Diphyllin Shows a Broad-Spectrum Antiviral Activity against Multiple Medically Important Enveloped RNA and DNA Viruses.

Diphyllin is a natural arylnaphtalide lignan extracted from tropical plants of particular importance in traditional Chinese medicine. This compound has been described as a potent inhibitor of vacuolar (H+)ATPases and hence of the endosomal acidification process that is required by numerous enveloped viruses to trigger their respective viral infection cascades after entering host cells by receptor-mediated endocytosis. Accordingly, we report here a revised, updated, and improved synthesis of diphyllin, and demonstrate its antiviral activities against a panel of enveloped viruses from Flaviviridae, Phenuiviridae, Rhabdoviridae, and Herpesviridae families. Diphyllin is not cytotoxic for Vero and BHK-21 cells up to 100 µM and exerts a sub-micromolar or low-micromolar antiviral activity against tick-borne encephalitis virus, West Nile virus, Zika virus, Rift Valley fever virus, rabies virus, and herpes-simplex virus type 1. Our study shows that diphyllin is a broad-spectrum host cell-targeting antiviral agent that blocks the replication of multiple phylogenetically unrelated enveloped RNA and DNA viruses. In support of this, we also demonstrate that diphyllin is more than just a vacuolar (H+)ATPase inhibitor but may employ other antiviral mechanisms of action to inhibit the replication cycles of those viruses that do not enter host cells by endocytosis followed by low pH-dependent membrane fusion.

V-ATPase blockade reduces renal gluconeogenesis and improves insulin secretion in type 2 diabetic rats.

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.

Impaired lysosomal acidification triggers iron deficiency and inflammation in vivo.

Lysosomal acidification is a key feature of healthy cells. Inability to maintain lysosomal acidic pH is associated with aging and neurodegenerative diseases. However, the mechanisms elicited by impaired lysosomal acidification remain poorly understood. We show here that inhibition of lysosomal acidification triggers cellular iron deficiency, which results in impaired mitochondrial function and non-apoptotic cell death. These effects are recovered by supplying iron via a lysosome-independent pathway. Notably, iron deficiency is sufficient to trigger inflammatory signaling in cultured primary neurons. Using a mouse model of impaired lysosomal acidification, we observed a robust iron deficiency response in the brain, verified by in vivo magnetic resonance imaging. Furthermore, the brains of these mice present a pervasive inflammatory signature associated with instability of mitochondrial DNA (mtDNA), both corrected by supplementation of the mice diet with iron. Our results highlight a novel mechanism linking impaired lysosomal acidification, mitochondrial malfunction and inflammation in vivo.

Effects of diphyllin as a novel V-ATPase inhibitor on TE-1 and ECA-109 cells.

Diphyllin is a natural component of traditional Chinese medicine, which effectively inhibits V-ATPase activity and affects the progression of cancer. However, few studies have been conducted on esophageal cancer, and the mechanisms remain to be elucidated. The present study revealedthat diphyllin inhibited proliferation and induced S arrest in esophageal cancer cell lines TE-1 and ECA-109. Further experiments revealed that diphyllin inhibited V-ATPase activity and decreased the mRNA expression of mammalian target of rapamycin complex 1 (mTORC1), hypoxia-inducible factor-1α (HIF-1α), and vascular endothelial growth factor (VEGF). The present study also revealed that diphyllin inhibited proliferation and reduced the formation of new blood vessels. Diphyllin inhibited blood metastasis by regulating the mTORC1/HIF-1α-/VEGF pathway, therefore it could be considered as a new V-ATPase inhibitor to treat esophageal cancer.

Intracerebroventricular infusion of the (Pro)renin receptor antagonist PRO20 attenuates deoxycorticosterone acetate-salt-induced hypertension.

