Archazolid and apicularen: Novel specific V-ATPase inhibitors
© Huss et al. 2005
Received: 07 March 2005
Accepted: 04 August 2005
Published: 04 August 2005
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© Huss et al. 2005
Received: 07 March 2005
Accepted: 04 August 2005
Published: 04 August 2005
V-ATPases constitute a ubiquitous family of heteromultimeric, proton translocating proteins. According to their localization in a multitude of eukaryotic membranes, they energize many different transport processes. Since their malfunction is correlated with various diseases in humans, the elucidation of the properties of this enzyme for the development of selective inhibitors and drugs is one of the challenges in V-ATPase research.
Archazolid A and B, two recently discovered cytotoxic macrolactones produced by the myxobacterium Archangium gephyra, and apicularen A and B, two novel benzolactone enamides produced by different species of the myxobacterium Chondromyces, exerted a similar inhibitory efficacy on a wide range of mammalian cell lines as the well established plecomacrolidic type V-ATPase inhibitors concanamycin and bafilomycin. Like the plecomacrolides both new macrolides also prevented the lysosomal acidification in cells and inhibited the V-ATPase purified from the midgut of the tobacco hornworm, Manduca sexta, with IC50 values of 20–60 nM. However, they did not influence the activity of mitochondrial F-ATPase or that of the Na+/K+-ATPase. To define the binding sites of these new inhibitors we used a semi-synthetic radioactively labelled derivative of concanamycin which exclusively binds to the membrane Vo subunit c. Whereas archazolid A prevented, like the plecomacrolides concanamycin A, bafilomycin A1 and B1, labelling of subunit c by the radioactive I-concanolide A, the benzolactone enamide apicularen A did not compete with the plecomacrolide derivative.
The myxobacterial antibiotics archazolid and apicularen are highly efficient and specific novel inhibitors of V-ATPases. While archazolid at least partly shares a common binding site with the plecomacrolides bafilomycin and concanamycin, apicularen adheres to an independent binding site.
Vacuolar-type ATPases (V-ATPases) are ubiquitous proton pumps in the endomembrane system of all eukaryotic cells and in plasma membranes of many animal cells where they energize transport processes across the membrane or regulate the pH of corresponding compartments . They are heteromultimeric enzymes consisting of a membrane bound, proton translocating Vo complex and a catalytic V1 complex which is oriented towards the cytosol. In recent years it became more and more evident that malfunction of the V-ATPase is correlated with a multitude of diseases such as osteopetrosis, male infertility or renal acidosis [2–4]. Therefore the V-ATPase turned out to be a subject for biomedical research and even was considered as a potential target for cancer drug therapy . In order to understand the development of these diseases and to design efficient drugs for their therapy it is necessary to gain a most comprehensive knowledge of the mode of action of the enzyme as well as of known V-ATPase inhibitors on the one hand, and, on the other hand, to search for novel potent and specific inhibitors with different inhibition characteristics.
The best examined and established specific V-ATPase inhibitors are the plecomacrolides bafilomycin  and concanamycin , which both take effect in nanomolar concentrations by binding to the Vo subunit c [8–10]. Recently various new inhibitors of V-ATPases such as the benzolactone enamides  or chondropsines  have been described (reviewed in ) but so far in no case the binding site has been determined. Only for the benzolactone enamide salicylihalamide it was shown that its binding site is different to that of plecomacrolides  and may reside somewhere between the Vo and the V1 complex . In the present report we introduce two types of antibiotics produced by myxobacteria, apicularens, new benzolactone enamides [15, 16] and archazolids, a novel class of macrolactones  which both represent highly potent and specific V-ATPase inhibitors, however, with different modes of action and different binding sites.
Growth inhibition of different mammalian cell lines by antibiotics
murine connective tissue
rat, embryogenic fibroblast cell line
human cervix carcinoma
(in presence of 11 μM verapamil)
human lung carcinoma
mouse, embryogenic fibroblast cell line
These findings confirmed our assumption that growth inhibition and the morphological changes induced by archazolid and by apicularen were due to their inhibitory effect on the V-ATPase. Apicularen B was slightly lower active compared to the other drugs, but the difference is much less than expected from the cell culture assays. This difference may result from a lower membrane permeability of apicularen B due to its additional N-acetyl-β-D-glucosamine residue.
