On‐Bead Two‐Color (OBTC) Cell Screen for Direct Identification of Highly Selective Cell Surface Receptor Ligands

D. Gomika Udugamasooriya1, Thomas Kodadek2

1 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, 2 Department of Cancer Biology, The Scripps Research Institute, Jupiter, Florida
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch110199
Online Posting Date:  March, 2012
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Abstract

Combinatorial library screens can identify a suitable ligand for a biological target of interest out of thousands or even millions of compounds, and can play a key role in the drug development process. While conventional high‐throughput cell screens based on functional assays require expensive robotics, simple on‐bead combinatorial assays for ligand binding to the target protein can be done far more cheaply. This unit describes one such assay, developed using combinatorial peptoid libraries for targeting integral membrane receptors or other cell surface‐exposed molecules. In addition to the reduced cost, a unique advantage of this assay is the direct identification of the most selective ligands for a cell surface receptor that is expressed in its natural environment. Curr. Protoc. Chem. Biol. 4:35‐48 © 2012 by John Wiley & Sons, Inc.

Keywords: combinatorial; cell screen; receptor; peptoid

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Direct Identification of Highly Selective Peptoids for Receptors of Interest (e.g., VEGFR2) Using the On‐Bead Two‐Color (OBTC) Assay
  • Support Protocol 1: Cell Labeling Procedure Using Qtraker Quantum Dots
  • Support Protocol 2: Preparation of Hit Beads for Edman Sequencing
  • Support Protocol 3: Preparation of Hit Beads for Mass Spectroscopic Sequencing
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Direct Identification of Highly Selective Peptoids for Receptors of Interest (e.g., VEGFR2) Using the On‐Bead Two‐Color (OBTC) Assay

  Materials
  • 100,000 (about 200 mg) TentaGel one‐bead one‐compound library beads (140‐ to 170‐µm; Rapp Polymere: TentaGel MB NH2, cat. no. MB 160 002)
  • Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, cat., no. 11965)
  • Dimethyl formamide (DMF; Sigma, cat. no. 227056)
  • Bovine serum albumin (BSA; Sigma, cat. no. A3803)
  • Fetal bovine serum (FBS; Invitrogen, cat. no. 16000‐044)
  • GIBCO cell dissociation buffer (Invitrogen, cat. no. 13151‐014) for adherent cells
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010‐023)
  • Two cell groups: e.g., PAE/KDR (VEGFR2 overexpressing) and parent (PAE) cell group (Sib Tech)
  • 5‐ml polypropylene tubes (Falcon, cat. no. 35 2063)
  • 35 × 10–mm tissue culture dishes (Falcon, cat. no. 35 3001)
  • 37°C, 5% CO 2 incubator
  • 15‐ml tubes (e.g., Falcon)
  • Centrifuge
  • Hemacytometer
  • 70‐µm cell strainer (BD Falcon, cat. no. 352350)
  • Double‐deck oscillating shaker
  • Light microscope
  • Fluorescence microscope equipped with a DAPI filter that has UV excitation and longpass emission (e.g., 11000V3 filter cube, Chroma Technology)
  • 20‐ and 100‐µl pipets
  • 1.5‐ml microcentrifuge tubes
  • Glass slides
  • Additional reagents and equipment for staining the cell groups with red and green fluorescence quantum dots ( protocol 2)

Support Protocol 1: Cell Labeling Procedure Using Qtraker Quantum Dots

  Materials
  • Qtraker 655 cell labeling kit (Invitrogen, cat. no.Q25021MP) containing:
    • Component A
    • Component B
  • Qtraker 565 cell labeling kit (Invitrogen, cat. no.Q25031MP) containing:
    • Component A
    • Component B
  • Dulbecco's modified eagle medium (DMEM; Invitrogen, cat. no. 11965)
  • Fetal bovine serum (FBS; Invitrogen, cat. no. 16000‐044)
  • Two cell groups: e.g., PAE/KDR (overexpressing VEGFR2) and parent (PAE) cell group (Sib Tech.)
  • 1.5‐ml microcentrifuge tubes
  • Vortex mixer
  • 37°C, 5% CO 2 cell incubator
  • Fluorescence microscope equipped with a DAPI filter that has UV excitation and longpass emission (e.g., 11000V3 filter cube, Chroma Technology)

Support Protocol 2: Preparation of Hit Beads for Edman Sequencing

  Materials
  • Hit beads with bound red cells (output from protocol 1)
  • 1% SDS solution [dissolve 1 g SDS (Sigma‐Aldrich, cat. no. L3771) in 100 ml distilled water]
  • Milli‐Q purified water
  • Dissecting microscope
  • Edman sequencing cartridge (Applied Biosystems)
  • 20‐µl pipets