We previously reported that binding of prorenin to the (pro)renin receptor (PRR) plays a major role in brain angiotensin II formation and the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Here, we designed and developed an antagonistic peptide, PRO20, to block prorenin binding to the PRR. Fluorescently labeled PRO20 bound to both mouse and human brain tissues with dissociation constants of 4.4 and 1.8 nmol/L, respectively. This binding was blocked by coincubation with prorenin and was diminished in brains of neuron-specific PRR-knockout mice, indicating specificity of PRO20 for PRR. In cultured human neuroblastoma cells, PRO20 blocked prorenin-induced calcium influx in a concentration- and AT(1) receptor-dependent manner. Intracerebroventricular infusion of PRO20 dose-dependently inhibited prorenin-induced hypertension in C57Bl6/J mice. Furthermore, acute intracerebroventricular infusion of PRO20 reduced blood pressure in both DOCA-salt and genetically hypertensive mice. Chronic intracerebroventricular infusion of PRO20 attenuated the development of hypertension and the increase in brain hypothalamic angiotensin II levels induced by DOCA-salt. In addition, chronic intracerebroventricular infusion of PRO20 improved autonomic function and spontaneous baroreflex sensitivity in mice treated with DOCA-salt. In summary, PRO20 binds to both mouse and human PRRs and decreases angiotensin II formation and hypertension induced by either prorenin or DOCA-salt. Our findings highlight the value of the novel PRR antagonist, PRO20, as a lead compound for a novel class of antihypertensive agents and as a research tool to establish the validity of brain PRR antagonism as a strategy for treating hypertension.

Inhibitory and combinatorial effect of diphyllin, a v-ATPase blocker, on influenza viruses.

An influenza pandemic poses a serious threat to humans and animals. Conventional treatments against influenza include two classes of pathogen-targeting antivirals: M2 ion channel blockers (such as amantadine) and neuraminidase inhibitors (such as oseltamivir). Examination of the mechanism of influenza viral infection has shown that endosomal acidification plays a major role in facilitating the fusion between viral and endosomal membranes. This pathway has led to investigations on vacuolar ATPase (v-ATPase) activity, whose role as a regulating factor on influenza virus replication has been verified in extensive genome-wide screenings. Blocking v-ATPase activity thus presents the opportunity to interfere with influenza viral infection by preventing the pH-dependent membrane fusion between endosomes and virions. This study aims to apply diphyllin, a natural compound shown to be as a novel v-ATPase inhibitor, as a potential antiviral for various influenza virus strains using cell-based assays. The results show that diphyllin alters cellular susceptibility to influenza viruses through the inhibition of endosomal acidification, thus interfering with downstream virus replication, including that of known drug-resistant strains. In addition, combinatorial treatment of the host-targeting diphyllin with pathogen-targeting therapeutics (oseltamivir and amantadine) demonstrates enhanced antiviral effects and cell protection in vitro.

V-ATPases in osteoclasts: structure, function and potential inhibitors of bone resorption.

The vacuolar-type H(+)-ATPase (V-ATPase) proton pump is a macromolecular complex composed of at least 14 subunits organized into two functional domains, V(1) and V(0). The complex is located on the ruffled border plasma membrane of bone-resorbing osteoclasts, mediating extracellular acidification for bone demineralization during bone resorption. Genetic studies from mice to man implicate a critical role for V-ATPase subunits in osteoclast-related diseases including osteopetrosis and osteoporosis. Thus, the V-ATPase complex is a potential molecular target for the development of novel anti-resorptive agents useful for the treatment of osteolytic diseases. Here, we review the current structure and function of V-ATPase subunits, emphasizing their exquisite roles in osteoclastic function. In addition, we compare several distinct classes of V-ATPase inhibitors with specific inhibitory effects on osteoclasts. Understanding the structure-function relationship of the osteoclast V-ATPase may lead to the development of osteoclast-specific V-ATPase inhibitors that may serve as alternative therapies for the treatment of osteolytic diseases.

Mechanisms of toxicity of Cleistanthus collinus: vacuolar ATPases are a putative target.

