title: Identification of ER-000444793, a Cyclophilin D-independent inhibitor of mitochondrial permeability transition, using a high-throughput screen in cryopreserved mitochondria.
abstract: Growing evidence suggests persistent mitochondrial permeability transition pore (mPTP) opening is a key pathophysiological event in cell death underlying a variety of diseases. While it has long been clear the mPTP is a druggable target, current agents are limited by off-target effects and low therapeutic efficacy. Therefore identification and development of novel inhibitors is necessary. To rapidly screen large compound libraries for novel mPTP modulators, a method was exploited to cryopreserve large batches of functionally active mitochondria from cells and tissues. The cryopreserved mitochondria maintained respiratory coupling and ATP synthesis, Ca2+uptake and transmembrane potential. A high-throughput screen (HTS), using an assay of Ca2+-induced mitochondrial swelling in the cryopreserved mitochondria identified ER-000444793, a potent inhibitor of mPTP opening. Further evaluation using assays of Ca2+-induced membrane depolarisation and Ca2+retention capacity also indicated that ER-000444793 acted as an inhibitor of the mPTP. ER-000444793 neither affected cyclophilin D (CypD) enzymatic activity, nor displaced of CsA from CypD protein, suggesting a mechanism independent of CypD inhibition. Here we identified a novel, CypD-independent inhibitor of the mPTP. The screening approach and compound described provides a workflow and additional tool to aid the search for novel mPTP modulators and to help understand its molecular nature.
authors.affiliation: UCL Collaboration Research Group, NGM-PCU, Eisai Ltd., Hatfield, UK.
authors.forename: Thomas
authors.initials: T
authors.lastname: Briston
authors.affiliation: UCL Collaboration Research Group, NGM-PCU, Eisai Ltd., Hatfield, UK.
authors.forename: Sian
authors.initials: S
authors.lastname: Lewis
authors.affiliation: UCL Collaboration Research Group, NGM-PCU, Eisai Ltd., Hatfield, UK.
authors.forename: Mumta
authors.initials: M
authors.lastname: Koglin
authors.affiliation: UCL Collaboration Research Group, NGM-PCU, Eisai Ltd., Hatfield, UK.
authors.forename: Kavita
authors.initials: K
authors.lastname: Mistry
authors.affiliation: Next Generation Systems CFU, Eisai Inc., Andover, MA, USA.
authors.forename: Yongchun
authors.initials: Y
authors.lastname: Shen
authors.affiliation: UCL Collaboration Research Group, NGM-PCU, Eisai Ltd., Hatfield, UK.
authors.forename: Naomi
authors.initials: N
authors.lastname: Hartopp
authors.affiliation: Next Generation Systems CFU, Eisai Co., Ltd, Tsukuba, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Katsumata
authors.affiliation: NGM-PCU, Eisai Co. Ltd., Tsukuba Research Laboratories, Tsukuba, Japan.
authors.forename: Hironori
authors.initials: H
authors.lastname: Fukumoto
authors.affiliation: Department of Cell and Developmental Biology, University College London, London, UK.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Duchen
authors.affiliation: Department of Cell and Developmental Biology, University College London, London, UK.|Department of Biomedical Sciences, University of Padua, Padua, Italy.
authors.forename: Gyorgy
authors.initials: G
authors.lastname: Szabadkai
authors.affiliation: UCL Collaboration Research Group, NGM-PCU, Eisai Ltd., Hatfield, UK.
authors.forename: James M
authors.initials: JM
authors.lastname: Staddon
authors.affiliation: UCL Collaboration Research Group, NGM-PCU, Eisai Ltd., Hatfield, UK.
authors.forename: Malcolm
authors.initials: M
authors.lastname: Roberts
authors.affiliation: UCL Collaboration Research Group, NGM-PCU, Eisai Ltd., Hatfield, UK.
authors.forename: Ben
authors.initials: B
authors.lastname: Powney
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D015925:Cryopreservation; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins / Q000502:physiology*; D004734:Energy Metabolism; D005260:Female; D006367:HeLa Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000037:antagonists & inhibitors*; D000083162:Mitochondrial Permeability Transition Pore; D011804:Quinolines / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000081406:Peptidyl-Prolyl Isomerase F; C000627684:ER-000444793; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D011804:Quinolines; D000255:Adenosine Triphosphate; D021983:Cyclophilins