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Bafilomycin A1

(Synonyms: NSC 381866) Catalog No.: GC17597

Bafilomycin A1, a macrolide antibiotic isolated from the Streptomyces species, is an inhibitor of vacuolar H ATPase (V-ATPase).

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Bafilomycin A1 Chemical Structure

Cas No.:88899-55-2

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Cell experiment [1, 2]:

Cell lines


Preparation Method

Circulating eosinophils were purified from donors with eosinophils >35 per μL of blood. Eosinophil preparations with purity >98% and viability ~98% were used.

Reaction Conditions

Eosinophils were cultured at 1 × 106/mL in complete medium (RPMI 1640 plus 10% fetal bovine serum) with IL3 (2 ng/mL) or IL5 (2 ng/mL) both cytokines from BD Biosciences for 20 h, and were then washed and cultured with complete medium (no IL3 or IL5) on coated heat-aggregated immunoglobulins-G (HA-IgG or IgG) or without IgG as previously described.


Bafilomycin-A1 could be used to show the impact of autophagolysosome formation on eosinophil cytolysis. Treated IL3- and IL5-primed eosinophils with bafilomycin-A1 before interaction with IgG, and then measured cytolysis using rhodamine-110 (R110). Treatment with bafilomycin-A1 increased IL3-primed and IL5-primed eosinophil lysis by 50% and 100%, respectively, suggesting that autophagolysosome formation protects from cytolysis for both IL3- and IL5-primed eosinophils.

Animal experiment [3]:

Animal models

Lck-mTOR + CLP mice, WT + CLP mice, Lck-TSC1 mice

Preparation Method

Crossed TSC1loxp/loxp and mTORloxp/loxp mice with Lck-Cre mice (Cre expression under the control of lymphocyte-specific protein tyrosine kinase, Lck) to obtain the F1 generation Lck-Cre:mTORloxp/− mice and Lck-Cre:TSC1loxp/− mice, respectively. We interbred F1 generation mice in each group and obtained F2 generation Lck-Cre: mTORloxp/loxp and Lck-Cre;TSC1loxp/loxp mice. Littermates lacking Lck-Cre served as controls. Then mice were anesthetized, and the abdomen was opened surgically and the cecum was ligated halfway between the distal pole and the base. The cecal stump was punctured once with a 22-gauge needle. Put back the cecum and closed the abdomen. Administered 1 mL saline subcutaneously to each animal for fluid resuscitation. Animals in the sham group underwent the same surgical procedure without ligation or puncture. Bafilomycin A1 was administered intraperitoneally 1 h after the CLP operation.

Dosage form

1 mg/kg


Bafilomycin A1 could specifically block fusion of autophagosomes and lysosomes and attenuate the protective effects of mTOR deletion against CD4 + T cell apoptosis. In Lck-mTOR + CLP mice treated with bafilomycin A1, the apoptosis rate of CD4 + T cells was higher compared with mice that did not receive bafilomycin A1 and lower compared with mice in WT + CLP + bafilomycin A1 group.


[1]. Esnault S, et al. IL-3 up-regulates and activates human eosinophil CD32 and αMβ2 integrin causing degranulation. Clin Exp Allergy. 2017 Apr;47(4):488-498.

[2]. Esnault S, et al. Autophagy Protects against Eosinophil Cytolysis and Release of DNA. Cells. 2022 Jun 2;11(11):1821.

[3]. Wang H, et al. mTOR deletion ameliorates CD4 + T cell apoptosis during sepsis by improving autophagosome-lysosome fusion. Apoptosis. 2022 Jun;27(5-6):401-408.


Bafilomycin A1, a macrolide antibiotic isolated from the Streptomyces species, is an inhibitor of vacuolar H ATPase (V-ATPase). Bafilomycin A1 has been used to study the autophagy as an inhibitor of autophagosomes-lysosomes fusion and as an inhibitor of lysosomal degradation.[1] It binds to the V0 sector subunit c of the V-ATPase complex and inhibits H translocation which cause an accumulation of H in the cytoplasm of treated cells. Bafilomycin A1 inhibits cell growth and leads to apoptosis and differentiation. These anticancer effects of Bafilomycin A1 are considered to be attributable to the intracellular acidosis caused by V-ATPase inhibition.[2]

