Skip to main content
SpringerLink
Log in

Development of a High-Throughput Cell-Based Assay for 11β-Hydroxysteroid Dehydrogenase Type 1 Using BacMam Technology

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Cortisol is an important glucocorticoid in humans that regulates many physiological processes. Human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts cortisone to cortisol in vivo and has emerged as an appealing therapeutic target for treating metabolic diseases. Here, we report a sensitive and robust high-throughput (HT) cell-based assay for screening 11β-HSD1 inhibitors. This assay utilizes a HEK293 cell line transduced by a BacMam virus expressing human 11β-HSD1. The enzyme activity in the cells was measured by quantifying cortisol levels released into the cell culture supernatant via a competitive homogenous time-resolved fluorescence (HTRF) method. We show that 11β-HSD1 activity in supernatant of BacMam-transduced HEK293 cells increases with 11β-HSD1 BacMam virus load in a dose-dependent manner, and is comparable to the enzyme activity detected in differentiated mouse adipocytes. In addition, we show that co-expression of hexose-6-phosphate dehydrogenase (H6PDH) is not required for the enzyme to function effectively as an oxo-reductase. This assay has been developed in low-volume 384-well format and it is sensitive, robust, and amenable to HT screening.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Sapolsky, R. M., Romero, L. M., & Munck, A. U. (2000). How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions [Review]. Endocrine Reviews, 21, 55–89.

    Article  PubMed  CAS  Google Scholar 

  2. Lakshmi, V., & Monder, C. (1988). Purification and characterization of the corticosteroid 11 beta-dehydrogenase component of the rat liver 11 beta-hydroxysteroid dehydrogenase complex. Endocrinology, 123, 2390–2398.

    PubMed  CAS  Google Scholar 

  3. Krozowski, Z., Li, K. X. Z., Koyama, K., Smith, R. E., Obeyesekere, V. R., Stein-Oakley, A., Sasano, H., Coulter, C., Cole, T., & Sheppard, K. E. (1999). The type I and type II 11 beta-hydroxysteroid dehydrogenase enzymes. The Journal of Steroid Biochemistry and Molecular Biology, 69, 391–401.

    Article  PubMed  CAS  Google Scholar 

  4. Bujalska, I. J., Draper, N., Michailidou, Z., Tomlinson, J. W., White, P. C., Chapman, K. E., Walker, E. A., & Stewart, P. M. (2005). Hexose-6-phosphate dehydrogenase confers oxo-reductase activity upon 11 beta-hydroxysteroid dehydrogenase type 1. Journal of Molecular Endocrinology, 34, 675–684.

    Article  PubMed  CAS  Google Scholar 

  5. Hewitt, K. N., Walker, E. A., & Stewart, P. M. (2005). Minireview: Hexose-6-phosphate dehydrogenase and redox control of 11{beta}-hydroxysteroid dehydrogenase type 1 activity. Endocrinology, 146, 2539–2543.

    Article  PubMed  CAS  Google Scholar 

  6. Barash, V., Erlich, T., & Bashan, N. (1990). Microsomal hexose-6-phosphate and 6-phosphogluconate dehydrogenases in extrahepatic tissues: human placenta and pig kidney cortex. Biochemistry International, 20, 267–274.

    PubMed  CAS  Google Scholar 

  7. Kulkarni, A. P., & Hodgson, E. (1982). Mouse liver microsomal hexose-6-phosphate dehydrogenase. NADPH generation and utilization in monooxygenation reactions. Biochemical Pharmacology, 31, 1131–1137.

    Article  PubMed  CAS  Google Scholar 

  8. Tomlinson, J. W., Walker, E. A., Bujalska, I. J., Draper, N., Lavery, G. G., Cooper, M. S., Hewison, M., & Stewart, P. M. (2004). 11beta-Hydroxysteroid dehydrogenase type 1: A tissue-specific regulator of glucocorticoid response. Endocrine Reviews, 25, 831–866.

    Article  PubMed  CAS  Google Scholar 

  9. Lavery, G. G., Walker, E. A., Draper, N., Jeyasuria, P., Marcos, J., Shackleton, C. H., Parker, K. L., White, P. C., & Stewart, P. M. (2006). Hexose-6-phosphate dehydrogenase knock-out mice lack 11 beta-hydroxysteroid dehydrogenase type 1-mediated glucocorticoid generation. The Journal of Biological Chemistry, 281, 6546–6551.

