A simple assay for inhibitors of mycobacterial oxidative phosphorylation.

Oxidative phosphorylation, the combined activities of the electron transport chain (ETC) and ATP synthase, has emerged as a valuable target for antibiotics to treat infection with Mycobacterium tuberculosis and related pathogens. In oxidative phosphorylation, the ETC establishes a transmembrane electrochemical proton gradient that powers ATP synthesis. Monitoring oxidative phosphorylation with luciferase-based detection of ATP synthesis or measurement of oxygen consumption can be technically challenging and expensive. These limitations reduce the utility of these methods for characterization of mycobacterial oxidative phosphorylation inhibitors. Here, we show that fluorescence-based measurement of acidification of inverted membrane vesicles (IMVs) can detect and distinguish between inhibition of the ETC, inhibition of ATP synthase, and nonspecific membrane uncoupling. In this assay, IMVs from Mycobacterium smegmatis are acidified either through the activity of the ETC or ATP synthase, the latter modified genetically to allow it to serve as an ATP-driven proton pump. Acidification is monitored by fluorescence from 9-amino-6-chloro-2-methoxyacridine, which accumulates and quenches in acidified IMVs. Nonspecific membrane uncouplers prevent both succinate- and ATP-driven IMV acidification. In contrast, the ETC Complex III2IV2 inhibitor telacebec (Q203) prevents succinate-driven acidification but not ATP-driven acidification, and the ATP synthase inhibitor bedaquiline prevents ATP-driven acidification but not succinate-driven acidification. We use the assay to show that, as proposed previously, lansoprazole sulfide is an inhibitor of Complex III2IV2, whereas thioridazine uncouples the mycobacterial membrane nonspecifically. Overall, the assay is simple, low cost, and scalable, which will make it useful for identifying and characterizing new mycobacterial oxidative phosphorylation inhibitors.

Phase 1, Single- and Multiple-Ascending-Dose, Food-Effect, and East Asian Subject Studies to Assess the Pharmacokinetics, Safety, and Tolerability of Bempedoic Acid, a Selective Inhibitor of Adenosine Triphosphate Citrate Lyase.

Bempedoic acid is an adenosine triphosphate citrate lyase inhibitor that lowers low-density lipoprotein cholesterol by inhibiting cholesterol synthesis and upregulating hepatic low-density lipoprotein receptor expression. After oral dosing, bempedoic acid was readily absorbed, attaining maximum concentrations with a median time of 3.5 hours, and may be taken without regard to food. Steady-state oral pharmacokinetics in healthy adults receiving bempedoic acid at the approved 180 mg/day dose were characterized by mean maximum concentration of 20.6 µg/mL, area under the concentration-time curve over 24 hours of 289 µg·h/mL, and elimination half-life of 21.1 hours. Multiple-dose pharmacokinetics were linear at bempedoic acid doses of 120-220 mg/day. Circulating concentrations of the active metabolite ESP15228 were 18.0% of bempedoic acid concentrations on average. Comparisons of bempedoic acid 180 mg/day pharmacokinetics after single and multiple dosing revealed no clinically meaningful differences between Japanese, Chinese, and Western subjects. Mean estimates of bempedoic acid elimination half-life in Japanese (25.2 hours) and Chinese (20.0 hours) subjects were comparable to Western subjects (23.9 hours) following 14 days of once-daily dosing. Bempedoic acid was generally safe and well tolerated up to a dose of 220 mg/day across the study populations described herein.

Structure of the complex between calmodulin and a functional construct of eukaryotic elongation factor 2 kinase bound to an ATP-competitive inhibitor.

