AlphaScreen-Based Assays: Ultra-High-Throughput Screening for Small-Molecule Inhibitors of Challenging Enzymes and Protein-Protein Interactions

  • Adam Yasgar
  • Ajit Jadhav
  • Anton Simeonov
  • Nathan P. CoussensEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1439)


AlphaScreen technology has been routinely utilized in high-throughput screening assays to quantify analyte accumulation or depletion, bimolecular interactions, and post-translational modifications. The high signal-to-background, dynamic range, and sensitivity associated with AlphaScreens as well as the homogenous assay format and reagent stability make the technology particularly well suited for high-throughput screening applications. Here, we describe the development of AlphaScreen assays to identify small-molecule inhibitors of enzymes and protein-protein interactions using the highly miniaturized 1536-well format. The subsequent implementation of counter assays to identify false-positive compounds is also discussed.

Key words

AlphaLISA AlphaScreen High-throughput screening Assay ELISA HTS Enzyme Protein-protein interaction 



This work was supported by the NCATS Division of Pre-Clinical Innovation Intramural Program.


  1. 1.
    Ullman EF, Kirakossian H, Singh S, Wu ZP, Irvin BR, Pease JS, Switchenko AC, Irvine JD, Dafforn A, Skold CN et al (1994) Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence. Proc Natl Acad Sci U S A 91(12):5426–5430CrossRefGoogle Scholar
  2. 2.
    Peppard J, Glickman F, He Y, Hu SI, Doughty J, Goldberg R (2003) Development of a high-throughput screening assay for inhibitors of aggrecan cleavage using luminescent oxygen channeling (AlphaScreen). J Biomol Screen 8(2):149–156. doi: 10.1177/1087057103252308 CrossRefGoogle Scholar
  3. 3.
    Eglen RM, Reisine T, Roby P, Rouleau N, Illy C, Bosse R, Bielefeld M (2008) The use of AlphaScreen technology in HTS: current status. Curr Chem Genomics 1:2–10. doi: 10.2174/1875397300801010002 CrossRefGoogle Scholar
  4. 4.
    Wan Y, Xhang X, Atherton JJ, Kostner K, Dimeski G, Punyadeera C (2015) A multimarker approach to diagnose and stratify heart failure. Int J Cardiol 181:369–375. doi: 10.1016/j.ijcard.2014.12.052 CrossRefGoogle Scholar
  5. 5.
    Zhang X, Dimeski G, Punyadeera C (2014) Validation of an immunoassay to measure plasminogen-activator inhibitor-1 concentrations in human saliva. Biochem Medica 24(2):258–265, 10.11613/BM.2014.028CrossRefGoogle Scholar
  6. 6.
    Zhang X, Wan Y, Cooper-White J, Dimeski G, Atherton J, Punyadeera C (2013) Quantification of D-dimer levels in human saliva. Bioanalysis 5(18):2249–2256. doi: 10.4155/bio.13.190 CrossRefGoogle Scholar
  7. 7.
    He A, Liu TC, Dong ZN, Ren ZQ, Hou JY, Li M, Wu YS (2013) A novel immunoassay for the quantization of CYFRA 21-1 in human serum. J Clin Lab Anal 27(4):277–283. doi: 10.1002/jcla.21597 CrossRefGoogle Scholar
  8. 8.
    Dynon K, Heng S, Puryer M, Li Y, Walton K, Endo Y, Nie G (2012) HtrA3 as an early marker for preeclampsia: specific monoclonal antibodies and sensitive high-throughput assays for serum screening. PLoS One 7(9):e45956. doi: 10.1371/journal.pone.0045956 CrossRefGoogle Scholar
  9. 9.
    Marchand C, Lea WA, Jadhav A, Dexheimer TS, Austin CP, Inglese J, Pommier Y, Simeonov A (2009) Identification of phosphotyrosine mimetic inhibitors of human tyrosyl-DNA phosphodiesterase I by a novel AlphaScreen high-throughput assay. Mol Cancer Ther 8(1):240–248. doi: 10.1158/1535-7163.MCT-08-0878 CrossRefGoogle Scholar
  10. 10.
    Dorjsuren D, Kim D, Maloney DJ, Wilson DM 3rd, Simeonov A (2011) Complementary non-radioactive assays for investigation of human flap endonuclease 1 activity. Nucleic Acids Res 39(2), e11. doi: 10.1093/nar/gkq1082 CrossRefGoogle Scholar
  11. 11.
    Leister KP, Huang R, Goodwin BL, Chen A, Austin CP, Xia M (2011) Two high throughput screen assays for measurement of TNF-alpha in THP-1 cells. Curr Chem Genomics 5:21–29. doi: 10.2174/1875397301105010021 CrossRefGoogle Scholar
