A Bead‐Based Proximity Assay for BRD4 Ligand Discovery

Justin M. Roberts1, James E. Bradner2

1 Department of Medical Oncology, Dana‐Farber Cancer Institute, Boston, Massachusetts, 2 Department of Medicine, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch150024
Online Posting Date:  December, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Bromodomain‐containing proteins have emerged as desirable targets for anti‐neoplastic and anti‐inflammatory drug discovery. Toward the development of selective inhibitors of the BET family of bromodomains, we optimized bead‐based assays to detect interactions between bromodomains and poly‐acetylated histone peptides. Donor and acceptor beads bound to target and ligand are brought into proximity by this protein‐protein interaction. After laser illumination, singlet oxygen evolved from donor beads travels to the spatially close acceptor beads, resulting in chemiluminesence. This AlphaScreen assay has proven amendable to high‐throughput screening, secondary validation, and specificity profiling during lead discovery and optimization. Here we report our protocol for assay development to measure inhibition of ligand binding to bromodomain‐containing protein 4 (BRD4). We discuss the discovery of an appropriate probe, optimization of bead, probe, and protein concentrations, and the derivation of protein‐probe inhibition curves. Finally, we explore the implementation of this technology for high‐throughput screening of potential BRD4 inhibitors. © 2015 by John Wiley & Sons, Inc.

Keywords: bromodomain; BRD4; JQ1; AlphaScreen; high‐throughput screening; protein‐protein interaction

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Ligand, Target, and Bead Concentration Optimization for Enhanced AlphaScreen Signal
  • Basic Protocol 2: Measurement of Competitive Binding to BRD4 Using AlphaScreen
  • Basic Protocol 3: Adaptation of the Competitive BRD4‐Binding Assay for HTS
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Ligand, Target, and Bead Concentration Optimization for Enhanced AlphaScreen Signal

  Materials
  • Protein stock solution: 10 μM BRD4(1)‐His 6 in AlphaScreen buffer solution
  • AlphaScreen buffer (see recipe)
  • Tagged probe stock solution: 100 μM Bio‐JQ1 in dimethyl sulfoxide (DMSO) (Filippakopoulos et al., ; Anders et al., )
  • Alpha bead stock solution: 10 μg/ml each of donor and acceptor beads from PerkinElmer's AlphaScreen Histidine (Nickel Chelate) Detection Kit (cat. no. 6760619R) in AlphaScreen buffer
  • 1.5‐ml microcentrifuge tubes
  • AlphaScreen 384‐well microplates (PerkinElmer, cat. no. 6005350)
  • Multichannel pipets
  • Centrifuge
  • Plate seals (e.g., Corning, 6570)
  • AlphaScreen capable plate reader (e.g., Envision, Enspire)
  • Graphing software (e.g., PRISM GraphPad)
  • Plastic reservoir (e.g., Corning, 4870)

Basic Protocol 2: Measurement of Competitive Binding to BRD4 Using AlphaScreen

  Materials
  • Dimethyl sulfoxide (DMSO)
  • Compound stock solutions: 10 mM in DMSO
  • Reagent Stock Solution: 40 nM BRD4(1)‐His 6, 40 nM Bio‐JQ1, and 10 μg/ml nickel coated acceptor beads from PerkinElmer's AlphaScreen Histidine (Nickel Chelate) Detection Kit (cat. no. 6760619R) in AlphaScreen buffer (see recipe)
  • Donor Bead Stock Solution: 10 μg/ml of streptavidin‐coated donor beads from PerkinElmer's AlphaScreen Histidine (Nickel Chelate) Detection Kit (cat. no. 6760619R) in AlphaScreen buffer (see recipe)
  • 384‐well compound storage plates (e.g., Greiner Bio One, cat. no. 784201)
  • Centrifuge
  • AlphaScreen 384‐well microplates (PerkinElmer, cat. no. 6005350)
  • Multichannel pipets
  • Plate seals (e.g., Corning, cat. no. 6570)
  • AlphaScreen capable reader (e.g., Envision, Enspire)
  • Graphing software (e.g., Excel, PRISM GraphPad)
  • Plastic reservoir (e.g., Corning, 4870)
  • Pin transfer apparatus (e.g., JANUS automated liquid handling workstation, PerkinElmer)
  • Compound pin transfer tool (e.g., V&P Scientific)