The calmodulin-activated α-kinase, eukaryotic elongation factor 2 kinase (eEF-2K), serves as a master regulator of translational elongation by specifically phosphorylating and reducing the ribosome affinity of the guanosine triphosphatase, eukaryotic elongation factor 2 (eEF-2). Given its critical role in a fundamental cellular process, dysregulation of eEF-2K has been implicated in several human diseases, including those of the cardiovascular system, chronic neuropathies, and many cancers, making it a critical pharmacological target. In the absence of high-resolution structural information, high-throughput screening efforts have yielded small-molecule candidates that show promise as eEF-2K antagonists. Principal among these is the ATP-competitive pyrido-pyrimidinedione inhibitor, A-484954, which shows high specificity toward eEF-2K relative to a panel of "typical" protein kinases. A-484954 has been shown to have some degree of efficacy in animal models of several disease states. It has also been widely deployed as a reagent in eEF-2K-specific biochemical and cell-biological studies. However, given the absence of structural information, the precise mechanism of the A-484954-mediated inhibition of eEF-2K has remained obscure. Leveraging our identification of the calmodulin-activatable catalytic core of eEF-2K, and our recent determination of its long-elusive structure, here we present the structural basis for its specific inhibition by A-484954. This structure, which represents the first for an inhibitor-bound catalytic domain of a member of the α-kinase family, enables rationalization of the existing structure-activity relationship data for A-484954 variants and lays the groundwork for further optimization of this scaffold to attain enhanced specificity/potency against eEF-2K.

Moxibustion attenuates neurogenic detrusor overactivity in spinal cord injury rats by inhibiting M2/ATP/P2X3 pathway.

PURPOSE: Activation of muscarinic receptors located in bladder sensory pathways is generally considered to be the primary contributor for driving the pathogenesis of neurogenic detrusor overactivity following spinal cord injury. The present study is undertaken to examine whether moxibustion improves neurogenic detrusor overactivity via modulating the abnormal muscarinic receptor pathway. MATERIALS AND METHODS: Female Sprague-Dawley rats were subjected to spinal cord injury with T9-10 spinal cord transection. Fourteen days later, animals were received moxibustion treatment for one week. Urodynamic parameters and pelvic afferents discharge were measured. Adenosine triphosphate (ATP) content in the voided cystometry fluid was determined. Expressions of M2, M3, and P2X3 receptors in the bladder mucosa were evaluated. RESULTS: Moxibustion treatment prevented the development of detrusor overactivity in spinal cord injury rats, with an increase in the intercontraction interval and micturition pressure threshold and a decrease in afferent activity during filling. The expression of M2 was markedly suppressed by moxibustion, accompanied by a reduction in the levels of ATP and P2X3. M2 receptor antagonist methoctramine hemihydrate had similar effects to moxibustion on bladder function and afferent activity, while the M2-preferential agonist oxotremorine methiodide abolished the beneficial effects of moxibustion. CONCLUSION: Moxibustion is a potential candidate for treating neurogenic bladder overactivity in a rat model of spinal cord injury, possibly through inhibiting the M2/ATP/P2X3 pathway.

Antifungal Activity and Action Mechanism Study of Coumarins from Cnidium monnieri Fruit and Structurally Related Compounds.

The increasing resistance of plant diseases caused by phytopathogenic fungi highlights the need for highly effective and environmentally benign agents. The antifungal activities of Cnidium monnieri fruit extracts and five isolated compounds as well as structurally related coumarins against five plant pathogenic fungi were evaluated. The acetone extract, which contained the highest amount of five coumarins, showed strongest antifungal activity. Among the coumarin compounds, we found that 4-methoxycoumarin exhibited stronger and broader antifungal activity against five phytopathogenic fungi, and was more potent than osthol. Especially, it could significantly inhibit the growth of Rhizoctonia solani mycelium with an EC50 value of 21 µg mL-1 . Further studies showed that 4-methoxycoumarin affected the structure and function of peroxisomes, inhibited the ß-oxidation of fatty acids, decreased the production of ATP and acetyl coenzyme A, and then accumulated ROS by damaging MMP and the mitochondrial function to cause the cell death of R. solani mycelia. 4-Methoxycoumarin presented antifungal efficacy in a concentration- dependent manner in vivo and could be used to prevent the potato black scurf. This study laid the foundation for the future development of 4-methoxycournamin as an alternative and friendly biofungicide.

