Label-Free Cell Phenotypic Identification of d-Luciferin as an Agonist for GPR35

  • Heidi Hu
  • Huayun Deng
  • Ye FangEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1461)


d-Luciferin (also known as beetle or firefly luciferin) is one of the most widely used bioluminescent reporters for monitoring in vitro or in vivo luciferase activity. The identification of several natural phenols and thieno[3,2-b]thiophene-2-carboxylic acid derivatives as agonists for GPR35, an orphan G protein-coupled receptor, had motivated us to examine the pharmacological activity of d-Luciferin, given that it also contains phenol and carboxylic acid moieties. Here, we describe label-free cell phenotypic assays that ascertain d-Luciferin as a partial agonist for GPR35. The agonistic activity of d-Luciferin at the GPR35 shall evoke careful interpretation of biological data when d-Luciferin or its analogues are used as probes.

Key words

Bioluminescence Dynamic mass redistribution G protein-coupled receptor GPR35 d-Luciferin Resonant waveguide grating biosensor 


  1. 1.
    Contag CH, Bachmann MH (2002) Advances in in vivo bioluminescence imaging of gene expression. Annu Rev Biomed Eng 4:235–260CrossRefPubMedGoogle Scholar
  2. 2.
    Negrin RS, Contag CH (2006) In vivo imaging using bioluminescence: a tool for probing graft-versus-host disease. Nat Rev Immunol 6:484–490CrossRefPubMedGoogle Scholar
  3. 3.
    Luker GD, Luker KE (2008) Optical imaging: current applications and future directions. J Nucl Med 49:1–4CrossRefPubMedGoogle Scholar
  4. 4.
    Keyaerts M, Caveliers V, Lahoutte T (2012) Bioluminescence imaging: looking beyond the light. Trends Mol Med 18:164–172CrossRefPubMedGoogle Scholar
  5. 5.
    Greer LF III, Szalay AA (2002) Imaging of light emission from the expression of luciferases in living cells and organisms. Luminescence 17:43–74CrossRefPubMedGoogle Scholar
  6. 6.
    Shinde R, Perkins J, Contag CH (2006) Luciferin derivatives for enhanced in vitro and in vivo bioluminescence assays. Biochemistry 45:11103–11112CrossRefPubMedGoogle Scholar
  7. 7.
    White EH, Rapaport E, Hopkins TA, Seliger HH (1969) Chemi- and bioluminescence of firefly luciferin. J Am Chem Soc 91:2178–2180CrossRefPubMedGoogle Scholar
  8. 8.
    Ando Y, Niwa K, Yamada N, Enomoto T, Irie T, Kubota H, Ohmiya Y, Akiyama H (2008) Firefly bioluminescence quantum yield and colour change by pH-sensitive green emission. Nat Photonics 2:44–47CrossRefGoogle Scholar
  9. 9.
    Nakatsu T, Ichiyama S, Hiratake J, Saldanha A, Kobashi N, Sakata K, Kato H (2006) Structural basis for the spectral difference in luciferase bioluminescence. Nature 440:372–376CrossRefPubMedGoogle Scholar
  10. 10.
    Jathoul AP, Grounds H, Anderson JC, Pule MA (2014) A dual-color far-red to near-infrared firefly luciferin analogue designed for multiparametric bioluminescence imaging. Angew Chem Int Ed Engl 53:13059–13063CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Fang Y, Ferrie AM, Fontaine NH, Yuen PK (2005) Characteristics of dynamic mass redistribution of EGF receptor signaling in living cells measured with label free optical biosensors. Anal Chem 77:5720–5725CrossRefPubMedGoogle Scholar
  12. 12.
    Fang Y, Ferrie AM, Fontaine NH, Mauro J, Balakrishnan J (2006) Resonant waveguide grating biosensor for living cell sensing. Biophys J 91:1925–1940CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Schröder R, Janssen N, Schmid J, Kebig A, Merten N, Hennen S, Müller A, Blättermann S, Mohr-Andrä M, Zahn S, Wenzel J, Smith NJ, Gomeza J, Drewke C, Milligan G, Mohr K, Kostenis E (2010) Deconvolution of complex G protein-coupled receptor signaling in live cells using dynamic mass redistribution measurements. Nat Biotechnol 28:943–949CrossRefPubMedGoogle Scholar
  14. 14.
    Verrier F, An S, Ferrie AM, Sun H, Kyoung M, Fang Y, Benkovic SJ (2011) GPCRs regulate the assembly of a multienzyme complex for purine biosynthesis. Nat Chem Biol 7:909–915CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Fang Y (2014) Label-free drug discovery. Front Pharmacol 5:52CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Fang Y (2015) Combining label-free cell phenotypic profiling with computational approaches for novel drug discovery. Expert Opin Drug Discov 10:331–343CrossRefPubMedGoogle Scholar
  17. 17.