Basic Protocol 3: Adaptation of the Competitive BRD4‐Binding Assay for HTS

  Materials
  • Dimethyl sulfoxide (DMSO)
  • Reagent Stock Solution: 40 nM BRD4(1)‐His 6, 40 nM Bio‐JQ1, 10 μg/ml nickel‐coated acceptor beads from PerkinElmer's AlphaScreen Histidine (Nickel Chelate) Detection Kit (cat. no. 6760619R) in AlphaScreen buffer (see recipe)
  • Commercial Compound Library
  • Donor Bead Stock Solution: 10 μg/ml of streptavidin‐coated donor beads from PerkinElmer's AlphaScreen Histidine (Nickel Chelate) Detection Kit (cat. no. 6760619R) in AlphaScreen buffer (see recipe)
  • Bio‐His 6 Stock Solution: 1 nM Bio‐His 6 and 10 μg/ml of nickel coated acceptor beads from PerkinElmer's AlphaScreen Histidine (Nickel Chelate) Detection Kit (cat. no. 6760619R) in AlphaScreen buffer (see recipe)
  • 384‐well compound storage plates (e.g., Greiner Bio One, cat. no. 784201)
  • Automated liquid dispenser for multiwell microplates (e.g., EL406, BioTek)
  • AlphaScreen 384‐well microplates (PerkinElmer, cat. no. 6005350)
  • Pin transfer apparatus (e.g., JANUS automated liquid handling workstation, PerkinElmer)
  • Compound pin transfer tool (e.g., V&P Scientific)
  • Plate seals (e.g., Corning cat. no. 6570)
  • Multichannel pipet
  • AlphaScreen‐capable reader (e.g., Envision, Enspire)
  • Graphing software (e.g., Excel, PRISM GraphPad)
  • Plastic reservoir (e.g., Corning, cat. no. 4870)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Anders, L., Guenther, M.G., Qi, J., and Fan, Z.P. 2013. Genome‐wide localization of small molecules. Nat. Biotechnol. 32:92‐96. doi: 10.1038/nbt.2776.
  Baell, J.B. and Holloway, G.A. 2010. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J. Med. Chem. 53:2719‐2740. doi: 10.1021/jm901137j.
  Chapuy, B., McKeown, M.R., Lin, C.Y., and Monti, S. 2013. Discovery and characterization of super‐enhancer‐associated dependencies in diffuse large B cell lymphoma. Cancer Cell. 24:777‐790. doi: 10.1016/j.ccr.2013.11.003.
  Ciceri, P., Müller, S., O'Mahony, A., Fedorov, O., Filippakopoulos, P., Hunt, J.P., Lasater, E.A., Pallares, G., Picaud, S., Wells, C., Martin, S., Wodicka, L.M., Shah, N.P., Treiber, D.K., Knapp, S. 2014. Dual kinase‐bromodomain inhibitors for rationally designed polypharmacology. Nat. Chem. Biol. 10:305‐312. doi: 10.1038/nCHeMBIO.1471
  Delmore, J.E., Issa, G.C., Lemieux, M.E., Rahl, P.B., Shi, J., Jacobs, H.M., Kastritis, E., Gilpatrick, T., Paranal, R.M., Qi, J., Chesi, M., Schinzel, A.C., McKeown, M.R., Heffernan, T.P., Vakoc, C.R., Bergsagel, P.L., Ghobrial, I.M., Richardson, P.G., Young, R.A., Hahn, W.C., Anderson, K.C., Kung, A.L., Bradner, J.E., and Mitsiades, C.S. 2011. BET bromodomain inhibition as a therapeutic strategy to target c‐Myc. Cell 146:904‐917. doi: 10.1016/j.cell.2011.08.017.
  Dey, A., Chitsaz, F., and Abbasi, A. 2003. The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis. Proc. Natl. Acad. Sci. 100:8758‐8763. doi: 10.1073/pnas.1433065100.
  Eglen, R.M., Reisine, T., Roby, P., Rouleau, N., Illy, C., Bossé, R., and Bielefeld, M. 2008. The use of AlphaScreen technology in HTS: Current status. Curr. Chem. Genomics 1:2‐10. doi: 10.2174/1875397300801010002.
  Filippakopoulos, P. and Knapp, S. 2014. Targeting bromodomains: Epigenetic readers of lysine acetylation. Nat. Rev. Drug Discov. 13:337‐356. doi: 10.1038/nrd4286.
  Filippakopoulos, P., Qi, J., Picaud, S., Shen, Y., Smith, W.B., Fedorov, O., Morse, E.M., Keates, T., Hickman, T.T., Felletar, I., Philpott, M., Munro, S., McKeown, M.R., Wang, Y., Christie, A.L., West, N., Cameron, M.J., Schwartz, B., Heightman, T.D., La Thangue, N., French, C.A., Wiest, O., Kung, A.L., Knapp, S., and Bradner, J.E. 2010. Selective inhibition of BET bromodomains. Nature 468:1067‐1073. doi: 10.1038/nature09504.
  French, C.A. 2012. Pathogenesis of NUT midline carcinoma. Ann. Revi. Pathol.: Mech. Dis. 7:247‐265. doi: 10.1146/annurev‐pathol‐011811‐132438.
  Huang, B., Yang, X.‐D.D., Zhou, M.‐M.M., Ozato, K., and Chen, L.‐F.F. 2009. Brd4 coactivates transcriptional activation of NF‐kappaB via specific binding to acetylated RelA. Mol. Cell. Biol. 29:1375‐1387. doi: 10.1128/MCB.01365‐08.
