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Meclizine Inhibits Mitochondrial Respiration through Direct Targeting of Cytosolic Phosphoethanolamine Metabolism*

  • Vishal M. Gohil
    Correspondence
    To whom correspondence may be addressed: Dept. of Biochemistry and Biophysics, Texas A&M University, ILSB Rm. No. 2146A, 301 Old Main Dr., College Station, TX 77843
    Footnotes
    Affiliations
    Department of Molecular Biology and Medicine, Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts 02114

    Broad Institute, Cambridge, Massachusetts 02142

    Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115

    Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843
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  • Lin Zhu
    Footnotes
    Affiliations
    Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario NIG 2W1, Canada
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  • Charli D. Baker
    Footnotes
    Affiliations
    Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843
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  • Valentin Cracan
    Affiliations
    Department of Molecular Biology and Medicine, Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts 02114

    Broad Institute, Cambridge, Massachusetts 02142

    Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115
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  • Abbas Yaseen
    Affiliations
    Department of Molecular Biology and Medicine, Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts 02114
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  • Mohit Jain
    Affiliations
    Department of Molecular Biology and Medicine, Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts 02114

    Broad Institute, Cambridge, Massachusetts 02142

    Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115
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  • Clary B. Clish
    Affiliations
    Broad Institute, Cambridge, Massachusetts 02142
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  • Paul S. Brookes
    Affiliations
    Department of Anesthesiology, University of Rochester Medical Center, Rochester, New York 14642
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  • Marica Bakovic
    Footnotes
    Affiliations
    Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario NIG 2W1, Canada
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  • Vamsi K. Mootha
    Correspondence
    To whom correspondence may be addressed: Dept. of Molecular Biology, Massachusetts General Hospital, 185 Cambridge St., 6th Floor, Boston, MA 02114
    Affiliations
    Department of Molecular Biology and Medicine, Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts 02114

    Broad Institute, Cambridge, Massachusetts 02142

    Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant 5K08HL107451 (to M. J.). This work was also supported by a grant from the American Diabetes Association/Smith Family Foundation (to V. K. M.). V. K. M. and V. M. G. are listed as inventors on a patent application filed by the Massachusetts General Hospital.
    This article contains supplemental Table 1.
    3 Supported by grants from the Canadian Institutes of Health Research and Ontario President Research Excellence Award.
    1 Supported by Welch Foundation Grant A-1810 and discretionary funds from Texas A&M University.
Open AccessPublished:October 19, 2013DOI:https://doi.org/10.1074/jbc.M113.489237
      We recently identified meclizine, an over-the-counter drug, as an inhibitor of mitochondrial respiration. Curiously, meclizine blunted respiration in intact cells but not in isolated mitochondria, suggesting an unorthodox mechanism. Using a metabolic profiling approach, we now show that treatment with meclizine leads to a sharp elevation of cellular phosphoethanolamine, an intermediate in the ethanolamine branch of the Kennedy pathway of phosphatidylethanolamine biosynthesis. Metabolic labeling and in vitro enzyme assays confirmed direct inhibition of the cytosolic enzyme CTP:phosphoethanolamine cytidylyltransferase (PCYT2). Inhibition of PCYT2 by meclizine led to rapid accumulation of its substrate, phosphoethanolamine, which is itself an inhibitor of mitochondrial respiration. Our work identifies the first pharmacologic inhibitor of the Kennedy pathway, demonstrates that its biosynthetic intermediate is an endogenous inhibitor of respiration, and provides key mechanistic insights that may facilitate repurposing meclizine for disorders of energy metabolism.

