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Phenotype-Based High-Content Screening Using Fluorescent Chemical Bioprobes: Lipid Droplets and Glucose Uptake Quantification in Live Cells

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Phenotypic Screening

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1787))

Abstract

Phenotypic screening in live cells has emerged as a promising strategy for drug discovery in pharmaceutical communities. For relevant phenotype-based screening setups, it is critical to develop adequate reporters in order to selectively visualize subcellular compartments or phenotypic changes that represent disease-related characteristics during compound screening. In this chapter, we introduce two phenotype-based high-content/high-throughput assays using fluorescent bioprobes that have been designed and refined to selectively stain cellular lipid droplets (LDs) and to show cellular glucose uptake. In conjunction with target identification process for the hit compounds from phenotypic screening, these fluorescent chemical probe-based screening techniques are expected to drive a great advancement for the discovery of novel first-in-class therapeutics.

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References

  1. Fishman MC, Porter JA (2005) Pharmaceuticals: a new grammar for drug discovery. Nature 437:491–493

    Article  CAS  PubMed Central  Google Scholar 

  2. Sams-Dodd F (2005) Target-based drug discovery: is something wrong? Drug Discov Today 10:139–147

    Article  CAS  PubMed Central  Google Scholar 

  3. Swinney DC (2013) Phenotypic vs. target-based drug discovery for first-in-class medicines. Clin Pharmacol Ther 93:299–301

    Article  CAS  PubMed Central  Google Scholar 

  4. Zheng W, Thorne N, McKew JC (2013) Phenotypic screens as a renewed approach for drug discovery. Drug Discov Today 18:1067–1073

    Article  CAS  PubMed Central  Google Scholar 

  5. Jo A, Jung J, Kim E, Park SB (2016) A high-content screening platform with fluorescent chemical probes for the discovery of first-in-class therapeutics. Chem Commun (Camb) 52:7433–7445

    Article  CAS  Google Scholar 

  6. Park H, Ha J, Koo JY, Park J, Park SB (2017) Label-free target identification using in-gel fluorescence difference via thermal stability shift. Chem Sci 8:1127–1133

    Article  CAS  PubMed Central  Google Scholar 

  7. Hart CP (2005) Finding the target after screening the phenotype. Drug Discov Today 10:513–519

    Article  CAS  PubMed Central  Google Scholar 

  8. Park J, Oh S, Park SB (2012) Discovery and target identification of an antiproliferative agent in live cells using fluorescence difference in two-dimensional gel electrophoresis. Angew Chem Int Ed Engl 51:5447–5451

    Article  CAS  PubMed Central  Google Scholar 

  9. Stoughton RB, Friend SH (2005) How molecular profiling could revolutionize drug discovery. Nat Rev Drug Discov 4:345–350

    Article  CAS  PubMed Central  Google Scholar 

  10. Zhang J, Campbell RE, Ting AY, Tsien RY (2002) Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 3:906–918

    Article  CAS  PubMed Central  Google Scholar 

  11. Bivona TG, Philips MR (2005) Analysis of Ras and Rap activation in living cells using fluorescent Ras binding domains. Methods 37:138–145

    Article  CAS  PubMed Central  Google Scholar 

  12. Xu W, Zeng Z, Jiang JH, Chang YT, Yuan L (2016) Discerning the chemistry in individual organelles with small-molecule fluorescent probes. Angew Chem Int Ed Engl 55:13658–13699

    Article  CAS  PubMed Central  Google Scholar 

  13. Lee MH, Kim JS, Sessler JL (2015) Small molecule-based ratiometric fluorescence probes for cations, anions, and biomolecules. Chem Soc Rev 44:4185–4191

    Article  CAS  PubMed Central  Google Scholar 

  14. Yin J, Hu Y, Yoon J (2015) Fluorescent probes and bioimaging: alkali metals, alkaline earth metals and pH. Chem Soc Rev 44:4619–4644

    Article  CAS  PubMed Central  Google Scholar 

  15. Martin S, Parton RG (2006) Lipid droplets: a unified view of a dynamic organelle. Nat Rev Mol Cell Biol 7:373–378

    Article  CAS  PubMed Central  Google Scholar 

  16. Walther TC, Farese RV (2012) Lipid droplets and cellular lipid metabolism. Annu Rev Biochem 81:687–714

    Article  CAS  PubMed Central  Google Scholar 

  17. Greenberg AS, Coleman RA, Kraemer FB, McManaman JL, Obin MS, Puri V, Yan QW, Miyoshi H, Mashek DG (2011) The role of lipid droplets in metabolic disease in rodents and humans. J Clin Invest 121:2102–2110