Identification of unprecedented ATP-competitive choline kinase inhibitors.

In this article we describe the identification of unprecedented ATP-competitive ChoKα inhibitors starting from initial hit NMS-P830 that binds to ChoKα in an ATP concentration-dependent manner. This result is confirmed by the co-crystal structure of NMS-P830 in complex with Δ75-ChoKα. NMS-P830 is able to inhibit ChoKα in cells resulting in the reduction of intracellular phosphocholine formation. A structure-based medicinal chemistry program resulted in the identification of selective compounds that have good biochemical activity, solubility and metabolic stability and are suitable for further optimization. The ChoKα inhibitors disclosed in this article demonstrate for the first time the possibility to inhibit ChoKα with ATP-competitive compounds.

Fragment-based lead discovery to identify novel inhibitors that target the ATP binding site of pyruvate dehydrogenase kinases.

A fragment-based lead discovery approach was applied to Pyruvate Dehydrogenase Kinases (PDHKs) to discover inhibitors against the ATP binding site with novel chemotypes. X-ray fragment screening toward PDHK4 provided a fragment hit 1 with a characteristic interaction in a deep pocket of the ATP binding site. While known inhibitors utilize several water molecules in a deep pocket to form water-mediated hydrogen bond interactions, the fragment hit binds deeper in the pocket with a hydrophobic group. Displacement of a remaining water molecule in the pocket led to the identification of lead compound 7 with a notable improvement in inhibition potency. This lead compound possessed high ligand efficiency (LE) and showed decent selectivity profile. Two additional lead compounds 10 and 13 with new scaffolds with tricyclic and bicyclic cores were generated by merging structural information of another fragment hit 2. The characteristic interaction of these novel inhibitors in a deep pocket provides new structural insights about PDHKs ATP binding site and opens a novel direction for the development of PDHKs inhibitors.

The nitric oxide-cyclic guanosine monophosphate pathway inhibits the bladder ATP release in response to a physiological or pathological stimulus.

The release of ATP from the epithelium of the urinary bladder (urothelium) in response to mechanical/chemical stimuli contributes to the visceral sensation in the micturition reflex. The nitric oxide (NO)-mediated induction of cyclic guanosine monophosphate (cGMP) has been detected in urothelial cells and may inhibit the micturition reflex. However, the function of the NO-cGMP pathway in the regulation of urothelial ATP release remains poorly understood in contrast to its effects on smooth muscles or primary afferent nerves. Therefore, we investigated the relevance of the NO-cGMP pathway to ATP release on the mucosal side in the present study. The administration of l-arginine (NO precursor) or NOC 12 (NO donor) significantly reduced ATP release to the mucosal side at a physiologically normal urine storage pressure (5 cmH2 O). L-NAME (NO synthase inhibitor) significantly increased the distention-induced release of ATP. The phosphodiesterase-5 inhibitor, sildenafil, which increases cGMP levels, inhibited distention-induced ATP release. Furthermore, sildenafil significantly reduced ATP release in response to the administration of lipopolysaccharide. These results suggest that the NO-cGMP pathway inhibited urothelial ATP release during the storage phase under both physiological and pathological conditions.

Inhibition of PI3K/mTOR/KATP channel blunts sodium thiosulphate preconditioning mediated cardioprotection against ischemia-reperfusion injury.