      Introduction

      Although mitochondrial respiration is crucial for cellular energetics and redox balance, during certain pathological conditions, respiration can actually contribute to pathogenesis (
      • Vafai S.B.
      • Mootha V.K.
      Mitochondrial disorders as windows into an ancient organelle.
      ). Attenuation of mitochondrial respiration has been proposed as a therapeutic strategy in a number of human disorders, including ischemia-reperfusion injury, neurodegeneration, autoimmune disease, and cancer (
      • Armstrong J.S.
      Mitochondrial medicine: pharmacological targeting of mitochondria in disease.
      ). Many naturally occurring as well as synthetic compounds targeting the mitochondrial respiratory chain are available, but their clinical utility is limited by their narrow therapeutic index (
      • Dykens J.A.
      • Will Y.
      The significance of mitochondrial toxicity testing in drug development.
      ); thus, there is an unmet need for discovering new classes of drugs that can safely modulate mitochondrial respiration.
      We recently identified meclizine, an over-the-counter anti-nausea drug, in a “nutrient-sensitized” chemical screen aimed at identifying compounds that attenuate mitochondrial respiration (
      • Gohil V.M.
      • Sheth S.A.
      • Nilsson R.
      • Wojtovich A.P.
      • Lee J.H.
      • Perocchi F.
      • Chen W.
      • Clish C.B.
      • Ayata C.
      • Brookes P.S.
      • Mootha V.K.
      Nutrient-sensitized screening for drugs that shift energy metabolism from mitochondrial respiration to glycolysis.
      ). In vivo follow-up studies demonstrated that meclizine could be protective against heart attack (
      • Gohil V.M.
      • Sheth S.A.
      • Nilsson R.
      • Wojtovich A.P.
      • Lee J.H.
      • Perocchi F.
      • Chen W.
      • Clish C.B.
      • Ayata C.
      • Brookes P.S.
      • Mootha V.K.
      Nutrient-sensitized screening for drugs that shift energy metabolism from mitochondrial respiration to glycolysis.
      ), stroke (
      • Gohil V.M.
      • Sheth S.A.
      • Nilsson R.
      • Wojtovich A.P.
      • Lee J.H.
      • Perocchi F.
      • Chen W.
      • Clish C.B.
      • Ayata C.
      • Brookes P.S.
      • Mootha V.K.
      Nutrient-sensitized screening for drugs that shift energy metabolism from mitochondrial respiration to glycolysis.
      ), and neurodegeneration (
      • Gohil V.M.
      • Offner N.
      • Walker J.A.
      • Sheth S.A.
      • Fossale E.
      • Gusella J.F.
      • MacDonald M.E.
      • Neri C.
      • Mootha V.K.
      Meclizine is neuroprotective in models of Huntington's disease.
      ) in animal models. Meclizine is a first generation piperazine class of H1-antihistamine that has been in use for decades for prophylaxis against nausea and vertigo (
      • Cohen B.
      • DeJong J.M.
      Meclizine and placebo in treating vertigo of vestibular origin. Relative efficacy in a double-blind study.
      ). Like many H1-antihistamines, meclizine has anticholinergic activity (
      • Kubo N.
      • Shirakawa O.
      • Kuno T.
      • Tanaka C.
      Antimuscarinic effects of antihistamines: quantitative evaluation by receptor-binding assay.
      ), and it has also been shown to target constitutive androstane receptors (
      • Huang W.
      • Zhang J.
      • Wei P.
      • Schrader W.T.
      • Moore D.D.
      Meclizine is an agonist ligand for mouse constitutive androstane receptor (CAR) and an inverse agonist for human CAR.
      ).
      Meclizine inhibition of respiration had not been reported before, and unlike classical respiratory inhibitors (e.g. antimycin and rotenone), meclizine did not inhibit respiration in isolated mitochondria but rather only in intact cells (
      • Gohil V.M.
      • Sheth S.A.
      • Nilsson R.
      • Wojtovich A.P.
      • Lee J.H.
      • Perocchi F.
      • Chen W.
      • Clish C.B.
      • Ayata C.
      • Brookes P.S.
      • Mootha V.K.
      Nutrient-sensitized screening for drugs that shift energy metabolism from mitochondrial respiration to glycolysis.
      ). We previously reported that this effect was independent of its antihistaminergic and anticholinergic activity (
      • Gohil V.M.
      • Sheth S.A.
      • Nilsson R.
      • Wojtovich A.P.
      • Lee J.H.
      • Perocchi F.
      • Chen W.
      • Clish C.B.
      • Ayata C.
      • Brookes P.S.
      • Mootha V.K.
      Nutrient-sensitized screening for drugs that shift energy metabolism from mitochondrial respiration to glycolysis.
      ), but the precise mechanism remained unknown. Curiously, the kinetics of meclizine-mediated inhibition of respiration were on the time scale of minutes (
      • Gohil V.M.
      • Sheth S.A.
      • Nilsson R.
      • Wojtovich A.P.
      • Lee J.H.
      • Perocchi F.
      • Chen W.
      • Clish C.B.
      • Ayata C.
      • Brookes P.S.
      • Mootha V.K.
      Nutrient-sensitized screening for drugs that shift energy metabolism from mitochondrial respiration to glycolysis.
      ), which is far too fast for a transcriptional mechanism but slower than direct inhibitors of the respiratory chain, suggesting that the inhibition arose from a potentially novel mechanism perhaps through intracellular accumulation of a meclizine-derived active metabolite or by perturbing metabolism.
      To gain insights into the mechanism of meclizine action, we performed global metabolic profiling of meclizine-treated cells to detect alterations in intracellular metabolites of intermediary metabolism. Metabolic profiling revealed a sharp increase in intracellular levels of phosphoethanolamine (PEtn),
      The abbreviations used are: PEtn
      phosphoethanolamine
      PE
      phosphatidylethanolamine
      PCho
      phosphocholine
      PCYT2
      CTP:phosphoethanolamine cytidylyltransferase
      Etn
      ethanolamine
      OCR
      oxygen consumption rate
      ECAR
      extracellular acidification rate.
      an intermediate in the CDP-ethanolamine (Etn) Kennedy pathway of phosphatidylethanolamine (PE) biosynthesis. Follow-up biochemical experiments confirmed the direct inhibition of CTP:phosphoethanolamine cytidylyltransferase (PCYT2), a rate-limiting enzyme of the CDP-Etn Kennedy pathway. The inhibition of PCYT2 results in the buildup of its substrate, PEtn, which itself directly inhibits mitochondrial respiration. Our work thus identifies a novel molecular target of meclizine and links the CDP-Etn Kennedy pathway to mitochondrial respiration.