    Article  CAS  PubMed Central  Google Scholar 

  18. Singh R, Cuervo AM (2011) Autophagy in the cellular energetic balance. Cell Metab 13:495–504

    Article  CAS  PubMed Central  Google Scholar 

  19. Singh R, Kaushik S, Wang Y, Xiang Y, Novak I, Komatsu M, Tanaka K, Cuervo AM, Czaja MJ (2009) Autophagy regulates lipid metabolism. Nature 458:1131–1135

    Article  CAS  PubMed Central  Google Scholar 

  20. Ward C, Martinez-Lopez N, Otten EG, Carroll B, Maetzel D, Singh R, Sarkar S, Korolchuk VI (2016) Autophagy, lipophagy and lysosomal lipid storage disorders. Biochim Biophys Acta 1861:269–284

    Article  CAS  PubMed Central  Google Scholar 

  21. Kondo Y, Kanzawa T, Sawaya R, Kondo S (2005) The role of autophagy in cancer development and response to therapy. Nat Rev Cancer 5:726–734

    Article  CAS  PubMed Central  Google Scholar 

  22. Rubinsztein DC, Codogno P, Levine B (2012) Autophagy modulation as a potential therapeutic target for diverse diseases. Nat Rev Drug Discov 11:709–U784

    Article  CAS  PubMed Central  Google Scholar 

  23. Cheng Y, Ren XC, Hait WN, Yang JM (2013) Therapeutic targeting of autophagy in disease: biology and pharmacology. Pharmacol Rev 65:1162–1197

    Article  CAS  PubMed Central  Google Scholar 

  24. Hansen TE, Johansen T (2011) Following autophagy step by step. BMC Biol 9:39

    Article  PubMed Central  Google Scholar 

  25. Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140:313–326

    Article  CAS  PubMed Central  Google Scholar 

  26. Kim E, Lee S, Park SB (2012) A Seoul-Fluor-based bioprobe for lipid droplets and its application in image-based high throughput screening. Chem Commun 48:2331–2333

    Article  CAS  Google Scholar 

  27. Lee S, Kim E, Park SB (2013) Discovery of autophagy modulators through the construction of a high-content screening platform via monitoring of lipid droplets. Chem Sci 4:3282–3287

    Article  CAS  Google Scholar 

  28. Lee Y, Na S, Lee S, Jeon NL, Park SB (2013) Optimization of Seoul-Fluor-based lipid droplet bioprobes and their application in microalgae for bio-fuel study. Mol Biosyst 9:952–956

    Article  CAS  PubMed Central  Google Scholar 

  29. Choi Y, Kim H, Shin YH, Park SB (2015) Diverse display of non-covalent interacting elements using pyrimidine-embedded polyheterocycles. Chem Commun 51:13040–13043

    Article  CAS  Google Scholar 

  30. Kim E, Koh M, Lim BJ, Park SB (2011) Emission wavelength prediction of a full-color-tunable fluorescent core skeleton, 9-aryl-1,2-dihydropyrrolo[3,4-b]indolizin-3-one. J Am Chem Soc 133:6642–6649

    Article  CAS  PubMed Central  Google Scholar 

  31. Hsu PP, Sabatini DM (2008) Cancer cell metabolism: Warburg and beyond. Cell 134:703–707

    Article  CAS  PubMed Central  Google Scholar 

  32. Heiden MGV, Cantley LC, Thompson CB (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324:1029–1033

    Article  Google Scholar 

  33. Lee CH, Olson P, Evans RM (2003) Minireview: lipid metabolism, metabolic diseases, and peroxisome proliferator-activated receptors. Endocrinology 144:2201–2207

    Article  CAS  PubMed Central  Google Scholar 

  34. Park J, Lee HY, Cho MH, Park SB (2007) Development of a Cy3-labeled glucose bioprobe and its application in bioimaging and screening for anticancer agents. Angew Chem Int Ed 46:2018–2022

    Article  CAS  Google Scholar 

  35. Lee HY, Lee JJ, Park J, Park SB (2011) Development of fluorescent glucose bioprobes and their application on real-time and quantitative monitoring of glucose uptake in living cells. Chemistry 17:143–150

    Article  CAS  PubMed Central  Google Scholar 

  36. Jo A, Park J, Park SB (2013) Exploiting the mechanism of cellular glucose uptake to develop an image-based high-throughput screening system in living cells. Chem Commun 49:5138–5140