Recent studies have shown that pre and postconditioning the heart with sodium thiosulfate (STS) attenuate ischemia-reperfusion (IR) injury. However, the underlying mechanism involved in the cardioprotective signaling pathway is not fully explored. This study examined the existing link of STS mediated protection (as pre and post-conditioning agents) with PI3K, mTOR, and mPTP signaling pathways using its respective inhibitors. STS was administered to the isolated perfused rat heart through Kreb's Heinselit buffer before ischemia (precondition: SIPC) and reperfusion (postcondition: SPOC) in the presence and absence of the PI3K, mTOR, and mPTP signaling pathway inhibitors (wortmannin, rapamycin, and glibenclamide respectively). SIPC failed to improve the IR injury-induced altered cardiac hemodynamics, increased infarct size, and the release of cardiac injury markers in the presence of these inhibitors. On the other hand, the SPOC protocol effectively rendered the cardioprotection even in the PI3K/mTOR/KATP inhibitors presence. Interestingly, the SIPC's identified mode of action viz reduction in oxidative stress and the preservation of mitochondrial function were lost in the inhibitors' presence. Based on the above results, we conclude that the underlying mechanism of SIPC mediated cardioprotection works via the PI3K/mTOR/KATP signaling pathway axis activation.

Acupuncture modulates extracellular ATP levels in peripheral sensory nervous system during analgesia of ankle arthritis in rats.

As an ancient analgesia therapy, acupuncture has been practiced worldwide nowadays. A good understanding of its mechanisms will offer a promise for its rational and wider application. As the first station of pain sensation, peripheral sensory ganglia express pain-related P2X receptors that are involved in the acupuncture analgesia mechanisms transduction pathway. While the role of their endogenous ligand, extracellular ATP (eATP), remains less studied. This work attempted to clarify whether acupuncture modulated eATP levels in the peripheral sensory nerve system during its analgesia process. Male Sprague-Dawley rats underwent acute inflammatory pain by injecting Complete Freund's Adjuvant in the unilateral ankle joint for 2 days. A twenty-minute acupuncture was applied to ipsilateral Zusanli acupoint. Thermal hyperalgesia and tactile allodynia were assessed on bilateral hind paws to evaluate the analgesic effect. eATP of bilateral isolated lumbar 4-5 dorsal root ganglia (DRGs) and sciatic nerves were determined by luminescence assay. Nucleotidases NTPDase-2 and -3 in bilateral ganglia and sciatic nerves were measured by real-time PCR to explore eATP hydrolysis process. Our results revealed that acute inflammation induced bilateral thermal hyperalgesia and ipsilateral tactile allodynia, which were accompanied by increased eATP levels and higher mechano-sensitivity of bilateral DRGs and decreased eATP levels of bilateral sciatic nerves. Acupuncture exerted anti-nociception on bilateral hind paws, reversed the increased eATP and mechanosensitivity of bilateral DRGs, and restored the decreased eATP of bilateral sciatic nerves. NTPDase-2 and -3 in bilateral ganglia and sciatic nerves were inconsistently modulated during this period. These observations indicate that eATP metabolism of peripheral sensory nerve system was simultaneously regulated during acupuncture analgesia, which might open a new frontier for acupuncture research.

A Genome-Wide Screen in Saccharomyces cerevisiae Reveals a Critical Role for Oxidative Phosphorylation in Cellular Tolerance to Lithium Hexafluorophosphate.

Lithium hexafluorophosphate (LiPF6) is one of the leading electrolytes in lithium-ion batteries, and its usage has increased tremendously in the past few years. Little is known, however, about its potential environmental and biological impacts. In order to improve our understanding of the cytotoxicity of LiPF6 and the specific cellular response mechanisms to it, we performed a genome-wide screen using a yeast (Saccharomyces cerevisiae) deletion mutant collection and identified 75 gene deletion mutants that showed LiPF6 sensitivity. Among these, genes associated with mitochondria showed the most enrichment. We also found that LiPF6 is more toxic to yeast than lithium chloride (LiCl) or sodium hexafluorophosphate (NaPF6). Physiological analysis showed that a high concentration of LiPF6 caused mitochondrial damage, reactive oxygen species (ROS) accumulation, and ATP content changes. Compared with the results of previous genome-wide screening for LiCl-sensitive mutants, we found that oxidative phosphorylation-related mutants were specifically hypersensitive to LiPF6. In these deletion mutants, LiPF6 treatment resulted in higher ROS production and reduced ATP levels, suggesting that oxidative phosphorylation-related genes were important for counteracting LiPF6-induced toxicity. Taken together, our results identified genes specifically involved in LiPF6-modulated toxicity, and demonstrated that oxidative stress and ATP imbalance maybe the driving factors in governing LiPF6-induced toxicity.

Inhibition of Mitochondrial ATP Synthesis and Regulation of Oxidative Stress Based on {SbW8 O30 } Determined by Single-Cell Proteomics Analysis.

The 10-nuclear heteroatom cluster modified {SbW8 O30 } was successfully synthesized and exhibited inhibitory activity (IC50 =0.29 µM). Based on proteomics analysis, Na4 Ni2 Sb2 W2 -SbW8 inhibited ATP production by affecting the expression of 16 related proteins, hindering metabolic functions in vivo and cell proliferation due to reactive oxygen species (ROS) stress. In particular, the low expression of FAD/FMN-binding redox enzymes (relative expression ratio of the experimental group to the control=0.43843) could be attributed to the redox mechanism of Na4 Ni2 Sb2 W2 -SbW8 , which was consistent with the effect of polyoxometalates (POMs) and FMN-binding proteins on ATP formation. An electrochemical study showed that Na4 Ni2 Sb2 W2 -SbW8 combined with FMN to form Na4 Ni2 Sb2 W2 -SbW8 -2FMN complex through a one-electron process of the W atoms. Na4 Ni2 Sb2 W2 -SbW8 acted as catalase and glutathione peroxidase to protect the cell from ROS stress, and the inhibition rates were 63.3 % at 1.77 µM of NADPH and 86.06 % at 10.62 µM of 2-hydroxyterephthalic acid. Overall, our results showed that POMs can be specific oxidative/antioxidant regulatory agents.

Sulfated Polysaccharides from Macroalgae Are Potent Dual Inhibitors of Human ATP-Hydrolyzing Ectonucleotidases NPP1 and CD39.

Extracellular ATP mediates proinflammatory and antiproliferative effects via activation of P2 nucleotide receptors. In contrast, its metabolite, the nucleoside adenosine, is strongly immunosuppressive and enhances tumor proliferation and metastasis. The conversion of ATP to adenosine is catalyzed by ectonucleotidases, which are expressed on immune cells and typically upregulated on tumor cells. In the present study, we identified sulfopolysaccharides from brown and red sea algae to act as potent dual inhibitors of the main ATP-hydrolyzing ectoenzymes, ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) and ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39), showing nano- to picomolar potency and displaying a non-competitive mechanism of inhibition. We showed that one of the sulfopolysaccharides tested as a representative example reduced adenosine formation at the surface of the human glioblastoma cell line U87 in a concentration-dependent manner. These natural products represent the most potent inhibitors of extracellular ATP hydrolysis known to date and have potential as novel therapeutics for the immunotherapy of cancer.

Multiparametric High-Content Assays to Measure Cell Health and Oxidative Damage as a Model for Drug-Induced Liver Injury.

Drug-induced liver injury is an important cause of non-approval in drug development and the withdrawal of already approved drugs from the market. Screening human hepatic cell lines for toxicity has been used extensively to predict drug-induced liver injury in preclinical drug development. Assessing hepatic-cell health with more diverse markers will increase the value of in vitro assays and help predict the mechanism of toxicity. We describe three live cell-based assays using HepG2 cells to measure cell health parameters indicative of hepatotoxicity. The first assay measures cellular ATP levels using luciferase. The second and third assays are multiparametric high-content screens covering a panel of cell health markers including cell count, mitochondrial membrane potential and structure, nuclear morphology, vacuolar density, and reactive oxygen species and glutathione levels. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Measurement of cellular ATP content Basic Protocol 2: High-content analysis assay to assess cell count, mitochondrial membrane potential and structure, and reactive oxygen species Basic Protocol 3: High-content analysis assay to assess nuclear morphology, vacuoles, and glutathione content Support Protocol 1: Subculturing and maintaining HepG2 cells Support Protocol 2: Plating HepG2 cell line Support Protocol 3: Transferring compounds by pin tool Support Protocol 4: Generating dose-response curves.