In vitro study of Bafilomycin A1 indicated that Bafilomycin A1 preferentially inhibits in vitro growth of pediatric B-cell acute lymphoblastic leukemia cells. A various of leukemia cell lines including 697, Nalm-6, RS4;11, NB4, HL-60, K562 and BV173 representing B-ALL (697, Nalm-6, RS4;11), acute myeloid leukemia (NB4, HL-60), and chronic myeloid leukemia (K562, BV173) were used to investigate the effect of Bafilomycin A1. Cells were cultured in the presence of increasing concentrations of Bafilomycin A1 (0 nM, 0.5 nM, 1 nM). Cell proliferation was measured using an MTT assay. Results showed that Bafilomycin A1 has frequently been used as an inhibitor to block the autophagosomes-lysosomes fusion, or to inhibit lysosomal activity at high doses (0.1–1 mμ). While low concentration of Bafilomycin A1 (1 nM) cpuld effectively and specifically inactivate autophagy and activate apoptosis at multiple targets in pediatric B-ALL cells.[3]

In vivo study demonstrated that Bafilomycin A1 can extend survival in the Bafilomycin A1-treated B-ALL mice with advanced disease compared with control mice. The red blood cell counts, hemoglobin concentration, and platelet counts in the B-ALL model group were extremely reduced, however they were normalized in the Bafilomycin A1-treated group. The mice in the B-ALL model group had severe hepatosplenomegaly compared with the control group. However, the size and weight of livers and spleens in the Bafilomycin A1-treated group were normalized compared with those in the disease model group. Results showed that Bafilomycin A1 dramatically inhibits pediatric B-ALL cell engraftment by targeting leukemia cells in vivo.[3]

[1]. Yamamoto A, et al. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct. 1998;23(1):33–42.
[2] Bowman EJ, et al. The bafilomycin/concanamycin binding site in subunit c of the V-ATPases from Neurospora crassa and Saccharomyces cerevisiae. J Biol Chem. 2004;279(32):33131–33138.
[3]. Yuan N, et al. Bafilomycin A1 targets both autophagy and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia. Haematologica. 2015 Mar;100(3):345-56.

Chemical Properties

Cas No. 88899-55-2 SDF
Synonyms NSC 381866
Chemical Name (3Z,5E,7R,8S,9S,11E,13E,15S,16R)-16-[(2S,3R,4S)-4-[(2R,4R,5S,6R)-2,4-dihydroxy-5-methyl-6-propan-2-yloxan-2-yl]-3-hydroxypentan-2-yl]-8-hydroxy-3,15-dimethoxy-5,7,9,11-tetramethyl-1-oxacyclohexadeca-3,5,11,13-tetraen-2-one
Formula C35H58O9 M.Wt 622.84
Solubility 5 mg/ml in DMSO or in menthanol Storage Desiccate at -20°C, protect from light
General tips For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months.
Shipping Condition Evaluation sample solution : ship with blue ice
All other available size: ship with RT , or blue ice upon request

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Research Update

Bafilomycin A1 disrupts autophagic flux by inhibiting both V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion

Autophagosome-lysosome fusion and autolysosome acidification constitute late steps in the autophagic process necessary to maintain functional autophagic flux and cellular homeostasis. Both of these steps are disrupted by the V-ATPase inhibitor bafilomycin A1, but the mechanisms potentially linking them are unclear. We recently revisited the role of lysosomal acidification in autophagosome-lysosome fusion, using an in vivo approach in Drosophila. By genetically depleting individual subunits of the V-ATPase, we confirmed its role in lysosomal acidification and autophagic cargo degradation. Surprisingly, vesicle fusion remained active in V-ATPase-depleted cells, indicating that autophagosome-lysosome fusion and autolysosome acidification are 2 separable processes. In contrast, bafilomycin A1 inhibited both acidification and fusion, consistent with its effects in mammalian cells. Together, these results imply that this drug inhibits fusion independently of its effect on V-ATPase-mediated acidification. We identified the ER-calcium ATPase Ca-P60A/dSERCA as a novel target of bafilomycin A1. Autophagosome-lysosome fusion was defective in Ca-P60A/dSERCA-depleted cells, and bafilomycin A1 induced a significant increase in cytosolic calcium concentration and disrupted Ca-P60A/SERCA-mediated fusion. Thus, bafilomycin A1 disrupts autophagic flux by independently inhibiting V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion.

Bafilomycin A1 targets both autophagy and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia

B-cell acute lymphoblastic leukemia is the most common type of pediatric leukemia. Despite improved remission rates, current treatment regimens for pediatric B-cell acute lymphoblastic leukemia are often associated with adverse effects and central nervous system relapse, necessitating more effective and safer agents. Bafilomycin A1 is an inhibitor of vacuolar H(+)-ATPase that is frequently used at high concentration to block late-phase autophagy. Here, we show that bafilomycin A1 at a low concentration (1 nM) effectively and specifically inhibited and killed pediatric B-cell acute lymphoblastic leukemia cells. It targeted both early and late stages of the autophagy pathway by activating mammalian target of rapamycin signaling and by disassociating the Beclin 1-Vps34 complex, as well as by inhibiting the formation of autolysosomes, all of which attenuated functional autophagy. Bafilomycin A1 also targeted mitochondria and induced caspase-independent apoptosis by inducing the translocation of apoptosis-inducing factor from mitochondria to the nucleus. Moreover, bafilomycin A1 induced the binding of Beclin 1 to Bcl-2, which further inhibited autophagy and promoted apoptotic cell death. In primary cells from pediatric patients with B-cell acute lymphoblastic leukemia and a xenograft model, bafilomycin A1 specifically targeted leukemia cells while sparing normal cells. An in vivo mouse toxicity assay confirmed that bafilomycin A1 is safe. Our data thus suggest that bafilomycin A1 is a promising candidate drug for the treatment of pediatric B-cell acute lymphoblastic leukemia.

Molecular basis of V-ATPase inhibition by bafilomycin A1

Pharmacological inhibition of vacuolar-type H+-ATPase (V-ATPase) by its specific inhibitor can abrogate tumor metastasis, prevent autophagy, and reduce cellular signaling responses. Bafilomycin A1, a member of macrolide antibiotics and an autophagy inhibitor, serves as a specific and potent V-ATPases inhibitor. Although there are many V-ATPase structures reported, the molecular basis of specific inhibitors on V-ATPase remains unknown. Here, we report the cryo-EM structure of bafilomycin A1 bound intact bovine V-ATPase at an overall resolution of 3.6-?. The structure reveals six bafilomycin A1 molecules bound to the c-ring. One bafilomycin A1 molecule engages with two c subunits and disrupts the interactions between the c-ring and subunit a, thereby preventing proton translocation. Structural and sequence analyses demonstrate that the bafilomycin A1-binding residues are conserved in yeast and mammalian species and the 7'-hydroxyl group of bafilomycin A1 acts as a unique feature recognized by subunit c.

Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion

Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for degradation. Autophagy is involved in the pathophysiology of numerous diseases and its modulation is beneficial for the outcome of numerous specific diseases. Several lysosomal inhibitors such as bafilomycin A1 (BafA1), protease inhibitors and chloroquine (CQ), have been used interchangeably to block autophagy in in vitro experiments assuming that they all primarily block lysosomal degradation. Among them, only CQ and its derivate hydroxychloroquine (HCQ) are FDA-approved drugs and are thus currently the principal compounds used in clinical trials aimed to treat tumors through autophagy inhibition. However, the precise mechanism of how CQ blocks autophagy remains to be firmly demonstrated. In this study, we focus on how CQ inhibits autophagy and directly compare its effects to those of BafA1. We show that CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle. Furthermore, CQ induces an autophagy-independent severe disorganization of the Golgi and endo-lysosomal systems, which might contribute to the fusion impairment. Strikingly, HCQ-treated mice also show a Golgi disorganization in kidney and intestinal tissues. Altogether, our data reveal that CQ and HCQ are not bona fide surrogates for other types of late stage lysosomal inhibitors for in vivo experiments. Moreover, the multiple cellular alterations caused by CQ and HCQ call for caution when interpreting results obtained by blocking autophagy with this drug.

SARS-CoV-2 Omicron variant shows less efficient replication and fusion activity when compared with Delta variant in TMPRSS2-expressed cells

The novel SARS-CoV-2 Omicron variant (B.1.1.529), first found in early November 2021, has sparked considerable global concern and it has >50 mutations, many of which are known to affect transmissibility or cause immune escape. In this study, we sought to investigate the virological characteristics of the Omicron variant and compared it with the Delta variant which has dominated the world since mid-2021. Omicron variant replicated more slowly than the Delta variant in transmembrane serine protease 2 (TMPRSS2)-overexpressing VeroE6 (VeroE6/TMPRSS2) cells. Notably, the Delta variant replicated well in Calu3 cell line which has robust TMPRSS2 expression, while the Omicron variant replicated poorly in this cell line. Competition assay showed that Delta variant outcompeted Omicron variant in VeroE6/TMPRSS2 and Calu3 cells. To confirm the difference in entry pathway between the Omicron and Delta variants, we assessed the antiviral effect of bafilomycin A1, chloroquine (inhibiting endocytic pathway), and camostat (inhibiting TMPRSS2 pathway). Camostat potently inhibited the Delta variant but not the Omicron variant, while bafilomycin A1 and chloroquine could inhibit both Omicron and Delta variants. Moreover, the Omicron variant also showed weaker cell-cell fusion activity when compared with Delta variant in VeroE6/TMPRSS2 cells. Collectively, our results suggest that Omicron variant infection is not enhanced by TMPRSS2 but is largely mediated via the endocytic pathway. The difference in entry pathway between Omicron and Delta variants may have an implication on the clinical manifestations or disease severity.

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