High‐Throughput, Cell‐Based Screens to Identify Small‐Molecule Inhibitors of Ricin Toxin and Related Category B Ribosome Inactivating Proteins (RIPs)

Paul G. Wahome1, Nicholas J. Mantis2

1 Division of Infectious Diseases, Wadsworth Center, New York State Department of Health, Albany, New York, 2 Department of Biomedical Sciences, University at Albany School of Public Health, Albany, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.23
DOI:  10.1002/0471140856.tx0223s55
Online Posting Date:  February, 2013
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Abstract

Ricin is a member of the ubiquitous ribosome‐inactivating protein (RIP) family of toxins. The Centers for Disease Control and Prevention (CDC) classify ricin and related toxins as Category B biothreat agents. There are currently no antidotes or therapeutics to counteract RIPs in humans. The discovery of effective small‐molecule inhibitors of RIPs is increasingly possible, however, due to the availability and accessibility of diverse small‐molecule chemical libraries coupled with robust robotics and automated screening methodologies. In this article, we describe a cell‐based, high‐throughput screening strategy and secondary assays that we have successfully used to identify compounds that target ricin toxin's enzymatic activity and intracellular trafficking, as well as stress‐activated signaling pathways associated with cell death. The methods described in the protocol are amenable to the other RIPs. Curr. Protoc. Toxicol. 55:2.23.1‐2.23.14. © 2013 by John Wiley & Sons, Inc.

Keywords: drug discovery; small‐molecules; high‐throughput; toxins; biodefense

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: A Cell‐Based, High‐Throughput Screen for the Identification of Ricin Toxin Inhibitors
  • Basic Protocol 2: Confirmation of the Primary Screen Hits Using a Scaled‐Down Cell‐Based Cytotoxicity Assay
  • Alternate Protocol 1: Confirmation of Ricin Inhibitors Using the MTS Assay
  • Basic Protocol 3: In Vitro Translation (IVT) Assay for Identifying Inhibitors of RTA
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: A Cell‐Based, High‐Throughput Screen for the Identification of Ricin Toxin Inhibitors

  Materials
  • Vero cells (African green monkey adult kidney cells; ATCC CCL‐81): a supply of Vero cells is required to initiate the screen; expand the number of Vero cells by culturing them in 150‐cm2 flasks 1 to 2 days before seeding them in microtiter plates—ensure that the growth of cells in separate flasks is synchronized and that the cells are not overgrown
  • Calcium‐ and magnesium‐free Dulbecco's PBS (CMF‐DPBS), pH 7.4 (Invitrogen)
  • Trypsin (0.05%)/EDTA (0.53 mM) in Hank's balanced salt solution (HBSS) without sodium carbonate, calcium, or magnesium (Mediatech)
  • Dulbecco's Modified Eagle Medium (DMEM; Invitrogen) containing 10% fetal bovine serum (FBS) and no antibiotics
  • 70% ethanol
  • Compound libraries (available through core facilities)
  • Dimethylsulfoxide (DMSO; Sigma‐Aldrich)
  • Ricin (Ricinus communis agglutinin II; 5 mg/ml; Vector Laboratories)
  • CellTiter‐Glo luciferase reagent (Promega)
  • Phosphate‐buffered saline (PBS), pH 7.4 (Mediatech)
  • Inverted light microscope
  • Sterile 250‐, 500‐ml, and 1‐liter conical flasks and beakers
  • Hot plate and magnetic stirrer
  • Polypropylene 8‐port manifold for liquid handling system (Cole‐Parmer)
  • Liquid handler/dispenser: e.g., Matrix liquid handler (Thermo Scientific)
  • Microtiter plates (96‐ and 384‐well, white flat‐bottom, sterile and polystyrene tissue‐culture treated; Corning)
  • Pin transfer robot equipped with 384 100‐nl pipetting heads
  • 0.6‐ and 1.5‐ml microcentrifuge (Eppendorf) tubes
  • 250‐ml, 500‐ml and 1‐liter conical flasks and beakers
  • SpectraMax L luminometer (Molecular Devices)
  • Additional reagents and equipment for mammalian cell culture, including trypsinization and cell counting ( appendix 3B)

Basic Protocol 2: Confirmation of the Primary Screen Hits Using a Scaled‐Down Cell‐Based Cytotoxicity Assay

  Materials
  • Vero cells (African green monkey adult kidney cells; ATCC CCL‐81): culture Vero cells in 75‐ or 150‐cm2 flasks for 2 to 3 days to allow the cells to form a confluent monolayer (≥90%)—if working with multiple flasks, ensure that the growth of cells in separate flasks is relatively synchronized and that the cells are not overgrown
  • Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) (Invitrogen)
  • Compounds to be tested
  • Dimethylsulfoxide (DMSO; Sigma‐Aldrich)
  • Ricin (Ricinus communis agglutinin II; 5 mg/ml; Vector Laboratories)
  • CellTiter‐Glo (Promega)
  • Phosphate‐buffered saline (PBS), pH 7.4 (Mediatech)
  • Microtiter plates (96‐well, clear bottom, sterile, and polystyrene tissue‐culture treated; 96‐well, opaque (e.g., white) flat‐bottom, sterile, and polystyrene tissue‐culture treated; 96‐well, ClearPro polypropylene; Corning)
  • Microtiter plate shaker
  • Inverted light microscope
  • SpectraMax L luminometer (Molecular Devices)
  • Additional reagents and equipment for trypsinizing cells ( protocol 1, steps 1 to 7)

Alternate Protocol 1: Confirmation of Ricin Inhibitors Using the MTS Assay

  • CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) kit (Promega)
  • Versamax Microplate Reader (Molecular Devices)

Basic Protocol 3: In Vitro Translation (IVT) Assay for Identifying Inhibitors of RTA

  Materials
  • Compounds to be tested
  • Dimethylsulfoxide (DMSO; Sigma‐Aldrich)
  • 1 mg/ml bovine serum albumin (BSA; Sigma‐Aldrich)
  • Ricin A subunit (RTA; 1 mg/ml; Vector Laboratories)
  • Phosphate buffered saline (PBS), pH 7.4 ( appendix 2A)
  • Retic Lysate IVT kit (Ambion)
  • Methionine (Sigma‐Aldrich)
  • RNasin ribonuclease inhibitor (Promega)
  • Luciferase (luc) control RNA (Promega)
  • RNase‐free H 2O (e.g., Invitrogen, Promega)
  • Bright‐Glo Luciferase Assay System (Promega, Carlsbad CA)
  • 0.6‐ and 1.5‐ml microcentrifuge (Eppendorf) tubes
  • Microtiter plates (96‐well, flat bottom, and white polystyrene)
  • Water bath set at 30°C
  • PerkinElmer Envision Luminometer
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Figures

Videos

Literature Cited

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Internet Resource
   http://www.promega.com/∼/media/files/resources/protocols/technical%20bulletins/0/celltiter%2096%20aqueous%20one%20solution%20cell%20proliferation%20assay%20system%20protocol.pdf?la=en
  Promega CellTiter96 AQueous One Solution Cell Proliferation Assay manual.
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