    Article  PubMed  CAS  Google Scholar 

  10. Jamieson, P. M., Chapman, K. E., Edwards, C. R., & Seckl, J. R. (1995). 11 beta-Hydroxysteroid dehydrogenase is an exclusive 11 beta-reductase in primary cultures of rat hepatocytes: Effect of physicochemical and hormonal manipulations. Endocrinology, 136, 4754–4761.

    Article  PubMed  CAS  Google Scholar 

  11. Wang, M. (2006). Inhibitors of 11b-hydroxysteroid dehydrogenase type 1 for the treatment of metabolic syndrome. Current Opinion in Investigational Drugs, 7, 319–323.

    PubMed  CAS  Google Scholar 

  12. Masuzaki, H., Paterson, J., Shinyama, H., Morton, N. M., Mullins, J. J., Seckl, J. R., & Flier, J. S. (2001). A transgenic model of visceral obesity and the metabolic syndrome. Science, 294, 2166–2170.

    Article  PubMed  CAS  Google Scholar 

  13. Fotsch, C., Askew, B. C., & Chen, J. J. (2005). 11 beta-Hydroxysteroid dehydrogenase-1 as a therapeutic target for metabolic diseases [Review]. Expert Opinion on Therapeutic Patents, 15, 289–303.

    Article  CAS  Google Scholar 

  14. Schweizer, R. A. S., Atanasov, A. G., Frey, B. M., & Odermatt, A. (2003). A rapid screening assay for inhibitors of 11 beta-hydroxysteroid dehydrogenases (11 beta-HSD): Flavanone selectively inhibits 11 beta-HSD1 reductase activity. Molecular and Cellular Endocrinology, 212, 41–49.

    Article  PubMed  CAS  Google Scholar 

  15. Barton, P. J., Clarke, D. S., Davies, C. D., Hargreaves, R. B., Pease, J. E., & Rankine, M.T. (2004). PCT/GB2003/003171(WO 2004/011410 A1), 1–147.

  16. Xu, R. D., Sang, B. C., Navre, M., & Kassel, D. B. (2006). Cell-based assay for screening 11 beta-hydroxysteroid dehydrogenase inhibitors using liquid chromatography/tandem mass spectrometry detection. Rapid Communications in Mass Spectrometry, 20, 1643–1647.

    Article  PubMed  CAS  Google Scholar 

  17. Enomoto, K., Araki, A., Nakajima, T., Ohta, H., Dohi, K., Preaudat, M., Seguin, P., Mathis, G., Suzuki, R., Kominami, G., et al. (2002). High-throughput miniaturized immunoassay for human interleukin-13 secreted from NK3.3 cells using homogenous time-resolved fluorescence. Journal of Pharmaceutical and Biomedical Analysis, 28, 73–79.

    Article  PubMed  CAS  Google Scholar 

  18. Mathis, G. (1993). Rare earth cryptates and homogeneous fluoroimmunoassays with human sera. Clinical Chemistry, 39, 1953–1959.

    PubMed  CAS  Google Scholar 

  19. Claret, E. J., Ouled-Diaf, J., & Seguin, P. (2003). Homogeneous time resolved fluorescence assay to measure histamine release. Combinatorial Chemistry & High Throughput Screening, 6, 789–794.

    CAS  Google Scholar 

  20. Enomoto, K., Aono, Y., Mitsugi, T., Takahashi, K., Suzuki, R., Preaudat, M., Mathis, G., Kominami, G., & Takemoto, H. (2000). High throughput screening for human interferon-gamma production inhibitor using homogenous time-resolved fluorescence. Journal of Biomolecular Screening, 5, 263–268.

    PubMed  CAS  Google Scholar 

  21. Clarke, E. E., & Shearman, M. S. (2000). Quantitation of amyloid-beta peptides in biological milieu using a novel homogeneous time-resolved fluorescence (HTRF) assay. Journal of Neuroscience Methods, 102, 61–68.

