In this study, we aimed to determine whether sensitizing NSAIDs, such as indomethacin, can modulate autophagy in gastric cancer epithelial cells. We observed that indomethacin caused lysosomal dysfunction in AGS cells and promoted the accumulation of autophagy substrates without altering mTOR activity. Indomethacin enhanced the inhibitory effects of the lysosomal-targeting drug chloroquine on lysosomal activity and autophagy, but had no effect when these two functions were maximally inhibited using another lysosomal inhibitor (bafloxacin B1). Indomethacin, alone or in combination with chloroquine, inhibited autophagy flux stimulated by the antitumor drug oxaliplatin and enhanced its toxicity, increasing apoptosis/necrosis rates and thus reducing cell viability. In summary, our results suggest that indomethacin enhances the sensitivity of gastric cancer cells to cytotoxic drugs by disrupting autophagy flux through interference with normal lysosomal function; this effect could be used to overcome cancer cell resistance to antitumor therapies. This study showed that bafloxacin B1 (BFM) exhibited antidiabetic effects in insulin-deficient streptozotocin (STZ) diabetic rats without causing significant changes in body weight or blood pressure. Since vacuolar H+-ATPase is also expressed in a variety of cells, including osteoclasts, lung cells, testicular cells, and neuroendocrine cells, the antidiabetic effect of BFM may depend on its blocking effect on non-renal cells. Recently, it has been reported that adult mice with conditional knockout of Atp6ap showed significantly reduced plasma glucose levels, but also exhibited abnormalities in the gut and hematopoietic cells. The limitation of this study was that one STZ rat treated with 200 nmol/kg body weight of BFM died from hypoglycemia during a 24-hour fast. Professor Ōmura reported that all mice survived after treatment with 0.6 mg/kg teratin, but died after treatment with more than 1.25 mg/kg teratin. The reduction in food intake may be related to the toxicity of BFM, and further research is needed to clarify the safety of BFM under longer treatment. [4]

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Bafilomycin B1 is a naturally occurring macrolide antibiotic isolated from Streptomyces sp. that acts as a potent and selective inhibitor of vacuolar H+-ATPases (V-ATPases). It also inhibits K+-dependent ATPase and has antimicrobial activity against Gram-positive bacteria and fungi. The compound is a valuable research tool for studying V-ATPase function, autophagy, and intracellular pH regulation. Bafilomycin A1, a closely related analog, has an IC50 of 0.44 nM for V-ATPase inhibition. Bafilomycin B1 is for research use only and is not approved for therapeutic applications.

C44H65NO13

815.985800000001

815.445

88899-56-3

88899-56-3 (Bafilomycin B1);88899-55-(Bafilomycin A1)

46881064

Off-white to yellow solid powder

1.2±0.1 g/cm3

939.4±65.0 °C at 760 mmHg

521.9±34.3 °C

0.0±0.6 mmHg at 25°C

1.563

Streptomyces halstedii K122

3.06

5

13

12

58

1670

12

C[C@H]1C/C(=C/C=C/[C@@H]([C@H](OC(=O)/C(=C/C(=C/[C@H]([C@H]1O)C)/C)/OC)[C@@H](C)[C@H]([C@H](C)[C@]2(C[C@H]([C@@H]([C@H](O2)C(C)C)C)OC(=O)/C=C/C(=O)NC3=C(CCC3=O)O)O)O)OC)/C

KFUFLYSBMNNJTF-ANDWMEETSA-N

InChI=1S/C44H65NO13/c1-23(2)41-28(7)35(56-37(49)18-17-36(48)45-38-31(46)15-16-32(38)47)22-44(53,58-41)30(9)40(51)29(8)42-33(54-10)14-12-13-24(3)19-26(5)39(50)27(6)20-25(4)21-34(55-11)43(52)57-42/h12-14,17-18,20-21,23,26-30,33,35,39-42,46,50-51,53H,15-16,19,22H2,1-11H3,(H,45,48)/b14-12+,18-17+,24-13+,25-20+,34-21-/t26-,27+,28-,29-,30-,33-,35+,39-,40+,41+,42+,44+/m0/s1

[(2R,4R,5S,6R)-2-hydroxy-2-[(2S,3R,4S)-3-hydroxy-4-[(2R,3S,4E,6E,9S,10S,11R,12E,14Z)-10-hydroxy-3,15-dimethoxy-7,9,11,13-tetramethyl-16-oxo-1-oxacyclohexadeca-4,6,12,14-tetraen-2-yl]pentan-2-yl]-5-methyl-6-propan-2-yloxan-4-yl] (E)-4-[(2-hydroxy-5-oxocyclopenten-1-yl)amino]-4-oxobut-2-enoate

Bafilomycin B1; 88899-56-3; CID 74030053; [2-Hydroxy-2-[3-hydroxy-4-(10-hydroxy-3,15-dimethoxy-7,9,11,13-tetramethyl-16-oxo-1-oxacyclohexadeca-4,6,12,14-tetraen-2-yl)pentan-2-yl]-5-methyl-6-propan-2-yloxan-4-yl] 4-[(2-hydroxy-5-oxocyclopenten-1-yl)amino]-4-oxobut-2-enoate; Bafilomycin B1 from Streptomyces sp

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)

DMSO : ~100 mg/mL (~122.55 mM)

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.)