    Deng H, Hu H, He M, Hu J, Niu W, Ferrie AM, Fang Y (2011) Discovery of 2-(4-methylfuran-2(5H)-ylidene)malononitrile and thieno[3,2-b]thiophene-2-carboxylic acid derivatives as G protein-coupled receptor 35 (GPR35) agonists. J Med Chem 54:7385–7396CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Deng H, Hu H, Ling S, Ferrie AM, Fang Y (2012) Discovery of natural phenols as G protein-coupled receptor-35 (GPR35) agonists. ACS Med Chem Lett 3:165–169CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    MacKenzie AE, Lappin JE, Taylor DL, Nicklin SA, Milligan G (2011) GPR35 as a novel therapeutic target. Front Endocrinol 2:68CrossRefGoogle Scholar
  20. 20.
    Deng H, Hu H, Fang Y (2012) Multiple tyrosine metabolites are GPR35 agonists. Sci Rep 2:373CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Divorty N, Mackenzie AE, Nicklin SA, Milligan G (2015) G protein-coupled receptor 35: an emerging target in inflammatory and cardiovascular disease. Front Pharmacol 6:41CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Shore DM, Reggio PH (2015) The therapeutic potential of orphan GPCRs, GPR35 and GPR55. Front Pharmacol 6:69CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Hu H, Deng H, Fang Y (2012) Label-free phenotypic profiling identified D-luciferin as a GPR35 agonist. PLoS One 7:e34934CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Thimm D, Funke M, Meyer A, Müller CE (2013) 6-Bromo-8-(4-[3H]methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid: a powerful tool for studying orphan G protein-coupled receptor GPR35. J Med Chem 56:7084–7099CrossRefPubMedGoogle Scholar
  25. 25.
    Ferrie AM, Wu Q, Fang Y (2010) Resonant waveguide grating imager for live cell sensing. Appl Phys Lett 97:223704CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Fang Y (2013) Troubleshooting and deconvoluting label-free cell phenotypic assays in drug discovery. J Pharmacol Toxicol Methods 67:69–81CrossRefPubMedGoogle Scholar
  27. 27.
    Ferrie AM, Sun H, Zaytseva N, Fang Y (2014) Divergent label-free cell phenotypic pharmacology of ligands at the overexpressed β2-adrenergic receptors. Sci Rep 4:3828CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Zhang X, Deng H, Xiao Y, Xue X, Ferrie AM, Tran E, Liang X, Fang Y (2014) Label-free cell phenotypic profiling identifies pharmacologically active compounds in two Traditional Chinese Medicinal plants. RSC Adv 4:26368–26377Google Scholar
  29. 29.
    Fang Y (2014) Label-free cell phenotypic drug discovery. Comb Chem High Throughput Screen 17:566–578CrossRefPubMedGoogle Scholar
  30. 30.
    Zhao P, Sharir H, Kapur A, Cowan A, Geller EB, Adler MW, Seltzman HH, Reggio PH, Heynen-Genel S, Sauer M, Chung TD, Bai Y, Chen W, Caron MG, Barak LS, Abood ME (2010) Targeting of the orphan receptor GPR35 by pamoic acid: a potent activator of ERK and β-arrestin2, with antinociceptive activity. Mol Pharmacol 78:560–568CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Taniguchi Y, Tonai-Kachi H, Shinjo K (2006) Zaprinast, a well-known cyclic guanosine monophosphate-specific phosphodiesterase inhibitor, is an agonist for GPR35. FEBS Lett 580:5003–5008CrossRefPubMedGoogle Scholar
  32. 32.
    Fang Y, Ferrie AM (2008) Label-free optical biosensor for ligand-directed functional selectivity acting on β2-adrenoceptor in living cells. FEBS Lett 582:558–564CrossRefPubMedGoogle Scholar
  33. 33.
    Fang Y, Ferrie AM, Tran E (2009) Resonant waveguide grating biosensor for whole cell GPCR assays. Methods Mol Biol 552:239–252CrossRefPubMedGoogle Scholar
  34. 34.
    Deng H, Wang C, Su M, Fang Y (2012) Probing biochemical mechanisms of action of muscarinic M3 receptor antagonists with label-free whole-cell assays. Anal Chem 84:8232–8239CrossRefPubMedGoogle Scholar
  35. 35.
    Deng H, Sun H, Fang Y (2013) Label-free cell phenotypic assessment of the biased agonism and efficacy of agonists at the endogenous muscarinic M3 receptors. J Pharmacol Toxicol Methods 68:323–333CrossRefPubMedGoogle Scholar
  36. 36.
    Fang Y (ed) (2015) Label-free biosensor methods in drug discovery, Methods in pharmacology and toxicity. Springer, New YorkGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Medical Laboratory Science, Jefferson College of Health Science/Carilion Roanoke Memorial HospitalQuest Diagnostic Lab, Carilion Clinic Health SystemRoanokeUSA
  2. 2.Biochemical Technologies, Science and Technology DivisionCorning IncorporatedCorningUSA

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