      DISCUSSION

      The current study provides the molecular basis for inhibition of respiration by meclizine, an over-the-counter antinausea and antivertigo drug. Using a combination of mass spectrometry, metabolic labeling, and in vitro biochemical assays, we found that PCYT2 is a direct target of meclizine. Our study highlights the use of metabolic profiling in deciphering mechanisms of drug action with important biological and clinical implications.
      To our knowledge, this study is the first to pharmacologically link the Kennedy pathway of PE biosynthesis to mitochondrial energy metabolism and is consistent with a previous report showing that mice heterozygous for the Pcyt2 gene have reduced energy production from fatty acid oxidation (
      • Fullerton M.D.
      • Hakimuddin F.
      • Bonen A.
      • Bakovic M.
      The development of a metabolic disease phenotype in CTP:phosphoethanolamine cytidylyltransferase-deficient mice.
      ). PE is an essential phospholipid present in eukaryotic membranes and is highly enriched in mitochondrial membranes. It has overlapping functions with cardiolipin (
      • Gohil V.M.
      • Thompson M.N.
      • Greenberg M.L.
      Synthetic lethal interaction of the mitochondrial phosphatidylethanolamine and cardiolipin biosynthetic pathways in Saccharomyces cerevisiae.
      ), a mitochondrion-specific phospholipid that is essential for optimal respiration (
      • Jiang F.
      • Ryan M.T.
      • Schlame M.
      • Zhao M.
      • Gu Z.
      • Klingenberg M.
      • Pfanner N.
      • Greenberg M.L.
      Absence of cardiolipin in the crd1 null mutant results in decreased mitochondrial membrane potential and reduced mitochondrial function.
      ). PE is synthesized by multiple biochemical pathways (
      • Vance J.E.
      Phosphatidylserine and phosphatidylethanolamine in mammalian cells: two metabolically related aminophospholipids.
      ,
      • Pavlovic Z.
      • Bakovic M.
      Regulation of phosphatidylethanolamine homeostasis—the critical role of CTP:phosphoethanolamine cytidylyltransferase (Pcyt2).
      ), including the phosphatidylserine decarboxylase-catalyzed mitochondrial pathway and the cytosolic/endoplasmic reticulum CDP-Etn Kennedy pathway. The mitochondrial pathway contributes the bulk of mitochondrial PE that is retained in this organelle and contributes to mitochondrial function (
      • Steenbergen R.
      • Nanowski T.S.
      • Beigneux A.
      • Kulinski A.
      • Young S.G.
      • Vance J.E.
      Disruption of the phosphatidylserine decarboxylase gene in mice causes embryonic lethality and mitochondrial defects.
      ,
      • Bleijerveld O.B.
      • Brouwers J.F.
      • Vaandrager A.B.
      • Helms J.B.
      • Houweling M.
      The CDP-ethanolamine pathway and phosphatidylserine decarboxylation generate different phosphatidylethanolamine molecular species.
      ). Recently, it has been shown that a decrease in mitochondrial PE by deletion of phosphatidylserine decarboxylase in the yeast Saccharomyces cerevisiae results in reduced respiration (
      • Böttinger L.
      • Horvath S.E.
      • Kleinschroth T.
      • Hunte C.
      • Daum G.
      • Pfanner N.
      • Becker T.
      Phosphatidylethanolamine and cardiolipin differentially affect the stability of mitochondrial respiratory chain supercomplexes.
      ). A similar reduction in mitochondrial respiration has been observed in mammalian cells where mitochondrial phosphatidylserine decarboxylase is depleted (
      • Tasseva G.
      • Bai H.D.
      • Davidescu M.
      • Haromy A.
      • Michelakis E.
      • Vance J.E.
      Phosphatidylethanolamine deficiency in mammalian mitochondria impairs oxidative phosphorylation and alters mitochondrial morphology.
      ). However, the non-mitochondrial CDP-Etn Kennedy pathway of PE synthesis has never been linked to a respiratory defect in either yeast or mammalian cells.