While the macrolactones (archazolid A and B) and the benzolactone enamides (apicularen A and B, salicylihalamide) were shown to be potent growth inhibitors of cultured mammalian cells, they all failed to inhibit bacterial growth, while only archazolid showed weak activity against fungi; in addition, the benzolactone enamide salicylihalamide effectively inhibited mammalian V-ATPases but not at all V-ATPases from fungi such as Saccharomyces cerevisiae or Neurospora crassa [11, 15, 17]. Since the tobacco hornworm V-ATPase is very sensitive to all these antibiotics and available in milligram amounts, it appears to be an appropriate model for further investigations on the mechanism of inhibition by archazolid and the benzolactone enamides.
Inhibition of Na+/K+-ATPase from pig kidney by antibiotics
specific activity μmol*mg-1 *min-1
control without inhibitors
vanadate 0.1 mM
vanadate 1 mM
ouabain 0.1 mM
ouabain 1 mM
concanamycin A 1 μM
apicularen A 1 μM
archazolid A 1 μM
Unlike archazolid, the benzolactone enamide apicularen A did not appear to interfere with labelling of subunit c by the concanolide A derivative because the signal obtained was almost as strong as in the control (Fig. 4). Therefore we suggest that apicularen binds to a site which is largely different from that for the plecomacrolides. This result is in agreement with our former observation that the benzolactone enamide salicylihalamide does not bind to subunit c and supports our conclusion that the sites and mechanisms of inhibition for benzolactone enamides are different from those for plecomacrolides .
The novel antibiotics archazolid and apicularen are highly efficient and specific novel inhibitors of V-ATPases. Despite the different structures of archazolid and the plecomacrolides they probably have a similar mode of inhibition and their binding sites in the V-ATPase have at least a considerable overlap. In contrast, apicularen which demonstrates the same inhibition efficacy does not interfere with I-concanolide A, thus suggesting a mode of inhibition different from that of the plecomacrolides.
The V-ATPase holoenzyme was purified as published elsewhere . Preparation of highly purified membranes containing Na+/K+-ATPase from pig kidney followed the protocol of Jørgensen  with the three main steps of differential centrifugation, incubation with SDS in the presence of ATP and sucrose density gradient centrifugation in a fixed angle rotor, and led to a specific enzyme activity which was in the same range as that reported by the authors. The resulting sample was stored frozen at -20°C. To prepare crude membrane extracts, the hearts of three mice were washed three times with an ice-cold buffer of pH 8.1 consisting of 0.25 M sucrose, 5 mM Tris-HCl, 5 mM EDTA, and 10 mM Pefabloc SC (Biomol), homogenized in 5 ml of this buffer and centrifuged at 4°C for 25 min at 233,000 × g max in a fixed angle rotor. The resulting pellet was resuspended in 5 ml of a buffer of pH 7.5, consisting of 5 mM Tris-MOPS (3-morpholinopropanesulfonic acid), 10 mM NaCl, 10 mM Pefabloc SC, 9.6 mM 2-mercaptoethanol, 0.53 mM EGTA and 0.1 % Triton X-100. After an aliquot for protein determination had been taken, 10% bovine serum albumin (final concentration) was added to the suspension which was then stored on ice until the activity assays were run. Beef heart mitochondria were isolated by differential centrifugation, following the protocol of Smith by using a blender to homogenize the heart mince . The initial homogenization buffer consisted of 250 mM saccharose, 10 mM KH2PO4, 10 mM Tris, 2 mM EGTA, 2 mM MgCl2, pH 7.4, and further isolation procedures were carried out in the same medium without EGTA . Submitochondrial particles were obtained by ultrasonic treatment of the mitochondria.
Labelled I-concanolide A was synthesized as described elsewhere . Archazolid A and B, apicularen A and B, concanamycin A and bafilomycin A1 and B1 were isolated according to published procedures [15–17, 26]. To avoid freeze-thaw cycles which have significant influence on the stability of the substances, aliquots of stock solutions in dimethyl sulfoxide were stored at -70°C and thawed only once immediately before use. The actual concentrations of the stock solutions were determined spectrophotometrically.
