Advertisement

Small-Molecule Inhibitors of Cyclophilins Block Opening of the Mitochondrial Permeability Transition Pore and Protect Mice From Hepatic Ischemia/Reperfusion Injury

      Background & Aims

      Hepatic ischemia/reperfusion injury is a complication of liver surgery that involves mitochondrial dysfunction resulting from mitochondrial permeability transition pore (mPTP) opening. Cyclophilin D (PPIF or CypD) is a peptidyl-prolyl cis-trans isomerase that regulates mPTP opening in the inner mitochondrial membrane. We investigated whether and how recently created small-molecule inhibitors of CypD prevent opening of the mPTP in hepatocytes and the resulting effects in cell models and livers of mice undergoing ischemia/reperfusion injury.

      Methods

      We measured the activity of 9 small-molecule inhibitors of cyclophilins in an assay of CypD activity. The effects of the small-molecule CypD inhibitors or vehicle on mPTP opening were assessed by measuring mitochondrial swelling and calcium retention in isolated liver mitochondria from C57BL/6J (wild-type) and Ppif–/– (CypD knockout) mice and in primary mouse and human hepatocytes by fluorescence microscopy. We induced ischemia/reperfusion injury in livers of mice given a small-molecule CypD inhibitor or vehicle before and during reperfusion and collected samples of blood and liver for histologic analysis.

      Results

      The compounds inhibited peptidyl-prolyl isomerase activity (half maximal inhibitory concentration values, 0.2–16.2 μmol/L) and, as a result, calcium-induced mitochondrial swelling, by preventing mPTP opening (half maximal inhibitory concentration values, 1.4–132 μmol/L) in a concentration-dependent manner. The most potent inhibitor (C31) bound CypD with high affinity and inhibited swelling in mitochondria from livers of wild-type and Ppif–/– mice (indicating an additional, CypD-independent effect on mPTP opening) and in primary human and mouse hepatocytes. Administration of C31 in mice with ischemia/reperfusion injury before and during reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage compared with vehicle.

      Conclusions

      Recently created small-molecule inhibitors of CypD reduced calcium-induced swelling in mitochondria from mouse and human liver tissues. Administration of these compounds to mice during ischemia/reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage. These compounds might be developed to protect patients from ischemia/reperfusion injury after liver surgery or for other hepatic or nonhepatic disorders related to abnormal mPTP opening.

      Graphical abstract

      Keywords

      Abbreviations used in this paper:

      ADP (adenosine diphosphate), ALT (alanine aminotransferase), AST (aspartate aminotransferase), ATP (adenosine triphosphate), CsA (cyclosporine A), Cyp (cyclophilin), IC50 (half maximal inhibitory concentration), mPTP (mitochondrial permeability transition pore), PPI (peptidyl prolyl cis-trans isomer), SMCypI (small-molecule cyclophilin inhibitor)
      To read this article in full you will need to make a payment
      AGA Member Login
      Login with your AGA username and password.
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Konishi T.
        • Lentsch A.B.
        Hepatic ischemia/reperfusion: mechanisms of tissue injury, repair, and regeneration.
        Gene Expr. 2017; 17: 277-287
        • Cannistra M.
        • Ruggiero M.
        • Zullo A.
        • et al.
        Hepatic ischemia reperfusion injury: a systematic review of literature and the role of current drugs and biomarkers.
        Int J Surg. 2016; 33: S57-S70
        • Go K.L.
        • Lee S.
        • Zendejas I.
        • et al.
        Mitochondrial dysfunction and autophagy in hepatic ischemia/reperfusion injury.
        Biomed Res Int. 2015; 2015: 183469
        • Halestrap A.P.
        What is the mitochondrial permeability transition pore?.
        J Mol Cell Cardiol. 2009; 46: 821-831
        • Friberg H.
        • Wieloch T.
        Mitochondrial permeability transition in acute neurodegeneration.
        Biochimie. 2002; 84: 241-250
        • Kim J.S.
        • He L.
        • Qian T.
        • et al.
        Role of the mitochondrial permeability transition in apoptotic and necrotic death after ischemia/reperfusion injury to hepatocytes.
        Curr Mol Med. 2003; 3: 527-535
        • Rauen U.
        • de Groot H.
        New insights into the cellular and molecular mechanisms of cold storage injury.
        J Investig Med. 2004; 52: 299-309
        • Halestrap A.P.
        A pore way to die: the role of mitochondria in reperfusion injury and cardioprotection.
        Biochem Soc Trans. 2010; 38: 841-860
        • Rao V.K.
        • Carlson E.A.
        • Yan S.S.
        Mitochondrial permeability transition pore is a potential drug target for neurodegeneration.
        Biochim Biophys Acta. 2014; 1842: 1267-1272
        • Jaeschke H.
        • McGill M.R.
        • Ramachandran A.
        Oxidant stress, mitochondria, and cell death mechanisms in drug-induced liver injury: lessons learned from acetaminophen hepatotoxicity.
        Drug Metab Rev. 2012; 44: 88-106
        • Giorgio V.
        • von Stockum S.
        • Antoniel M.
        • et al.
        Dimers of mitochondrial ATP synthase form the permeability transition pore.
        Proc Natl Acad Sci U S A. 2013; 110: 5887-5892
        • Wang P.
        • Heitman J.
        The cyclophilins.
        Genome Biol. 2005; 6: 226
        • Davis T.L.
        • Walker J.R.
        • Campagna-Slater V.
        • et al.
        Structural and biochemical characterization of the human cyclophilin family of peptidyl-prolyl isomerases.
        PLoS Biol. 2010; 8e1000439
        • Javadov S.
        • Kuznetsov A.
        Mitochondrial permeability transition and cell death: the role of cyclophilin D.
        Front Physiol. 2013; 4: 76
        • Griffiths E.J.
        • Halestrap A.P.
        Protection by cyclosporin A of ischemia/reperfusion-induced damage in isolated rat hearts.
        J Mol Cell Cardiol. 1993; 25: 1461-1469
        • Matsuda S.
        • Koyasu S.
        Mechanisms of action of cyclosporine.
        Immunopharmacology. 2000; 47: 119-125
        • Azzolin L.
        • Antolini N.
        • Calderan A.
        • et al.
        Antamanide, a derivative of Amanita phalloides, is a novel inhibitor of the mitochondrial permeability transition pore.
        PLoS One. 2011; 6e16280
        • Piot C.
        • Croisille P.
        • Staat P.
        • et al.
        Effect of cyclosporine on reperfusion injury in acute myocardial infarction.
        N Engl J Med. 2008; 359: 473-481
        • Ahmed-Belkacem A.
        • Colliandre L.
        • Ahnou N.
        • et al.
        Fragment-based discovery of a new family of non-peptidic small-molecule cyclophilin inhibitors with potent antiviral activities.
        Nat Commun. 2016; 7: 12777
        • Pons J.L.
        • Labesse G.
        @TOME-2: a new pipeline for comparative modeling of protein-ligand complexes.
        Nucleic Acids Res. 2009; 37: W485-W491
        • Miteva M.A.
        • Guyon F.
        • Tuffery P.
        Frog2: efficient 3D conformation ensemble generator for small compounds.
        Nucleic Acids Res. 2010; 38: W622-W627
        • Fontaine E.
        • Eriksson O.
        • Ichas F.
        • et al.
