The bafilomycins are a family of macrolide antibiotics produced from a variety of Streptomycetes. Their chemical structure is defined by a 16-membered lactone ring scaffold. Bafilomycins exhibit a wide range of biological activity, including anti-tumor, anti-parasitic, immunosuppressant and anti-fungal activity. The most used bafilomycin is bafilomycin A1, a potent inhibitor of cellular autophagy. Bafilomycins have also been found to act as ionophores, transporting potassium K+ across biological membranes and leading to mitochondrial damage and cell death.
Bafilomycin A1 specifically targets the vacuolar-type H+ -ATPase (V-ATPase) enzyme, a membrane-spanning proton pump that acidifies either the extracellular environment or intracellular organelles such as the lysosome of animal cells or the vacuole of plants and fungi. At higher micromolar concentrations, bafilomycin A1 also acts on P-type ATPases, which have a phosphorylated transitional state.
Bafilomycin A1 serves as an important tool compound in many in vitro research applications; however, its clinical use is limited by a substantial toxicity profile.
Discovery and history Bafilomycin A1, B1 and C1 were first isolated from Streptomyces griseus in 1983. During a screen seeking to identify microbial secondary metabolites whose activity mimicked that of two cardiac glycosides, bafilomycin C1 was identified as an inhibitor of P-ATPase with a ki of 11 μM. Bafilomycin C1 was found to have activity against Caenorhabditis elegans, ticks, and tapeworms, in addition to stimulating the release of γ-aminobutyruc acid (GABA) from rat synaptosomes. Independently, bafilomycin A1 and other derivatives were isolated from S. griseus and shown to have antibiotic activity against some yeast, Gram-positive bacteria and fungi. Bafilomycin A1 was also shown to have an anti-proliferative effect on concanavalin-A-stimulated T cells. However, its high toxicity has prevented use in clinical trials.
Two years later, bafilomycins D and E were also isolated from S. griseus. In 2010, 9-hydroxy-bafilomycin D, 29-hydroxy-bafilomycin D and a number of other bafilomycins were identified from the endophytic microorganism Streptomyces sp. YIM56209. From 2004 to 2011, bafilomycins F-K were isolated from other Streptomyces sp.
As one of the first identified and most commonly used, bafilomycin A1 is of particular importance, especially as its structure serves as the core of all other bafilomycins. With its large structure, bafilomycin has multiple chiral centers and functional groups, which makes modifying its structure difficult, a task that has been attempted to reduce the compound's associated toxicity.
Target
Within the cell, bafilomycin A1 specifically interacts with the proton pump V-ATPase. This large protein depends on Adenosine triphosphate (ATP) hydrolysis to pump protons across a biological membrane. When bafilomycin and other inhibitors of V-ATPase, such as concanamycin, were first discovered in the 1980s they were used to establish the presence of V-ATPase in specialized cells types and tissues, characterizing the proton pump's distribution. Structurally, V-ATPase consists of 13 distinct subunits that together make up the membrane spanning Vo and cytosolic V1 domains of the enzyme. The V1 domain in the cytosol is made up of subunits A through H whereas the Vo domain is made up of subunits a, d, e, c, and c".
V-ATPase mechanism of action In order to move protons across the membrane, a proton first enters subunit a within the Vo domain through a cytoplasmic hemichannel. This allows conserved glutamic acid residues within the proteolipid ring of Vo subunits c and c" to become protonated. Adenosine triphosphate (ATP) is then hydrolyzed by the V1 domain of the enzyme, enabling both the rotation of the central stalk of the pump, made up of subunits D, F and d, and the rotation of the proteolipid ring. This rotation puts the protonated glutamic acid residues in contact with a luminal hemichannel located in subunit a. Within subunit a, arginine residues serve to stabilize the deprotonated form of glutamic acid and allow the release of their protons. This rotation and proton transfer brings the protons through the pump and across the membrane.
Bafilomycin–V-ATPase interaction For more than ten years after bafilomycin was discovered as a V-ATPase inhibitor, the site of its interaction with V-ATPase was unclear. Beginning studies used the chromaffin granule V-ATPase to suggest that bafilomycin interacted with the Vo domain. Two further studies confirmed this hypothesis using V-ATPase from bovine clathrin coated vesicles. They showed that application of bafilomycin inhibited proton flow through Vo and that this inhibition could be overcome by adding back the Vo domain to the coated vesicles. Further narrowing bafilomycin's interaction site, they found that specific addition of just Vo subunit a could restore function. This suggested bafilomycin interacted specifically with subunit a of V-ATPase; however, another study contradicted this finding. A group found that by using a bafilomycin affinity chromatography column V-ATPase could be purified, and that addition of DCCD, an inhibitor of the Vo c subunit, drastically decreased bafilomycin's affinity for V-ATPase. This suggested that bafilomycin interacted more strongly with subunit c of the Vo domain. It was further found that amino acid changes within subunit a could also lower V-ATPase-Bafilomycin interaction, indicating a minor role of subunit a in bafilomycin binding in addition to subunit c. An analysis of nine mutations that conferred resistance to bafilomycin showed all of them to change amino acids in the Vo c subunit. These data suggested that the bafilomycin binding site was on the outer surface of the Vo domain, at the interface between two c subunits. This binding site has recently been described in high resolution by two groups that used cryo electron microscopy to obtain structures of the V-ATPase bound to bafilomycin.