      How does direct inhibition of PCYT2 lead to attenuation of mitochondrial respiration? Two possibilities exist. 1) The accumulation of upstream metabolites (PEtn) could interfere with mitochondrial respiration, or 2) depletion of downstream metabolites (PE) could alter mitochondrial membrane structure, thereby inhibiting respiration. We favor the first hypothesis, which is supported by our observation that the intracellular concentration of PEtn in meclizine-teated fibroblasts increased to a level sufficient to inhibit mitochondrial respiration (Figs. 1D and 5, A and B). Notably, our in vitro data on PEtn inhibition of mitochondrial respiration is consistent with a previous study that showed that both Etn and PEtn inhibit respiration in isolated mitochondria (
      • Modica-Napolitano J.S.
      • Renshaw P.F.
      Ethanolamine and phosphoethanolamine inhibit mitochondrial function in vitro: implications for mitochondrial dysfunction hypothesis in depression and bipolar disorder.
      ). The second mechanism seems less likely because the bulk of mitochondrial PE is synthesized in situ by the action of phosphatidylserine decarboxylase. Moreover, we did not observe any decrease in cellular or mitochondrial PE of the meclizine-treated fibroblasts, discounting the second possibility (Fig. 6). The synthetic interaction between meclizine and Etn in MCH58 cells further buttresses the first model because addition of Etn to meclizine-treated cells exacerbated, rather than alleviated, the cell viability as measured by ATP levels (Fig. 7).
      According to our model, meclizine itself has no effect on mitochondria but rather blocks PCYT2, leading to the accumulation of PEtn, which is itself an endogenous inhibitor of respiration. Although multiple lines of evidence support our model, questions still remain. First, the genetic depletion of PCYT2 by RNAi did not completely phenocopy the effect of meclizine on mitochondrial respiration, although this could be due to incomplete knockdown and the lack of sustained accumulation of PEtn (Fig. 4E). The inability of genetic silencing to fully phenocopy drug treatment is not uncommon (
      • Weiss W.A.
      • Taylor S.S.
      • Shokat K.M.
      Recognizing and exploiting differences between RNAi and small-molecule inhibitors.
      ) and in principle could be due to the presence of residual enzyme activity of PCYT2. Second, overexpression of PCYT2 did not confer resistance to the effect of meclizine on respiration (data not shown), which could be due to tight regulation of its intracellular levels. Given that meclizine is known to target multiple cellular proteins (
      • Cohen B.
      • DeJong J.M.
      Meclizine and placebo in treating vertigo of vestibular origin. Relative efficacy in a double-blind study.
      ,
      • Kubo N.
      • Shirakawa O.
      • Kuno T.
      • Tanaka C.
      Antimuscarinic effects of antihistamines: quantitative evaluation by receptor-binding assay.
      ,
      • Huang W.
      • Zhang J.
      • Wei P.
      • Schrader W.T.
      • Moore D.D.
      Meclizine is an agonist ligand for mouse constitutive androstane receptor (CAR) and an inverse agonist for human CAR.
      ), we cannot exclude additional mechanisms that may underlie its impact on respiration.
      Regardless, we have clearly shown that meclizine inhibits PCYT2 and causes an increase in cytosolic PEtn to a level sufficient to inhibit mitochondrial respiration. To our knowledge, such a mechanism of respiratory inhibition has never been described before. The mechanism of PEtn-mediated inhibition of respiration appears to be distinct from canonical inhibitors of respiration, including inhibitors of electron transport (rotenone and antimycin), uncouplers (carbonyl cyanide 3-chlorophenylhydrazone and dinitrophenol), and ATP synthesis (oligomycin). PEtn addition to mitochondria resulted in reduced oxygen consumption, diminished membrane potential, and a decrease in NADH levels (Fig. 5), whereas treatment with rotenone or antimycin would have increased NADH levels, carbonyl cyanide 3-chlorophenylhydrazone would have increased oxygen consumption, and oligomycin would have increased membrane potential. The inhibition does not appear to be substrate-specific as we observed inhibition of respiration using complex I- or complex II-linked substrates. The precise mechanism by which accumulation of cytosolic PEtn inhibits respiration is currently not known, but our bioenergetics measurements suggest a mechanism whereby PEtn may interfere with the generation of reducing equivalents that feed into the respiratory chain.
