title: A Combination of Screening and Computational Approaches for the Identification of Novel Compounds That Decrease Mast Cell Degranulation.
abstract: High-content screening of compound libraries poses various challenges in the early steps in drug discovery such as gaining insights into the mode of action of the selected compounds. Here, we addressed these challenges by integrating two biological screens through bioinformatics and computational analysis. We screened a small-molecule library enriched in amphiphilic compounds in a degranulation assay in rat basophilic leukemia 2H3 (RBL-2H3) cells. The same library was rescreened in a high-content image-based endocytosis assay in HeLa cells. This assay was previously applied to a genome-wide RNAi screen that produced quantitative multiparametric phenotypic profiles for genes that directly or indirectly affect endocytosis. By correlating the endocytic profiles of the compounds with the genome-wide siRNA profiles, we identified candidate pathways that may be inhibited by the compounds. Among these, we focused on the Akt pathway and validated its inhibition in HeLa and RBL-2H3 cells. We further showed that the compounds inhibited the translocation of the Akt-PH domain to the plasma membrane. The approach performed here can be used to integrate chemical and functional genomics screens for investigating the mechanism of action of compounds.
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors.forename: Marisa P
authors.initials: MP
authors.lastname: McShane
authors.affiliation: JADO Technologies GmbH, Dresden, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Friedrichson
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors.forename: Angelika
authors.initials: A
authors.lastname: Giner
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany University of Fribourg, Department of Medicine-Anatomy, Fribourg, Switzerland.
authors.forename: Felix
authors.initials: F
authors.lastname: Meyenhofer
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors.forename: Rico
authors.initials: R
authors.lastname: Barsacchi
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Bickle
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany zerial@mpi-cbg.de.
authors.forename: Marino
authors.initials: M
authors.lastname: Zerial
mesh_terms: D000818:Animals; D015550:Cell Degranulation / Q000187:drug effects*; D002460:Cell Line; D055808:Drug Discovery / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004705:Endocytosis / Q000187:drug effects; D015870:Gene Expression; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays; D006801:Humans; D008407:Mast Cells / Q000187:drug effects* / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics; D054852:Small Molecule Libraries
chemical_list: D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries
keywords: Akt; mast cell; phosphorylation; screening
references.citation: EMBO J. 1996 Dec 2;15(23):6541-51
references.pmid: 8978681
references.citation: Nucleic Acids Res. 2013 Jul;41(Web Server issue):W77-83
references.pmid: 23703215
references.citation: Curr Biol. 1999 Apr 22;9(8):433-6
references.pmid: 10226029
references.citation: Nat Protoc. 2014 Feb;9(2):474-90
references.pmid: 24481274
references.citation: J Dermatolog Treat. 2013 Aug;24(4):244-9
references.pmid: 22385017
references.citation: Nat Med. 2012 May;18(5):693-704
references.pmid: 22561833
references.citation: J Biomol Screen. 2014 Jun;19(5):640-50
references.pmid: 24710339
references.citation: Nat Rev Drug Discov. 2013 Aug;12(8):569
references.pmid: 23903212
references.citation: Nat Rev Drug Discov. 2011 Jul;10(7):507-19
references.pmid: 21701501
references.citation: Science. 2005 Feb 18;307(5712):1098-101
references.pmid: 15718470
references.citation: PLoS One. 2009;4(5):e5604
references.pmid: 19440386
references.citation: Eur J Med Chem. 2010 Jun;45(6):2700-4
references.pmid: 20153565
references.citation: Nat Chem Biol. 2008 Sep;4(9):538-47
references.pmid: 18641634
references.citation: Anticancer Drugs. 2003 Feb;14(2):167-73
references.pmid: 12569304
references.citation: Cell Host Microbe. 2013 Feb 13;13(2):129-42
references.pmid: 23414754
references.citation: Adv Immunol. 2008;98:85-120
references.pmid: 18772004
references.citation: J Biomol Screen. 2013 Dec;18(10):1186-92
references.pmid: 23983234
references.citation: Nucleic Acids Res. 2013 Jan;41(Database issue):D1021-6
references.pmid: 23193271
references.citation: Int Immunol. 2013 Apr;25(4):215-20
references.pmid: 23143475
references.citation: Curr Biol. 1998 Mar 12;8(6):343-6
references.pmid: 9512420
references.citation: Science. 2006 Sep 29;313(5795):1929-35
references.pmid: 17008526
references.citation: Bioorg Med Chem Lett. 2003 Sep 15;13(18):3111-4
references.pmid: 12941345
references.citation: Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W741-8
references.pmid: 15980575
references.citation: J Biol Chem. 2004 Sep 24;279(39):41189-96
references.pmid: 15262962
references.citation: J Invest Dermatol. 2009 Feb;129(2):496-8
references.pmid: 18769454
references.citation: Nature. 2010 Mar 11;464(7286):243-9
references.pmid: 20190736