title: Human Adenine Nucleotide Translocase (ANT) Modulators Identified by High-Throughput Screening of Transgenic Yeast.
abstract: Transport of ADP and ATP across mitochondria is one of the primary points of regulation to maintain cellular energy homeostasis. This process is mainly mediated by adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane. There are four human ANT isoforms, each having a unique tissue-specific expression pattern and biological function, highlighting their potential as drug targets for diverse clinical indications, including male contraception and cancer. In this study, we present a novel yeast-based high-throughput screening (HTS) strategy to identify compounds inhibiting the function of ANT. Yeast strains generated by deletion of endogenous proteins with ANT activity followed by insertion of individual human ANT isoforms are sensitive to cell-permeable ANT inhibitors, which reduce proliferation. Screening hits identified in the yeast proliferation assay were characterized in ADP/ATP exchange assays employing recombinant ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis as well as by oxygen consumption rate in mammalian cells. Using this approach, closantel and CD437 were identified as broad-spectrum ANT inhibitors, whereas leelamine was found to be a modulator of ANT function. This yeast "knock-out/knock-in" screening strategy is applicable to a broad range of essential molecular targets that are required for yeast survival.
authors.affiliation: Otsuka Maryland Medicinal Laboratories, Inc., Rockville, MD, USA.
authors.forename: Yujian
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Institute for Therapeutics Discovery & Development, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Defeng
authors.initials: D
authors.lastname: Tian
authors.affiliation: Otsuka Maryland Medicinal Laboratories, Inc., Rockville, MD, USA.
authors.forename: Hironori
authors.initials: H
authors.lastname: Matsuyama
authors.affiliation: Department of Pediatrics, Osaka City University Graduate School of Medicine, Abeno-ku, Osaka, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Hamazaki
authors.affiliation: Otsuka Maryland Medicinal Laboratories, Inc., Rockville, MD, USA.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Shiratsuchi
authors.affiliation: Department of Pathology, University of Florida College of Medicine, Gainesville, FL, USA.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Terada
authors.affiliation: Institute for Therapeutics Discovery & Development, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Derek J
authors.initials: DJ
authors.lastname: Hook
authors.affiliation: Institute for Therapeutics Discovery & Development, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Walters
authors.affiliation: Institute for Therapeutics Discovery & Development, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Gunda I
authors.initials: GI
authors.lastname: Georg
authors.affiliation: Institute for Therapeutics Discovery & Development, University of Minnesota, Minneapolis, MN, USA hawkinso@umn.edu.
authors.forename: Jon E
authors.initials: JE
authors.lastname: Hawkinson
mesh_terms: D045784:Abietanes / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D001692:Biological Transport; D049109:Cell Proliferation / Q000187:drug effects; D057166:High-Throughput Screening Assays; D006801:Humans; D013294:Lactococcus lactis / Q000187:drug effects / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D000226:Mitochondrial ADP, ATP Translocases / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D030781:Organisms, Genetically Modified; D012176:Retinoids / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D012458:Salicylanilides / Q000494:pharmacology; D054852:Small Molecule Libraries / Q000494:pharmacology*; D019076:Transgenes
chemical_list: D045784:Abietanes; C099555:CD 437; D012176:Retinoids; D012458:Salicylanilides; D054852:Small Molecule Libraries; C000600819:dehydroabietylamine; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases; C023342:closantel
keywords: ADP/ATP exchange; Lactococcus lactis; adenine nucleotide translocase; high throughput screening; yeast mitochondria