Standard V-ATPase assays with a final volume of 160 μl and a pH of 8.1 consisted of 3–4 μg of protein, 50 mM Tris-MOPS, 3 mM 2-mercaptoethanol, 1 mM MgCl2, 20 mM KCl, 0.003% C12E10, 20 mM NaCl, and 3 mM Tris-HCl. After 5 min of pre-incubation at 30°C with or without inhibitors, 1 mM Tris-ATP was added and after incubation for 2 min the reaction was stopped by placing the tube in liquid nitrogen. Assays using Na+/K+-ATPase were performed in 160 μl at pH 7.5 and contained 0.5 μg of protein, 50 mM Tris-MOPS, 5 mM imidazole, 0.2 mM EDTA, 4 mM MgCl2, 20 mM KCl and 100 mM NaCl. After 5 min of pre-incubation at 37°C with or without inhibitors, 3 mM Tris-ATP was added and after 1 min of incubation the reaction was stopped by placing the tube in liquid nitrogen. Assays using crude membranes from mouse heart had a volume of 160 μl and a pH of 7.5, and consisted of 10 μg of membrane protein, 50 mM Tris-MOPS, 3 mM 2-mercaptoethanol, 1 mM MgCl2, 20 mM KCl, 100 mM NaCl, 0.02% Triton X-100 and 0.3 mg/ml BSA. After 5 min of pre-incubation at 30°C with or without inhibitors, 1 mM Tris-ATP was added and after an incubation time of 10 min the reaction was stopped by placing the tubes in liquid nitrogen. ATPase assays with submitochondrial particles from beef heart were performed in a final volume of 1 ml and a pH of 8.0. The samples contained 9–12 μg protein, 50 mM Tris, 50 mM KCl and 2.5 mM MgCl2. After 5 min of preincubation with or without inhibitors, 5 mM ATP was added, and after an additional incubation time of 15 min the reaction was stopped by the addition of 0.4 ml of 20 % TCA.
Inorganic phosphate produced in the assays of V-ATPase, Na+/K+-ATPase and mouse heart F-ATPase was measured according the protocol of Wieczorek et al. , while the determination of inorganic phosphate produced in the assays of beef heart F-ATPase followed the method of Fiske and Subarrow  using ascorbic acid as reducing agent.
Twenty micrograms of the samples were pre-incubated with the inhibitors for 1 h on ice. The labelled I-concanolide A was then added to give a final concentration of 10 μM. Controls were run without effectors. Mixtures with volumes of 40 μl were incubated for an additional 1 h on ice and then treated for 3 min with UV light (366 nm) on ice. After UV irradiation, 10 μl of 5-fold sample buffer  was added, the mixture was heated for 45 s at 95°C, cooled on ice, and subjected to Tricine-SDS-PAGE (16.5% T, 3% C separating gel and 10%T, 3% C spacer gel ), followed by Coomassie staining. The gels were sealed in plastic wrap before they were exposed to a phosphoscreen for up to 72 h and analyzed with the aid of a phosphoimager (Molecular Dynamics).
Cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). Cell lines 3Y1 and M1 (embryonal fibroblasts from 129/Ola × C57BL/6 mice, ) were a generous gift from Dr. S. Miyamoto, Fukoka, Japan, and Dr. P. F. Mühlradt, Braunschweig, Germany. All cell lines were cultivated under conditions recommended by the supplier. Growth inhibition was measured in microtiterplates. Aliquots of 120 μl of the suspended cells (50,000/ml) were given to 60 μl of a serial dilution of the inhibitor. After 5 days, growth was determined using the MTT assay .
PtK2 (ATCC CCL-56) or KB-3-1 cells were grown on glass coverslips (13 mm diameter) in four-well-plates. Exponentially growing cells were incubated with the inhibitors for 4 hours and stained for lysosomes with 50 nM LysoTracker Red DND-99 and for mitochondria with 75 nM MitoTracker Green FM (both from Molecular Probes) at 37°C for 30 min. The nuclei were stained using Hoechst 33258 (5 μg/ml). The coverslips were mounted upside down in PBS, fixed with nail polish, and observed under the fluorescent microscope.
Fifth instar larvae of M. sexta (Lepidoptera, Sphingidae), weighing 6–8 g, were reared under long day conditions (16 h of light) at 27°C using a synthetic diet modified according to Bell et al .
We like to thank Nina Dankers, Martin Dransmann, Petra Haunhorst, Birte Engelhardt and Bettina Hinkelmann for their excellent technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 431: H.W.) and the Fonds der Chemischen Industrie (A.Z.).
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