    Article  CAS  Google Scholar 

  37. Park J, Um JI, Jo A, Lee J, Jung DW, Williams DR, Park SB (2014) Impact of molecular charge on GLUT-specific cellular uptake of glucose bioprobes and in vivo application of the glucose bioprobe, GB2-Cy3. Chem Commun 50:9251–9254

    Article  CAS  Google Scholar 

  38. Fuhrmann GF, Dernedde S, Frenking G (1992) Phloretin keto-enol-tautomerism and inhibition of glucose-transport in human erythrocytes (including effects of phloretin on anion transporter). Biochim Biophys Acta 1110:105–111

    Article  CAS  PubMed Central  Google Scholar 

  39. Schenone M, Dancik V, Wagner BK, Clemons PA (2013) Target identification and mechanism of action in chemical biology and drug discovery. Nat Chem Biol 9:232–240

    Article  CAS  PubMed Central  Google Scholar 

  40. Park J, Koh M, Koo JY, Lee S, Park SB (2016) Investigation of specific binding proteins to photoaffinity linkers for efficient deconvolution of target protein. ACS Chem Biol 11:44–52

    Article  CAS  PubMed Central  Google Scholar 

  41. Lee S, Nam Y, Koo JY, Lim D, Park J, Ock J, Kim J, Suk K, Park SB (2014) A small molecule binding HMGB1 and HMGB2 inhibits microglia-mediated neuroinflammation. Nat Chem Biol 10:1055–1060

    Article  CAS  PubMed Central  Google Scholar 

  42. Levoin N, Calmels T, Poupardin-Olivier O, Labeeuw O, Danvy D, Robert P, Berrebi-Bertrand I, Ganellin CR, Schunack W, Stark H, Capet M (2008) Refined docking as a valuable tool for lead optimization: application to histamine H-3 receptor antagonists. Arch Pharm 341:610–623

    Article  CAS  Google Scholar 

  43. Bollini M, Domaoal RA, Thakur VV, Gallardo-Macias R, Spasov KA, Anderson KS, Jorgensen WL (2011) Computationally-guided optimization of a docking hit to yield catechol diethers as potent anti-HIV agents. J Med Chem 54:8582–8591

    Article  CAS  PubMed Central  Google Scholar 

  44. Koh M, Park J, Koo JY, Lim D, Cha MY, Jo A, Choi JH, Park SB (2014) Phenotypic screening to identify small-molecule enhancers for glucose uptake: target identification and rational optimization of their efficacy. Angew Chem Int Ed 53:5102–5106

    Article  CAS  Google Scholar 

  45. Bae H, Jang JY, Choi SS, Lee JJ, Kim H, Jo A, Lee KJ, Choi JH, Suh SW, Park SB (2016) Mechanistic elucidation guided by covalent inhibitors for the development of anti-diabetic PPAR gamma ligands. Chem Sci 7:5523–5529

    Article  CAS  Google Scholar 

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Acknowledgment

This work was supported by the National Creative Research Initiative Grant (NRF-2014R1A3A2030423) and the Bio & Medical Technology Development Program (2012M3A9C4048780) through the National Research Foundation of Korea (NRF) funded by the Korean Government (Ministry of Science and ICT​). This research was also supported by Basic Science Research Program (NRF-2009-0093826, 2016R1A6A3A01011351) through NRF funded by the Korean Ministry of Education.

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Authors and Affiliations

  1. Department of Chemistry, Seoul National University, Seoul, South Korea

    Young-Hee Shin & Seung Bum Park

  2. Department of Biophysics and Chemical Biology, Seoul National University, Seoul, South Korea

    Seung Bum Park

Authors
  1. Young-Hee Shin
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  2. Seung Bum Park
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Corresponding author

Correspondence to Seung Bum Park .

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Editors and Affiliations

  1. Chemical Biology and Therapeutics Science Program, The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Bridget Wagner

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Shin, YH., Park, S.B. (2018). Phenotype-Based High-Content Screening Using Fluorescent Chemical Bioprobes: Lipid Droplets and Glucose Uptake Quantification in Live Cells. In: Wagner, B. (eds) Phenotypic Screening. Methods in Molecular Biology, vol 1787. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7847-2_17

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  • DOI: https://doi.org/10.1007/978-1-4939-7847-2_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7846-5

  • Online ISBN: 978-1-4939-7847-2

  • eBook Packages: Springer Protocols

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