Support Protocol 3: Preparation of Hit Beads for Mass Spectroscopic Sequencing

  Materials
  • Hit beads with bound red cells (output from protocol 1)
  • 1% SDS solution [dissolve 1 g SDS (Sigma‐Aldrich, cat. no. L3771) in 100 ml distilled water]
  • Milli‐Q purified water
  • GIBCO cell dissociation buffer (Invitrogen, cat. no. 13151‐014)
  • 1:1 (v/v) Acetonitrile:water mixture
  • CNBr cleavage cocktail: Cyanogen bromide (CNBr) 30 mg/ml in 5:4:1 acetonitrile:acetic acid:water mixture
  • Dissection microscope
  • SpeedVac system
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Figures

Videos

Literature Cited

Literature Cited
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   Alluri, P.G., Reddy, M.M., Bachhawat‐Sikder, K., Olivos, H.J., and Kodadek, T. 2003. Isolation of protein ligands from large peptoid libraries. J. Am. Chem. Soc. 125:13995‐14004.
   Astle, J.M., Udugamasooriya, D.G., Smallshaw, J.E., and Kodadek, T. 2008. A VEGFR2 antagonist and other peptoids evade immune recognition. Int. J. Pept. Res. Ther. 14:223‐227.
   Black, C.B., Duensing, T.D., Trinkle, L.S., and Dunlay, R.T. 2011. Cell‐based screening using high‐throughput flow cytometry. Assay Drug Dev. Technol. 9:13‐20.
   Cooper, M.A. 2004. Advances in membrane receptor screening and analysis. J. Mol. Recognit. 17:286‐315.
   De Leon‐Rodriguez, L.M., Lubag, A., Udugamasooriya, D.G., Proneth, B., Brekken, R.A., Sun, X., Kodadek, T., and Dean Sherry, A. 2010. MRI detection of VEGFR2 in vivo using a low molecular weight peptoid‐(Gd)8‐dendron for targeting. J. Am. Chem. Soc. 132:12829‐12831.
   Gocke, A.R., Udugamasooriya, D.G., Archer, C.T., Lee, J., and Kodadek, T. 2009. Isolation of antagonists of antigen‐specific autoimmune T cell proliferation. Chem. Biol. 16:1133‐1139.
   Lim, H.S., Archer, C.T., and Kodadek, T. 2007. Identification of a peptoid inhibitor of the proteasome 19S regulatory particle. J. Am. Chem. Soc. 129:7750‐7751.
   Lynn, K.D., Udugamasooriya, D.G., Roland, C.L., Castrillon, D.H., Kodadek, T.J., and Brekken, R.A. 2010. GU81, a VEGFR2 antagonist peptoid, enhances the anti‐tumor activity of doxorubicin in the murine MMTV‐PyMT transgenic model of breast cancer. BMC Cancer 10:397.
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   Olivos, H.J., Bachhawat‐Sikder, K., and Kodadek, T. 2003. Quantum dots as a visual aid for screening bead‐bound combinatorial libraries. Chembiochem 4:1242‐1245.
   Osmond, R.I., Crouch, M.F., and Dupriez, V.J. 2010. An emerging role for kinase screening in GPCR drug discovery. Curr. Opin. Mol. Ther. 12:305‐315.
   Paulick, M.G., Hart, K.M., Brinner, K.M., Tjandra, M., Charych, D.H., and Zuckermann, R.N. 2006. Cleavable hydrophilic linker for one‐bead‐one‐compound sequencing of oligomer libraries by tandem mass spectrometry. J. Comb. Chem. 8:417‐426.
   Peng, L., Liu, R., Marik, J., Wang, X., Takada, Y., and Lam, K.S. 2006. Combinatorial chemistry identifies high‐affinity peptidomimetics against alpha4beta1 integrin for in vivo tumor imaging. Nature Chem. Biol. 2:381‐389.
   Roland, C.L., Lynn, K.D., Toombs, J.E., Dineen, S.P., Udugamasooriya, D.G., and Brekken, R.A. 2009. Cytokine levels correlate with immune cell infiltration after anti‐VEGF therapy in preclinical mouse models of breast cancer. PLoS One 4:e7669.
   Simpson, L.S., Burdine, L., Dutta, A.K., Feranchak, A.P., and Kodadek, T. 2009. Selective toxin sequestrants for the treatment of bacterial infections. J. Am. Chem. Soc. 131:5760‐5762.
   Udugamasooriya, D.G., Dineen, S.P., Brekken, R.A., and Kodadek, T. 2008a. A peptoid “antibody surrogate” that antagonizes VEGF receptor 2 activity. J. Am. Chem. Soc. 130:5744‐5752.
   Udugamasooriya, D.G., Dunham, G., Ritchie, C., Brekken, R.A., and Kodadek, T. 2008b. The pharmacophore of a peptoid VEGF receptor 2 antagonist includes both side chain and main chain residues. Bioorg. Med. Chem. Lett. 18:5892‐5894.
   Udugamasooriya, D.G., Ritchie, C., Brekken, R.A., and Kodadek, T. 2008c. A peptoid antagonist of VEGF receptor 2 recognizes a ‘hotspot’ in the extracellular domain distinct from the hormone‐binding site. Bioorg. Med. Chem. 16:6338‐6343.
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