title: Novel class of potential therapeutics that target ricin retrograde translocation.
abstract: Ricin toxin, an A-B toxin from Ricinus communis, induces cell death through the inhibition of protein synthesis. The toxin binds to the cell surface via its B chain (RTB) followed by its retrograde trafficking through intracellular compartments to the ER where the A chain (RTA) is transported across the membrane and into the cytosol. Ricin A chain is transported across the ER membrane utilizing cellular proteins involved in the disposal of aberrant ER proteins by a process referred to as retrograde translocation. Given the current lack of therapeutics against ricin intoxication, we developed a high-content screen using an enzymatically attenuated RTA chimera engineered with a carboxy-terminal enhanced green fluorescent protein (RTA(E177Q)egfp) to identify compounds that target RTA retrograde translocation. Stabilizing RTA(E177Q)egfp through the inclusion of proteasome inhibitor produced fluorescent peri-nuclear granules. Quantitative analysis of the fluorescent granules provided the basis to discover compounds from a small chemical library (2080 compounds) with known bioactive properties. Strikingly, the screen found compounds that stabilized RTA molecules within the cell and several compounds limited the ability of wild type RTA to suppress protein synthesis. Collectively, a robust high-content screen was developed to discover novel compounds that stabilize intracellular ricin and limit ricin intoxication.
authors.forename: Veronika
authors.initials: V
authors.lastname: Redmann
authors.forename: Thomas
authors.initials: T
authors.lastname: Gardner
authors.forename: Zerlina
authors.initials: Z
authors.lastname: Lau
authors.forename: Keita
authors.initials: K
authors.lastname: Morohashi
authors.forename: Dan
authors.initials: D
authors.lastname: Felsenfeld
authors.affiliation: Icahn School of Medicine at Mount Sinai, Department of Microbiology, One Gustave L. Levy Place, New York, NY 10029, USA. domenico.tortorella@mssm.edu.
authors.forename: Domenico
authors.initials: D
authors.lastname: Tortorella
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D012276:Ricin / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D012278:Ricinus / Q000737:chemistry; D054852:Small Molecule Libraries / Q000737:chemistry*; D014118:Toxins, Biological / Q000037:antagonists & inhibitors / Q000737:chemistry
chemical_list: D054852:Small Molecule Libraries; D014118:Toxins, Biological; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012276:Ricin; D046988:Proteasome Endopeptidase Complex