P2X3-Containing Receptors as Targets for the Treatment of Chronic Pain.

Current therapies for the treatment of chronic pain provide inadequate relief for millions of suffering patients, demonstrating the need for better therapies that will treat pain effectively and improve the quality of patient's lives. Better understanding of the mechanisms that mediate chronic pain is critical for developing drugs with improved clinical outcomes. Adenosine triphosphate (ATP) is a key modulator in nociceptive pathways. Release of ATP from injured tissue or sympathetic efferents has sensitizing effects on sensory neurons in the periphery, and presynaptic vesicular release of ATP from the central terminals can increase glutamate release thereby potentiating downstream central sensitization mechanisms, a condition thought to underlie many chronic pain conditions. The purinergic receptors on sensory nerves primarily responsible for ATP signaling are P2X3 and P2X2/3. Selective knockdown experiments, or inhibition with small molecules, demonstrate P2X3-containing receptors are key targets to modulate nociceptive signals. Preclinical studies have identified that P2X3-containing receptors are critical for sensory transduction for bladder function, and clinical studies have shown promise in treatment for bladder pain and pain associated with osteoarthritis. Further clinical characterization of antagonists to P2X3-containing receptors may lead to improved therapies in the treatment of chronic pain.

Intrinsic Apyrase-Like Activity of Cerium-Based Metal-Organic Frameworks (MOFs): Dephosphorylation of Adenosine Tri- and Diphosphate.

Apyrase is an important family of extracellular enzymes that catalyse the hydrolysis of high-energy phosphate bonds (HEPBs) in ATP and ADP, thereby modulating many physiological processes and driving life activities. Herein, we report an unexpected discovery that cerium-based metal-organic frameworks (Ce-MOFs) of UiO-66(Ce) have intrinsic apyrase-like activity for ATP/ADP-related physiological processes. The abundant CeIII /CeIV couple sites of Ce-MOFs endow them with the ability to selectively catalyse the hydrolysis of HEPBs of ATP and ADP under physiological conditions. Compared to natural enzymes, they could resist extreme pH and temperature, and present a broad range of working conditions. Based on this finding, a significant inhibitory effect on ADP-induced platelet aggregation was observed upon exposing the platelet-rich plasma (PRP) to the biomimetic UiO-66(Ce) films, prefiguring their wide application potentials in medicine and biotechnology.

ATP depletion induced cochlear hair cells death through histone deacetylation in vitro.

Previous studies have shown histone modifications being present in cochlear hair cells in animal models of hearing loss. Our past studies have shown that ATP depletion, histone deacetylase (HDAC) upregulation, and histone deacetylation occur in cochlea after noise exposure, and these are linked to hair cell death. Whether ATP depletion correlates with the expression level of HDACs and acetylation of histones is still unknown. In this study, we investigated the changes in the expression of HDACs and the level of histone acetylation in cochlear hair cells using an ATP-depleted explant culture of mouse organ of Corti. We found that the expression of HDAC3 and HDAC6 increased and hair cells were lost after oligomycin A (OA) treatment. Meanwhile, the acetylation level of histone H2B reduced. However, when oligomycin was combined with an HDAC inhibitor, trichostatin A (TSA), the acetylation level of histone H3 was restored. Moreover, combined treatment of oligomycin and TSA or sodium butyrate (NaB) attenuated oligomycin-induced cochlear hair cell loss. In conclusion, our results indicated that ATP depletion led to histone deacetylation and eventually resulted in hair cell death.

Small Molecule Inhibition of CPS1 Activity through an Allosteric Pocket.