  Johnston, P.A. 2011. Redox cycling compounds generate H2O2 in HTS buffers containing strong reducing reagents—real hits or promiscuous artifacts? Curr. Opin. Chem. Biol. 15:174‐182. doi: 10.1016/j.cbpa.2010.10.022.
  Jung, M., Philpott, M., Müller, S., and Schulze, J. 2014. Affinity Map of Bromodomain Protein 4 (BRD4) Interactions with the Histone H4 Tail and the Small Molecule Inhibitor JQ1. J. Biol. Chem. 289. doi: 10.1074/jbc.M113.523019.
  Matzuk, M.M., McKeown, M.R., Filippakopoulos, P., Li, Q., Ma, L., Agno, J.E., Lemieux, M.E., Picaud, S., Yu, R.N., Qi, J., Knapp, S., and Bradner, J.E. 2012. Small‐molecule inhibition of BRDT for male contraception. Cell 150:673‐684. doi: 10.1016/j.cell.2012.06.045.
  McGovern, S.L., Caselli, E., and Grigorieff, N. 2002. A common mechanism underlying promiscuous inhibitors from virtual and high‐throughput screening. J. Med. Chem. 45:1712‐1722. doi: 10.1021/jm010533y.
  McKeown, M.R., Shaw, D.L., Fu, H., Liu, S., Xu, X., Marineau, J.J., Huang, Y., Zhang, X., Buckley, D.L., Kadam, A., Zhang, Z., Blacklow, S.C., Qi, J., Zhang, W., and Bradner, J.E. 2014. Biased multicomponent reactions to develop novel bromodomain inhibitors. J. Med. Chem. 57:9019‐9027. doi: 10.1021/jm501120z.
  Philpott, M., Yang, J., Tumber, T., Fedorov, O., Uttarkar, S., Filippakopoulos, P., Picaud, S., Keates, T., Felletar, I., Ciulli, A., Knapp, S., and Heightman, T.D. 2011. Bromodomain‐peptide displacement assays for interactome mapping and inhibitor discovery. Mol. Biosyst. 7:2899‐2908. doi: 10.1039/C1MB05099K.
  Quinn, A.M., Bedford, M.T., Espejo, A., Spannhoff, A., Austin, C.P., Oppermann, U., and Simeonov, A. 2010. A homogeneous method for investigation of methylation‐dependent protein–protein interactions in epigenetics. Nucleic Acids Res. 38:e11. doi: 10.1093/nar/gkp899.
  Rishton, G.M. 1997. Reactive compounds and in vitro false positives in HTS. Drug Discovery Today. 2:382‐384. doi: 10.1016/S1359‐6446(97)01083‐0.
  Ryan, A.J., Gray, N.M., and Lowe, P.N. 2003. Effect of detergent on “promiscuous” inhibitors. J. Med. Chem. 46:3448‐3451. doi: 10.1021/jm0340896.
  Ullman, E.F., Kirakossian, H., and Singh, S. 1994. Luminescent oxygen channeling immunoassay: Measurement of particle binding kinetics by chemiluminescence. Proc. Natl. Acad. Sci. 19:5426‐5430. doi: 10.1073/pnas.91.12.5426.
  Ullman, E.F., Kirakossian, H., Switchenko, A.C., Ishkanian, J., Ericson, M., Wartchow, C.A., Pirio, M., Pease, J., Irvin, B.R., Singh, S., Singh, R., Patel, R., Dafforn, A., Davalian, D., Skold, C., Kurn, N., and Wagner, D.B. 1996. Luminescent oxygen channeling assay (LOCI): Sensitive, broadly applicable homogeneous immunoassay method. Clin. Chem. 42:1518‐1526.
  Yang, Z., Yik, J.H., Chen, R., He, N., Jang, M.K., Ozato, K., and Zhou, Q. 2005. Recruitment of P‐TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4. Mol. cell 19:535‐545. doi: 10.1016/j.molcel.2005.06.029.
  Yi, J.S., Fderation, A.J., Qi, J., Dhe‐Paganon, S., Hadler, M., Xu, X., Pierre, R., Varca, A.C., Wu, L., and Marineau, J.J. 2015. Structure‐Guided DOT1L probe optimization by label‐free ligand displacement. ACS Chem. Biol. 10:667‐674. doi: 10.1021/cb500796d.
  Zhang, J.H. and Chung, T.D.Y. 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4:67‐73. doi: 10.1177/108705719900400206.
  Zeng, L., and Zhou, M.M. 2002. Bromodomain: An acetyl‐lysine binding domain. FEBS Lett. 513:124‐128.
  Zuber, J., Shi, J., Wang, E., Rappaport, A.R., Herrmann, H., Sison, E., A., Magoon, D., Qi, J., Blatt, K., Wunderlich, M., Taylor, M.J., Johns, C., Chicas, A., Mulloy, J.C., Kogan, S.C., Brown, P., Valent, P., Bradner, J.E., Lowe, S.W., and Vakoc, C.R. 2011. RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature 478:524‐528. doi: 10.1038/nature10334.
Internet Resources
  http://www.perkinelmer.com/
  PerkinElmer homepage. All of the necessary reagents, as well as background and FAQs on Alpha technology and steps to creating a successful assay can be found here.
  http://www.urmc.rochester.edu/hts/_source/AlphaScreenPracticalGuide.pdf
  “A practical guide to working with AlphaScreen” posted by the University of Rochester's High Throughput Screening Core. This document gives a detailed background on working with the assay, including an exhaustive troubleshooting guide.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library