Ingestion of Cleistanthus collinus, a shrub native to South India, either intentionally or accidentally, is a common cause of death in the area. Consumption of a boiled decoction of leaves is highly toxic, but medical management of patients is mainly supportive because the molecular mechanisms of toxin action are unknown. Distal renal tubular acidosis is one of the symptoms of poisoning in patients and adenosine triphosphate (ATP) requiring proton pumps is important for acid secretion in the kidney. Hence, we hypothesized that these may be putative targets for C. collinus action and we tested this by exposing rat renal brush border membrane (BBM) as well as cultured kidney cells to a boiled decoction of C. collinus. Exposure to the C. collinus decoction resulted in significant inhibition of vacuolar type H(+)-ATPase (V-ATPase) activity in renal BBM as well as blocking of the proton pump in renal BBM vesicles. C. collinus decoction was also found to inhibit acidification of intracellular organelles in cells in culture, similar to the effect seen with either bafilomycin or concanamycin - specific inhibitors of the V-ATPase. This was accompanied by a decrease in V-ATPase activity, but an increase in protein levels. These results demonstrate that the V-ATPase in renal cells is a putative target for the toxins in C. collinus and the inhibition of this important proton pump probably plays a role in the development of distal renal tubular acidosis and subsequent renal failure seen in poisoned patients.

Acidic NAADP-releasable Ca(2+) compartments in the megakaryoblastic cell line MEG01.

BACKGROUND: A novel family of intracellular Ca(2+)-release channels termed two-pore channels (TPCs) has been presented as the receptors of NAADP (nicotinic acid adenine dinucleotide phosphate), the most potent Ca(2+) mobilizing intracellular messenger. TPCs have been shown to be exclusively localized to the endolysosomal system mediating NAADP-evoked Ca(2+) release from the acidic compartments. OBJECTIVES: The present study is aimed to investigate NAADP-mediated Ca(2+) release from intracellular stores in the megakaryoblastic cell line MEG01. METHODS: Changes in cytosolic and intraluminal free Ca(2+) concentrations were registered by fluorimetry using fura-2 and fura-ff, respectively; TPC expression was detected by PCR. RESULTS: Treatment of MEG01 cells with the H(+)/K(+) ionophore nigericin or the V-type H(+)-ATPase selective inhibitor bafilomycin A1 revealed the presence of acidic Ca(2+) stores in these cells, sensitive to the SERCA inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). NAADP releases Ca(2+) from acidic lysosomal-like Ca(2+) stores in MEG01 cells probably mediated by the activation of TPC1 and TPC2 as demonstrated by TPC1 and TPC2 expression silencing and overexpression. Ca(2+) efflux from the acidic lysosomal-like Ca(2+) stores or the endoplasmic reticulum (ER) results in ryanodine-sensitive activation of Ca(2+)-induced Ca(2+) release (CICR) from the complementary Ca(2+) compartment. CONCLUSION: Our results show for the first time NAADP-evoked Ca(2+) release from acidic compartments through the activation of TPC1 and TPC2, and CICR, in a megakaryoblastic cell line.

Identification of inhibitors of vacuolar proton-translocating ATPase pumps in yeast by high-throughput screening flow cytometry.

Fluorescence intensity of the pH-sensitive carboxyfluorescein derivative 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was monitored by high-throughput flow cytometry in living yeast cells. We measured fluorescence intensity of BCECF trapped in yeast vacuoles, acidic compartments equivalent to lysosomes where vacuolar proton-translocating ATPases (V-ATPases) are abundant. Because V-ATPases maintain a low pH in the vacuolar lumen, V-ATPase inhibition by concanamycin A alkalinized the vacuole and increased BCECF fluorescence. Likewise, V-ATPase-deficient mutant cells had greater fluorescence intensity than wild-type cells. Thus, we detected an increase of fluorescence intensity after short- and long-term inhibition of V-ATPase function. We used yeast cells loaded with BCECF to screen a small chemical library of structurally diverse compounds to identify V-ATPase inhibitors. One compound, disulfiram, enhanced BCECF fluorescence intensity (although to a degree beyond that anticipated for pH changes alone in the mutant cells). Once confirmed by dose-response assays (EC(50)=26 microM), we verified V-ATPase inhibition by disulfiram in secondary assays that measured ATP hydrolysis in vacuolar membranes. The inhibitory action of disulfiram against V-ATPase pumps revealed a novel effect previously unknown for this compound. Because V-ATPases are highly conserved, new inhibitors identified could be used as research and therapeutic tools in cancer, viral infections, and other diseases where V-ATPases are involved.