Overall, bafilomycin binds with nanomolar efficiency to the Vo c subunit of the V-ATPase complex and inhibits proton translocation. Although the interaction between bafilomycin and V-ATPase is not covalent, its low dissociation constant of about 10 nM describes the strength of its interaction and can make the effects of bafilomycin difficult to reverse.
V-ATPase localization and function V-ATPase is ubiquitous in mammalian cells and plays an important role in many cellular processes. It is localized to the trans-golgi network and the cellular organelles that are derived from it, including lysosomes, secretory vesicles and endosomes. V-ATPase can also be found within the plasma membrane. In mammals, location of the V-ATPase can be linked to the specific isoform of subunit a that the complex has. Isoforms a1 and a2 target V-ATPase intracellularly, to synaptic vesicles and endosomes respectively. Subunits a3 and a4, however, mediate V-ATPase localization to the plasma membrane in osteoclasts (a3) and renal intercalated cells (a4). If located at the lysosomal membrane, this results in the acidification of the lysosome as lumenal pH is lowered, enabling activity of lysosomal hydrolases. When V-ATPase is located at the plasma membrane, proton extrusion through the pump causes the acidification of the extracellular space, which is utilized by specialized cells such as osteoclasts, epididymal clear cells, and renal epithelial intercalated cells.
Intracellular function As it promotes the acidification of lysosomes, endosomes, and secretory vesicles, V-ATPase contributes to processes including: vesicular/protein trafficking receptor recycling endocytosis protein degradation autophagy cell signaling
With its role in lysosomal acidification, V-ATPase is also crucial in driving the transport of ions and small molecules into the cytoplasm, particularly calcium and amino acids. Additionally, its acidification of endosomes is critical in receptor endocytosis as low pH tends to drive ligand release as well as receptor cleavage which contributes to signaling events, such as through the release of the intracellular domain of Notch.
Plasma membrane function When at the plasma membrane, V-ATPase function is critical in the acidification of the extracellular environment, which is seen with osteoclasts and epididymal clear cells. When present at the plasma membrane in renal epithelial intercalated cells, V-ATPase is important for acid secretion, which contributes to the acidification of urine. In response to reduced plasma pH, increased levels of V-ATPase are typically trafficked to the plasma membrane in these cells by phosphorylation of the pump by Protein Kinase A (PKA).
V-ATPase in disease Clinically, dysfunction of V-ATPase has been correlated with several diseases in humans. Some of these diseases include male infertility, osteopetrosis, and renal acidosis. Additionally, V-ATPase can be found at the plasma membrane of some invasive cancer cells including breast, prostate and liver cancer, among others. In human lung cancer samples, V-ATPase expression was correlated with drug resistance. A large number of V-ATPase subunit mutations have also been identified in a number of cancers, including follicular lymphomas.
Cellular action As the target of Bafilomycin V-ATPase, is involved in many aspects of cellular function, Bafilomycin treatment greatly alters cellular processes.
Inhibition of autophagy Bafilomycin A1 is most known for its use as an autophagy inhibitor. Autophagy is the process by which the cell degrades its own organelles and some proteins through the formation of autophagosomes. Autophagosomes then fuse with lysosomes facilitating the degradation of engulfed cargo by lysosomal proteases. This process is critical in maintaining the cell's store of amino acids and other nutrients during times of nutrient deprivation or other metabolic stresses. Bafilomycin interferes with this process by inhibiting the acidification of the lysosome through its interaction with V-ATPase. Lack of lysosomal acidification prevents the activity of lysosomal proteases like cathepsins so that engulfed cargo can no longer be degraded.
Since V-ATPase is widely distributed within the cell, Bafilomycin is only specific as an autophagy inhibitor for a short amount of time. Other effects are seen outside this short window, including interference in the trafficking of endosomes and proteasomal inhibition.
In addition to blocking the acidification of the lysosome, Bafilomycin has been reported to block the fusion of autophagosomes with lysosomes. This was initially found in a paper by Yamamoto, et al. in which the authors used bafilomycin A1 to treat rat hepatoma H-4-II-E cells. By electron microscopy, they saw a blockage of autophagosome-lysosome fusion after using bafilomycin at a concentration of 100 nM for 1 hour. This has been confirmed by other studies, particularly two that found decreased colocalization of mitochondria and lysosomes by fluorescence microscopy following a 12-24 hour treatment with 100 or 400 nM Bafilomycin. However, further studies have failed to see this inhibition of fusion with similar bafilomycin treatments. These contradictory results have been explained by time differences among treatments as well as use of different cell lines. The effect of Bafilomycin on autophagosome-lysosome fusion is complex and time dependent in each cell line.
In neurons, an increase in the autophagosome marker LC3-II has been seen with Bafilomycin treatment. This occurs as autophagosomes fail to fuse with lysosomes, which normally stimulates the degradation of LC3-II.
Induction of apoptosis