        Regulation of the permeability transition pore in skeletal muscle mitochondria. Modulation by electron flow through the respiratory chain complex I.
        J Biol Chem. 1998; 273: 12662-12668
        • Seglen P.O.
        Preparation of isolated rat liver cells.
        Methods Cell Biol. 1976; 13: 29-83
        • Petronilli V.
        • Miotto G.
        • Canton M.
        • et al.
        Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence.
        Biophys J. 1999; 76: 725-734
        • Petronilli V.
        • Penzo D.
        • Scorrano L.
        • et al.
        The mitochondrial permeability transition, release of cytochrome C and cell death. Correlation with the duration of pore openings in situ.
        J Biol Chem. 2001; 276: 12030-12034
        • Bankhead P.
        • Loughrey M.B.
        • Fernandez J.A.
        • et al.
        QuPath: open source software for digital pathology image analysis.
        Sci Rep. 2017; 7: 16878
        • Baines C.P.
        • Kaiser R.A.
        • Purcell N.H.
        • et al.
        Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death.
        Nature. 2005; 434: 658-662
        • Walter L.
        • Miyoshi H.
        • Leverve X.
        • et al.
        Regulation of the mitochondrial permeability transition pore by ubiquinone analogs. A progress report.
        Free Radic Res. 2002; 36: 405-412
        • Li B.
        • Chauvin C.
        • De Paulis D.
        • et al.
        Inhibition of complex I regulates the mitochondrial permeability transition through a phosphate-sensitive inhibitory site masked by cyclophilin D.
        Biochim Biophys Acta. 2012; 1817: 1628-1634
        • Nakagawa T.
        • Shimizu S.
        • Watanabe T.
        • et al.
        Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death.
        Nature. 2005; 434: 652-658
        • Hansson M.J.
        • Mattiasson G.
        • Mansson R.
        • et al.
        The nonimmunosuppressive cyclosporin analogs NIM811 and UNIL025 display nanomolar potencies on permeability transition in brain-derived mitochondria.
        J Bioenerg Biomembr. 2004; 36: 407-413
        • Waldmeier P.C.
        • Feldtrauer J.J.
        • Qian T.
        • et al.
        Inhibition of the mitochondrial permeability transition by the nonimmunosuppressive cyclosporin derivative NIM811.
        Mol Pharmacol. 2002; 62: 22-29
        • Nevers Q.
        • Ruiz I.
        • Ahnou N.
        • et al.
        Characterization of the anti-hepatitis C virus activity of new nonpeptidic small-molecule cyclophilin inhibitors with the potential for broad anti-Flaviviridae activity.
        Antimicrob Agents Chemother. 2018; 62 (e00126–18)
        • Shanmughapriya S.
        • Rajan S.
        • Hoffman N.E.
        • et al.
        SPG7 is an essential and conserved component of the mitochondrial permeability transition pore.
        Mol Cell. 2015; 60: 47-62
        • Glantzounis G.K.
        • Salacinski H.J.
        • Yang W.
        • et al.
        The contemporary role of antioxidant therapy in attenuating liver ischemia-reperfusion injury: a review.
        Liver Transpl. 2005; 11: 1031-1047
        • Lefkowitch J.H.
        Scheuer’s liver biopsy interpretation.
        Elsevier, Edinburgh, UK2016
        • Neil D.A.
        • Hubscher S.G.
        Delay in diagnosis: a factor in the poor outcome of late acute rejection of liver allografts.
        Transplant Proc. 2001; 33: 1525-1526
        • Hubscher S.G.
        Histological findings in liver allograft rejection: new insights into the pathogenesis of hepatocellular damage in liver allografts.
        Histopathology. 1991; 18: 377-383
        • Khettry U.
        • Backer A.
        • Ayata G.
        • et al.
        Centrilobular histopathologic changes in liver transplant biopsies.
        Hum Pathol. 2002; 33: 270-276
        • Datta G.
        • Fuller B.J.
        • Davidson B.R.
        Molecular mechanisms of liver ischemia reperfusion injury: insights from transgenic knockout models.
        World J Gastroenterol. 2013; 19: 1683-1698
        • Saeed W.K.
        • Jun D.W.
        • Jang K.
        • et al.
        Does necroptosis have a crucial role in hepatic ischemia-reperfusion injury?.
        PLoS One. 2017; 12e0184752
        • Arab H.A.
        • Sasani F.
        • Rafiee M.H.
        • et al.
        Histological and biochemical alterations in early-stage lobar ischemia-reperfusion in rat liver.
        World J Gastroenterol. 2009; 15: 1951-1957
        • King A.L.
        • Swain T.M.
        • Dickinson D.A.
        • et al.
        Chronic ethanol consumption enhances sensitivity to Ca2+-mediated opening of the mitochondrial permeability transition pore and increases cyclophilin D in liver.
        Am J Physiol Gastrointest Liver Physiol. 2010; 299: G954-G966