Inhibition of bone resorption in cultures of mouse calvariae by apicularen A.

Apicularens A and B were isolated from the myxobacterial genus Chondromyces apiculatus JW184. Apicularen A inhibited bafilomycin A1-sensitive ATP-dependent proton transport into microsome vesicles more potently than apicularen B. Bone resorption in cultures of mouse calvariae induced by human parathyroid hormone (PTH) or interleukin-1beta (IL-1beta) was inhibited by apicularen A at 10 and 100 nM, while apicularen B had no effect. The bisphosphonate incadronate inhibited bone resorption at 100 nM, being less effective than apicularen A. Our findings indicate that apicularen A inhibits bone resorption induced by PTH or IL-1beta more potently than apicularen B, probably due to inhibition of the V-ATPase.

FR177995, a novel vacuolar ATPase inhibitor, exerts not only an inhibitory effect on bone destruction but also anti-immunoinflammatory effects in adjuvant-induced arthritic rats.

There is considerable evidence that osteoclasts are involved in the pathogenesis of juxta-articular bone destruction in rheumatoid arthritis. Vacuolar ATPases (V-ATPases), which are highly expressed in the ruffled border membrane of osteoclasts, play a central role in the process of bone resorption, and V-ATPase inhibitors are effective in preventing bone destruction in several animal models of lytic bone diseases. Here, we evaluated for the first time the effects of V-ATPase inhibition in rats with adjuvant-induced arthritis (AIA) using FR177995, a novel V-ATPase inhibitor. FR177995 completely inhibited H(+) transport driven by V-ATPase, but exerted no effect on the H(+) transport activities of F- and P-ATPase, indicating that FR177995 is a specific inhibitor of V-ATPase. FR177995 acted directly on osteoclastic bone resorption and equally inhibited in vitro bone resorption stimulated by IL-1, IL-6 or PTH. In addition, FR177995 dose-dependently reduced retinoic acid-induced hypercalcemia in thyroparathyroidectomized-ovariectomized rats. When FR177995 was administered to AIA rats once a day, the loss of femoral bone mineral density was significantly improved. Moreover, indicators of cartilage damage (arthritis score and glycosaminoglycan content in the femoral condyles) and inflammation parameters (paw swelling volume, erythrocyte sedimentation rate and plasma sialic acid level) were found to be unexpectedly ameliorated. These results strongly suggest that V-ATPase may be an interesting drug target in the treatment of rheumatoid arthritis.

A vacuolar ATPase inhibitor, FR167356, prevents bone resorption in ovariectomized rats with high potency and specificity: potential for clinical application.

UNLABELLED: FR167356, a novel inhibitor of vacuolar ATPase, has high potency against osteoclast V-ATPase and low potency against lysosomal V-ATPase. FR167356 is the first compound of this nature to be tested. It has the potential to be useful for clinical application. INTRODUCTION: It has been suggested that the key issue regarding the therapeutic usefulness of V-ATPase inhibitors is their selectivity. MATERIALS AND METHODS: In in vitro and in vivo studies, we compared FR167356 with other vacuolar ATPase (V-ATPase) inhibitors, bafilomycin A1 and SB242784. H+ transport by various membrane vesicles was assayed by measuring uptake of acridine orange. Inhibitory activity against in vitro bone resorption was examined by measuring the Ca2+ release from cultured calvariae. In vivo, hypercalcemia was induced by retinoic acid in thyroparathyroidectomized-ovariectomized rats, and the effect on serum Ca2+ level was assessed. Ovariectomized rats were treated with FR167356 or SB242784. One week after surgery, free deoxypyridinoline levels in 24-h urine samples, which were collected from 6 h after administration of FR167356, were measured by ELISA. After 4 weeks of treatment, plasma biochemical parameters were analyzed. BMD of the distal femur metaphysis was measured with pQCT. Histomorphometric analysis of the proximal tibias was performed. Blood gases of rats treated with FR167356 were measured with a blood gas analyzer for estimating the effect of FR167356 on in vivo function of renal V-ATPase. RESULTS: FR167356, which is distinctly different from other V-ATPase inhibitors, has a high potency against osteoclast V-ATPase and low potency against lysosomal V-ATPase. Similarly, FR167356 inhibited bone resorption in vitro when stimulated by PTH, IL-1, and IL-6. FR167356 reduced retinoic acid-induced hypercalcemia in thyroparathyroidectomized-ovariectomized rats in a dose-dependent manner. Moreover, FR167356 was shown to restore BMD of ovariectomized rats caused by the inhibition of bone resorption. Ovariectomized rats treated with FR167356 did not show adverse symptoms, whereas SB242784 caused a decrease in body weight gain and significant changes in two plasma biochemical parameters. Interestingly, FR167356 treatment did not affect blood acid-base balance; however, FR167356 inhibited renal V-ATPase with a similar potency as for osteoclast V-ATPase inhibition. CONCLUSION: Comparison of FR167356 with SB242784 implies that the characteristics of FR167356 may be more appropriate for clinical application as a V-ATPase inhibitor.