  12. 12.
    Dehdashti SJ, Zheng W, Gever JR, Wilhelm R, Nguyen DT, Sittampalam G, McKew JC, Austin CP, Prusiner SB (2013) A high-throughput screening assay for determining cellular levels of total tau protein. Curr Alzheimer Res 10(7):679–687CrossRefGoogle Scholar
  13. 13.
    Liu F, Chen X, Allali-Hassani A, Quinn AM, Wigle TJ, Wasney GA, Dong A, Senisterra G, Chau I, Siarheyeva A, Norris JL, Kireev DB, Jadhav A, Herold JM, Janzen WP, Arrowsmith CH, Frye SV, Brown PJ, Simeonov A, Vedadi M, Jin J (2010) Protein lysine methyltransferase G9a inhibitors: design, synthesis, and structure activity relationships of 2,4-diamino-7-aminoalkoxy-quinazolines. J Med Chem 53(15):5844–5857. doi: 10.1021/jm100478y CrossRefGoogle Scholar
  14. 14.
    Quinn AM, Allali-Hassani A, Vedadi M, Simeonov A (2010) A chemiluminescence-based method for identification of histone lysine methyltransferase inhibitors. Mol Biosyst 6(5):782–788. doi: 10.1039/b921912a CrossRefGoogle Scholar
  15. 15.
    Quinn AM, Bedford MT, Espejo A, Spannhoff A, Austin CP, Oppermann U, Simeonov A (2010) A homogeneous method for investigation of methylation-dependent protein-protein interactions in epigenetics. Nucleic Acids Res 38(2):e11. doi: 10.1093/nar/gkp899 CrossRefGoogle Scholar
  16. 16.
    Yi F, Zhu P, Southall N, Inglese J, Austin CP, Zheng W, Regan L (2009) An AlphaScreen-based high-throughput screen to identify inhibitors of Hsp90-cochaperone interaction. J Biomol Screen 14(3):273–281. doi: 10.1177/1087057108330114 CrossRefGoogle Scholar
  17. 17.
    Yi F, Regan L (2008) A novel class of small molecule inhibitors of Hsp90. ACS Chem Biol 3(10):645–654. doi: 10.1021/cb800162x CrossRefGoogle Scholar
  18. 18.
    Srinivasan P, Yasgar A, Luci DK, Beatty WL, Hu X, Andersen J, Narum DL, Moch JK, Sun H, Haynes JD, Maloney DJ, Jadhav A, Simeonov A, Miller LH (2013) Disrupting malaria parasite AMA1-RON2 interaction with a small molecule prevents erythrocyte invasion. Nat Commun 4:2261. doi: 10.1038/ncomms3261 CrossRefGoogle Scholar
  19. 19.
    Cunningham L, Finckbeiner S, Hyde RK, Southall N, Marugan J, Yedavalli VR, Dehdashti SJ, Reinhold WC, Alemu L, Zhao L, Yeh JR, Sood R, Pommier Y, Austin CP, Jeang KT, Zheng W, Liu P (2012) Identification of benzodiazepine Ro5-3335 as an inhibitor of CBF leukemia through quantitative high throughput screen against RUNX1-CBFbeta interaction. Proc Natl Acad Sci U S A 109(36):14592–14597. doi: 10.1073/pnas.1200037109 CrossRefGoogle Scholar
  20. 20.
    Chen CZ, Sobczak K, Hoskins J, Southall N, Marugan JJ, Zheng W, Thornton CA, Austin CP (2012) Two high-throughput screening assays for aberrant RNA-protein interactions in myotonic dystrophy type 1. Anal Bioanal Chem 402(5):1889–1898. doi: 10.1007/s00216-011-5604-0 CrossRefGoogle Scholar
  21. 21.
    Baell JB, Holloway GA (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(7):2719–2740. doi: 10.1021/jm901137j CrossRefGoogle Scholar
  22. 22.
    Brooks HB, Geeganage S, Kahl SD, Montrose C, Sittampalam S, Smith MC, Weidner JR (2004) Basics of enzymatic assays for HTS. In: Sittampalam GS, Gal-Edd N, Arkin M et al (eds) Assay Guidance Manual. Eli Lilly & Company and the National Center for Advancing Translational Sciences, Bethesda, MDGoogle Scholar
  23. 23.
    Newton P, Harrison P, Clulow S (2008) A novel method for determination of the affinity of protein: protein interactions in homogeneous assays. J Biomol Screen 13(7):674–682. doi: 10.1177/1087057108321086 CrossRefGoogle Scholar
  24. 24.
    Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4(2):67–73CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Adam Yasgar
    • 1
  • Ajit Jadhav
    • 1
  • Anton Simeonov
    • 1
  • Nathan P. Coussens
    • 1
    Email author
  1. 1.National Center for Advancing Translational SciencesNational Institutes of HealthRockvilleUSA

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