Carbamoyl phosphate synthetase 1 (CPS1) catalyzes the first step in the ammonia-detoxifying urea cycle, converting ammonia to carbamoyl phosphate under physiologic conditions. In cancer, CPS1 overexpression supports pyrimidine synthesis to promote tumor growth in some cancer types, while in others CPS1 activity prevents the buildup of toxic levels of intratumoral ammonia to allow for sustained tumor growth. Targeted CPS1 inhibitors may, therefore, provide a therapeutic benefit for cancer patients with tumors overexpressing CPS1. Herein, we describe the discovery of small-molecule CPS1 inhibitors that bind to a previously unknown allosteric pocket to block ATP hydrolysis in the first step of carbamoyl phosphate synthesis. CPS1 inhibitors are active in cellular assays, blocking both urea synthesis and CPS1 support of the pyrimidine biosynthetic pathway, while having no activity against CPS2. These newly discovered CPS1 inhibitors are a first step toward providing researchers with valuable tools for probing CPS1 cancer biology.

SERCA and P-glycoprotein inhibition and ATP depletion are necessary for celastrol-induced autophagic cell death and collateral sensitivity in multidrug-resistant tumor cells.

Multidrug resistance (MDR) represents an obstacle in anti-cancer therapy. MDR is caused by multiple mechanisms, involving ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp), which reduces intracellular drug levels to sub-therapeutic concentrations. Therefore, sensitizing agents retaining effectiveness against apoptosis- or drug-resistant cancers are desired for the treatment of MDR cancers. The sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pump is an emerging target to overcome MDR, because of its continuous expression and because the calcium transport function is crucial to the survival of tumor cells. Previous studies showed that SERCA inhibitors exhibit anti-cancer effects in Bax-Bak-deficient, apoptosis-resistant and MDR cancers, whereas specific P-gp inhibitors reverse the MDR phenotype of cancer cells by blocking efflux of chemotherapeutic agents. Here, we unraveled SERCA and P-gp as double targets of the triterpenoid, celastrol to reverse MDR. Celastrol inhibited both SERCA and P-gp to stimulate calcium-mediated autophagy and ATP depletion, thereby induced collateral sensitivity in MDR cancer cells. In vivo studies further confirmed that celastrol suppressed tumor growth and metastasis by SERCA-mediated calcium mobilization. To the best of our knowledge, our findings demonstrate collateral sensitivity in MDR cancer cells by simultaneous inhibition of SERCA and P-gp for the first time.

Ouabain impairs cancer metabolism and activates AMPK-Src signaling pathway in human cancer cell lines.

In addition to the well-known cardiotonic effects, cardiac glycosides (CGs) produce potent anticancer effects with various molecular mechanisms. We previously show that ouabain induces autophagic cell death in human lung cancer cells by regulating AMPK-mediated mTOR and Src-mediated ERK1/2 signaling pathways. However, whether and how AMPK and Src signaling interacts in ouabain-treated cancer cells remains unclear. Given the pivotal role of AMPK in metabolism, whether ouabain affects cancer cell metabolism remains elusive. In this study we showed that treatment with ouabain (25 nM) caused simultaneous activation of AMPK and Src signaling pathways in human lung cancer A549 cells and human breast cancer MCF7 cells. Cotreatment with AMPK inhibitor compound C or siRNA greatly abrogates ouabain-induced Src activation, whereas cotreatment with Src inhibitor PP2 has little effect on ouabain-induced AMPK activity, suggesting that AMPK served as an upstream regulator of the Src signaling pathway. On the other hand, ouabain treatment greatly depletes ATP production in A549 and MCF7 cells, and supplement of ATP (100 µM) blocked ouabain-induced AMPK activation. We further demonstrated that ouabain greatly inhibited the mitochondrial oxidative phosphorylation (OXPHOS) in the cancer cells, and exerted differential metabolic effects on glycolysis depending on cancer cell type. Taken together, this study reveals that the altered cancer cell metabolism caused by ouabain may contribute to AMPK activation, as well as its cytotoxicity towards cancer cells.