Interaction of inhibitors of the vacuolar H(+)-ATPase with the transmembrane Vo-sector.

The macrolide antibiotic concanamycin A and a designed derivative of 5-(2-indolyl)-2,4-pentadienamide (INDOL0) are potent inhibitors of vacuolar H(+)-ATPases, with IC(50) values in the low and medium nanomolar range, respectively. Interaction of these V-ATPase inhibitors with spin-labeled subunit c in the transmembrane V(o)-sector of the ATPase was studied by using the transport-active 16-kDa proteolipid analogue of subunit c from the hepatopancreas of Nephrops norvegicus. Analogous experiments were also performed with vacuolar membranes from Saccharomyces cerevisiae. Membranous preparations of the Nephrops 16-kDa proteolipid were spin-labeled either on the unique cysteine C54, with a nitroxyl maleimide, or on the functionally essential glutamate E140, with a nitroxyl analogue of dicyclohexylcarbodiimide (DCCD). These residues were previously demonstrated to be accessible to lipid. Interaction of the inhibitors with these lipid-exposed residues was studied by using both conventional and saturation transfer EPR spectroscopy. Immobilization of the spin-labeled residues by the inhibitors was observed on both the nanosecond and microsecond time scales. The perturbation by INDOL0 was mostly greater than that by concanamycin A. Qualitatively similar but quantitatively greater effects were obtained with the same spin-label reagents and vacuolar membranes in which the Nephrops 16-kDa proteolipid was expressed in place of the native vma3p proteolipid of yeast. The spin-label immobilization corresponds to a direct interaction of the inhibitors with these intramembranous sites on the protein. A mutational analysis on transmembrane segment 4 known to give resistance to concanamycin A also gave partial resistance to INDOL0. The results are consistent with transmembrane segments 2 and 4 of the 16-kDa putative four-helix bundle, and particularly the functionally essential protonation locus, being involved in the inhibitor binding sites. Inhibition of proton transport may also involve immobilization of the overall rotation of the proteolipid subunit assembly.

Elicitor-activated phospholipase A(2) generates lysophosphatidylcholines that mobilize the vacuolar H(+) pool for pH signaling via the activation of Na(+)-dependent proton fluxes.

The elicitation of phytoalexin biosynthesis in cultured cells of California poppy involves a shift of cytoplasmic pH via the transient efflux of vacuolar protons. Intracellular effectors of vacuolar proton transport were identified by a novel in situ approach based on the selective permeabilization of the plasma membrane for molecules of < or = 10 kD. Subsequent fluorescence imaging of the vacuolar pH correctly reported experimental changes of activity of the tonoplast proton transporters. Lysophosphatidylcholine (LPC) caused a transient increase of the vacuolar pH by increasing the Na(+) sensitivity of a Na(+)-dependent proton efflux that was inhibited by amiloride. In intact cells, yeast elicitor activated phospholipase A(2), as demonstrated by the formation of LPC from fluorescent substrate analogs, and caused a transient increase of endogenous LPC, as determined by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. It is suggested that LPC generated by phospholipase A(2) at the plasma membrane transduces the elicitor-triggered signal into the activation of a tonoplast H(+)/Na(+) antiporter.