      Importantly, our work identifies for the first time an inhibitor of the CDP-Etn Kennedy pathway with therapeutic potential. This pathway has been implicated in a range of human disorders, including cancer (
      • Ferreira A.K.
      • Meneguelo R.
      • Pereira A.
      • Mendonça Filho O.
      • Chierice G.O.
      • Maria D.A.
      Anticancer effects of synthetic phosphoethanolamine on Ehrlich ascites tumor: an experimental study.
      ) and ischemia-reperfusion injury of brain and heart (
      • Kelly R.F.
      • Lamont K.T.
      • Somers S.
      • Hacking D.
      • Lacerda L.
      • Thomas P.
      • Opie L.H.
      • Lecour S.
      Ethanolamine is a novel STAT-3 dependent cardioprotective agent.
      ), and infectious disorders, including African sleeping sickness and malaria (
      • Gibellini F.
      • Hunter W.N.
      • Smith T.K.
      The ethanolamine branch of the Kennedy pathway is essential in the bloodstream form of Trypanosoma brucei.
      ,
      • Maheshwari S.
      • Lavigne M.
      • Contet A.
      • Alberge B.
      • Pihan E.
      • Kocken C.
      • Wengelnik K.
      • Douguet D.
      • Vial H.
      • Cerdan R.
      Biochemical characterization of Plasmodium falciparum CTP:phosphoethanolamine cytidylyltransferase shows that only one of the two cytidylyltransferase domains is active.
      ). Our own previous work has shown that meclizine, through blunting of respiration, is cytoprotective against ischemic injury to the brain and the heart (
      • Gohil V.M.
      • Sheth S.A.
      • Nilsson R.
      • Wojtovich A.P.
      • Lee J.H.
      • Perocchi F.
      • Chen W.
      • Clish C.B.
      • Ayata C.
      • Brookes P.S.
      • Mootha V.K.
      Nutrient-sensitized screening for drugs that shift energy metabolism from mitochondrial respiration to glycolysis.
      ) as well as polyglutamine toxicity observed in Huntington disease (
      • Gohil V.M.
      • Offner N.
      • Walker J.A.
      • Sheth S.A.
      • Fossale E.
      • Gusella J.F.
      • MacDonald M.E.
      • Neri C.
      • Mootha V.K.
      Meclizine is neuroprotective in models of Huntington's disease.
      ). Currently, effective therapies are not available for these disorders, making meclizine an attractive drug for repurposing. An intriguing question is to what extent the antivertigo and antinausea effects of meclizine may be occurring through targeting of PCYT2. A recent pharmacokinetics study on human subjects given an oral dose of 25 mg showed a peak plasma concentration of 80 ng/ml (∼0.2 μm) (
      • Wang Z.
      • Lee B.
      • Pearce D.
      • Qian S.
      • Wang Y.
      • Zhang Q.
      • Chow M.S.
      Meclizine metabolism and pharmacokinetics: formulation on its absorption.
      ), which is almost 70-fold below the minimum concentration required for inhibition of respiration in cell lines we have tested (
      • Gohil V.M.
      • Sheth S.A.
      • Nilsson R.
      • Wojtovich A.P.
      • Lee J.H.
      • Perocchi F.
      • Chen W.
      • Clish C.B.
      • Ayata C.
      • Brookes P.S.
      • Mootha V.K.
      Nutrient-sensitized screening for drugs that shift energy metabolism from mitochondrial respiration to glycolysis.
      ); thus, currently approved doses are unlikely to be active on mitochondrial respiration. We anticipate that identification of PCYT2 as a direct molecular target of meclizine may help to guide its clinical development for new uses. It is notable that PEtn, which accumulates following inhibition, can be detected noninvasively using 31P NMR, providing a facile marker of pharmacodynamics.

      Acknowledgments

      We thank Michelle Yu for assistance with measurements of bioenergetics. We thank Eric Shoubridge and Marcy MacDonald for providing MCH58 and mouse striatal cell lines, respectively.

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