title: Structure-activity analysis of niclosamide reveals potential role for cytoplasmic pH in control of mammalian target of rapamycin complex 1 (mTORC1) signaling.
abstract: Mammalian target of rapamycin complex 1 (mTORC1) signaling is frequently dysregulated in cancer. Inhibition of mTORC1 is thus regarded as a promising strategy in the treatment of tumors with elevated mTORC1 activity. We have recently identified niclosamide (a Food and Drug Administration-approved antihelminthic drug) as an inhibitor of mTORC1 signaling. In the present study, we explored possible mechanisms by which niclosamide may inhibit mTORC1 signaling. We tested whether niclosamide interferes with signaling cascades upstream of mTORC1, the catalytic activity of mTOR, or mTORC1 assembly. We found that niclosamide does not impair PI3K/Akt signaling, nor does it inhibit mTORC1 kinase activity. We also found that niclosamide does not interfere with mTORC1 assembly. Previous studies in helminths suggest that niclosamide disrupts pH homeostasis of the parasite. This prompted us to investigate whether niclosamide affects the pH balance of cancer cells. Experiments in both breast cancer cells and cell-free systems demonstrated that niclosamide possesses protonophoric activity in cells and in vitro. In cells, niclosamide dissipated protons (down their concentration gradient) from lysosomes to the cytosol, effectively lowering cytoplasmic pH. Notably, analysis of five niclosamide analogs revealed that the structural features of niclosamide required for protonophoric activity are also essential for mTORC1 inhibition. Furthermore, lowering cytoplasmic pH by means other than niclosamide treatment (e.g. incubation with propionic acid or bicarbonate withdrawal) recapitulated the inhibitory effects of niclosamide on mTORC1 signaling, lending support to a possible role for cytoplasmic pH in the control of mTORC1. Our data illustrate a potential mechanism for chemical inhibition of mTORC1 signaling involving modulation of cytoplasmic pH.
authors.affiliation: Department of Biochemistry and Molecular Biology, 2350 Health Sciences Mall, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada; Department of Biochemistry, 1160 Pine Avenue West, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, Quebec H3A 1A3, Canada. Electronic address: bd.fonseca@mcgill.ca.
authors.forename: Bruno D
authors.initials: BD
authors.lastname: Fonseca
authors.affiliation: Department of Biochemistry and Molecular Biology, 2350 Health Sciences Mall, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Graham H
authors.initials: GH
authors.lastname: Diering
authors.affiliation: Department of Biochemistry, 1160 Pine Avenue West, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, Quebec H3A 1A3, Canada.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Bidinosti
authors.affiliation: Department of Biochemistry and Molecular Biology, 2350 Health Sciences Mall, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Kush
authors.initials: K
authors.lastname: Dalal
authors.affiliation: Department of Biochemistry, 1160 Pine Avenue West, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, Quebec H3A 1A3, Canada.
authors.forename: Tommy
authors.initials: T
authors.lastname: Alain
authors.affiliation: Department of Biochemistry and Molecular Biology, 2350 Health Sciences Mall, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Aruna D
authors.initials: AD
authors.lastname: Balgi
authors.affiliation: Departments of Chemistry and Earth and Ocean Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
authors.forename: Roberto
authors.initials: R
authors.lastname: Forestieri
authors.affiliation: Departments of Chemistry and Earth and Ocean Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
authors.forename: Matt
authors.initials: M
authors.lastname: Nodwell
authors.affiliation: Department of Biochemistry, 1160 Pine Avenue West, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, Quebec H3A 1A3, Canada.
authors.forename: Charles V
authors.initials: CV
authors.lastname: Rajadurai
authors.affiliation: Department of Biochemistry and Molecular Biology, 2350 Health Sciences Mall, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Gunaratnam
authors.affiliation: Institute of Medical Genetics, Wales College of Medicine, Cardiff University, Heath Park Way, Cardiff CF14 4XN, United Kingdom.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Tee
authors.affiliation: Department of Biochemistry and Molecular Biology, 2350 Health Sciences Mall, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Franck
authors.initials: F
authors.lastname: Duong
authors.affiliation: Departments of Chemistry and Earth and Ocean Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Andersen
authors.affiliation: Department of Physiology, 3655 Promenade Sir William Osler, Bellini Building, McGill University, Montreal, Quebec H3G 1Y6, Canada.
authors.forename: John
authors.initials: J
authors.lastname: Orlowski
authors.affiliation: Department of Biochemistry and Molecular Biology, 2350 Health Sciences Mall, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Numata
authors.affiliation: Department of Biochemistry, 1160 Pine Avenue West, Rosalind and Morris Goodman Cancer Centre, McGill University, Montreal, Quebec H3A 1A3, Canada.
authors.forename: Nahum
authors.initials: N
authors.lastname: Sonenberg
authors.affiliation: Department of Biochemistry and Molecular Biology, 2350 Health Sciences Mall, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. Electronic address: michelr@mail.ubc.ca.
authors.forename: Michel
authors.initials: M
authors.lastname: Roberge
mesh_terms: D000818:Animals; D000969:Antinematodal Agents / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D000076222:Mechanistic Target of Rapamycin Complex 1; D051379:Mice; D018345:Mice, Knockout; D046912:Multiprotein Complexes; D009534:Niclosamide / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects* / Q000235:genetics; D058570:TOR Serine-Threonine Kinases
chemical_list: D000969:Antinematodal Agents; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes; D009534:Niclosamide; D019869:Phosphatidylinositol 3-Kinases; D011506:Proteins; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases