Antamanide, a Derivative of Amanita phalloides, Is a Novel Inhibitor of the Mitochondrial Permeability Transition Pore

Is a Novel Inhibitor of the Mitochondrial Permeability Transition Pore. PLoS ONE 6(1): e16280. doi:10.1371/journal.pone.0016280 Antamanide, a Derivative of Amanita phalloides , Is a Novel Inhibitor of the Mitochondrial Permeability Transition Pore Luca Azzolin 0 Nicola Antolini 0 Andrea Calderan 0 Paolo Ruzza 0 Marco Sciacovelli 0 Oriano Marin 0 Stefano Mammi 0 Paolo Bernardi 0 Andrea Rasola 0 Alicia Kowaltowski, Instituto de Qumica - Universidade de Sao Paulo, Brazil 0 1 Department of Biomedical Sciences University of Padova and CNR Institute of Neuroscience , Padova , Italy , 2 Department of Chemical Sciences, University of Padova and CNR Institute of Biomolecular Chemistry , Padova , Italy , 3 Department of Biological Chemistry, University of Padova , Padova , Italy , 4 Venetian Institute of Molecular Medicine, University of Padova , Padova , Italy Antamanide is a cyclic decapeptide derived from the fungus Amanita phalloides. Here we show that antamanide inhibits the mitochondrial permeability transition pore, a central effector of cell death induction, by targeting the pore regulator cyclophilin D. Indeed, (i) permeability transition pore inhibition by antamanide is not additive with the cyclophilin D-binding drug cyclosporin A, (ii) the inhibitory action of antamanide on the pore requires phosphate, as previously shown for cyclosporin A; (iii) antamanide is ineffective in mitochondria or cells derived from cyclophilin D null animals, and (iv) abolishes CyP-D peptidyl-prolyl cis-trans isomerase activity. Permeability transition pore inhibition by antamanide needs two critical residues in the peptide ring, Phe6 and Phe9, and is additive with ubiquinone 0, which acts on the pore in a cyclophilin D-independent fashion. Antamanide also abrogates mitochondrial depolarization and the ensuing cell death caused by two well-characterized pore inducers, clotrimazole and a hexokinase II N-terminal peptide. Our findings have implications for the comprehension of cyclophilin D activity on the permeability transition pore and for the development of novel pore-targeting drugs exploitable as cell death inhibitors. - Funding: This work was made possible by grants from Progetti di Ricerca di Interesse Nazionale del Ministero dellUniversita` e della Ricerca (to P.B.), from Associazione Italiana Ricerca sul Cancro (P.B., project number 8722), and from Progetti di Ateneo dellUniversita` di Padova (A.R.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Antamanide (AA) is a monocyclic, homodetic decapeptide isolated from the poisonous mushroom Amanita phalloides [1]. AA has been extensively studied for its strong antidote activity against phallotoxins and amatoxins, which are extremely toxic peptides isolated from the same fungus [2]. It was also reported that AA inhibits tumor cell growth in vitro [3], displays an antitumor action in an animal model [4], and attenuates IL-2-induced multisystem organ edema [5]. Little is known about the biochemical mechanisms underlying these diverse bioactivities. To explain its antitoxic activity, it was proposed that AA competitively antagonizes a hepatocyte membrane transporter for the phallotoxin phalloidin and for the amatoxin alpha-amanitin [6,7]. This transporter was later identified as a member of the organic aniontransporting polypeptide family [8,9]. Notably, cell uptake of phalloidin was also inhibited by the immunosuppressive drugs rapamycin, FK506 or cyclosporin A (CsA) [8], and AA itself acts as an immunosuppressant [10,11]. These observations strongly suggest that AA could interact with the immunophilins FK506BP or cyclophilin (CyP) A, which are the protein targets of rapamycin/FK506 and CsA, respectively [12,13]. CyP-A is a component of the CyP protein family, whose members display peptidyl-prolyl cis-trans isomerase activity [14] and are characterized by a high degree of sequence conservation and by a differential subcellular distribution [15]. We therefore reasoned that if the AA target was the cytosolic CyP-A, the drug could also act on other members of this protein family. Indeed, such a pleiotropic effect is well-characterized for CsA, as CsA also targets the mitochondria-restricted CyP-D [1618]. CyP-D displays an important role in the cell response to a variety of noxious stimuli, as it modulates a channel located in the inner mitochondrial membrane, the permeability transition pore (PTP) [19,20], whose prolonged opening irreversibly commits cells to death [21]. PTP dysregulation is emerging as a common feature in a variety of pathologies endowed with either an excess of cell death, such as neurodegenerative disease or muscular dystrophies, or with an aberrant hyperactivation of survival pathways, as in cancer [21,22]. CsA inhibits PTP opening through binding to CyP-D [21]. Therefore, it constitutes an interesting molecule for the treatment of degenerative diseases [23,24]. Nonetheless, due to its immunosuppressant activity, to its side effects [25] and to its inability to pass the blood-brain barrier [24], CsA analogues with a higher selectivity for CyP-D are under intense scrutiny [23,2629]. Here we demonstrate that, similar to CsA, AA targets CyP-D leading to PTP inhibition and to cell protection from insults that cause pore opening. AA could be exploited as a lead compound for a new class of PTP-inhibiting drugs. AA inhibits the PTP in isolated mitochondria AA is the cyclodecapeptide c(Val-Pro-Pro-Ala-Phe-Phe-ProPro-Phe-Phe) (Figure 1A). To evaluate its effect on the PTP, we performed Ca2+ retention capacity (CRC) assays on isolated mouse liver mitochondria (MLM). Notably, when mitochondria were incubated in a phosphate-containing medium, AA inhibited pore opening, similar to the PTP inhibitors CsA or Ubiquinone 0 (Ub0; Figure 1B,C). PTP inhibition by AA was not additive with that of CsA, whose molecular target is CyP-D, while AA did increase inhibition by Ub0, which is independent of CyP-D (Figure 1C). We had shown that the effect of CsA, but not of Ub0, is abolished by substituting phosphate with arsenate [30]. Likewise, AA inhibition of the PTP was abrogated in the presence of arsenate (Figure 1D). To dissect AA potency as a PTP inhibitor and the residues involved in its activity, we performed a concentration-response CRC experiment on MLM treated with AA or with a panel of derivatives (Figure 2A). We found that the effect of AA reached a plateau at a concentration of about 20 mM, and that changing amino acids in position 6 or 9 completely abolished pore inhibition (Figure 2B,C). Figure 1. Effect of AA on PTP opening in isolated mouse liver mitochondria. A, chemical structure of AA. B, D, Ca2+ retention capacity (CRC) either in phosphate (Pi) buffer (B) or in arsenate (Asi) buffer (D). Calcium Green-5N fluorescence is reported as arbitrary units on the y a (...truncated)