    Article  PubMed  CAS  Google Scholar 

  22. Mathis, G. (1999). HTRF(R) technology. Journal of Biomolecular Screening, 4, 309–314.

    Article  PubMed  CAS  Google Scholar 

  23. Kolb, A. (1997). A homogenous, time-resolved fluorescence method for drug discovery. In Devlin, J. P., & Kolb, A. (Eds.), High throughput screening: The discovery of bioactive substance (pp. 345–360). Marcel Dekker.

  24. Grepin, C., & Pernelle, C. (2000). High-throughput screening evolution of homogeneous time resolved fluorescence (HTRF) technology for HTS. Drug Discovery Today, 5(5), 212–214.

    Article  PubMed  Google Scholar 

  25. Mathis, G. (1995). Probing molecular interactions with homogeneous techniques based on rare earth cryptates and fluorescence energy transfer. Clinical Chemistry, 41, 1391–1397.

    PubMed  CAS  Google Scholar 

  26. Gu, X., Dragovic, J., Koo, G. C., Koprak, S. L., LeGrand, C., Mundt, S.S., Shah, K., Springer, M. S., Tan, E.Y., Thieringer, R., et al. (2005). Discovery of 4-heteroarylbicyclo[2.2.2]octyltriazoles as potent and selective inhibitors of 11 beta-HSD1: Novel therapeutic agents for the treatment of metabolic syndrome. Bioorganic & Medicinal Chemistry Letters, 15, 5266–5269.

    Article  CAS  Google Scholar 

  27. Condreay, J. P., Witherspoon, S. M., Clay, W. C., & Kost, T. A. (1999). Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector. Proceedings of the National Academy of Science of the United States of America, 96, 127–132.

    Article  CAS  Google Scholar 

  28. Fornwald, J. A., Wang, D., Lu, Q., Wang, D., & Ames, R. S. (2007). Gene expression in mammalian cells using BacMam, a modified baculovirus system. In D. W. Murhammer (Ed.), Methods in molecular biology, baculovirus and insect cell expression protocols (Vol. 338, pp. 95–114). Totowa, NJ: Humana Press.

    Google Scholar 

  29. Liu, Y., Sun, W. L., Sun, Y., Hu, G., & Ding, G. X. (2006). Role of 11-beta-hydroxysteroid dehydrogenase type 1 in differentiation of 3T3-L1 cells and in rats with diet-induced obesity. Acta Pharmacologica Sinica, 27, 588–596.

    Article  PubMed  CAS  Google Scholar 

  30. Lysko, P. G., Weinstock, J., Webb, C. L., Brawner, M. E., & Elshourbagy, N. A. (1999). Identification of a small-molecule, nonpeptide macrophage scavenger receptor antagonist. The Journal of Pharmacology and Experimental Therapeutics, 289, 1277–1285.

    PubMed  CAS  Google Scholar 

  31. Ames, R., Fornwald, J., Nuthulaganti, P., Trill, J., Foley, J., Buckley, P., Kost, T., Wu, Z., & Romanos, M. (2004). BacMam recombinant baculoviruses in G protein-coupled receptor drug discovery. Receptors & Channels, 10, 99–107.

    Article  CAS  Google Scholar 

  32. Condreay, J. P., Ames, R. S., Hassan, N. J., Kost, T. A., Merrihew, R. V., Mossakowska, D. E., Pountney, D. J., & Romanos, M. A. (2006). Baculoviruses and mammalian cell-based assays for drug screening. Advances in Virus Research, 68, 255–286.

    Article  PubMed  CAS  Google Scholar 

  33. Scott, M. J., Modha, S. S., Rhodes, A. D., Broadway, N. M., Hardwicke, P. I., Zhao, H. J., Kennedy-Wilson, K. M., Sweitzer, S. M., & Martin, S. L. (2007). Efficient expression of secreted proteases via recombinant BacMam virus. Protein Expression and Purification, 52, 104–116.

    Article  PubMed  CAS  Google Scholar 

  34. Kost, T. A., Condreay, J. P., Ames, R. S., Rees, S., & Romanos, M. A. (2007). Implementation of BacMam virus gene delivery technology in a drug discovery setting. Drug Discovery Today, 12, 396–403.

    Article  PubMed  CAS  Google Scholar 

  35. Ames, R. S., Kost, T. A., & Condreay, J. P. (2007). BacMam technology and its application to drug discovery. Expert Opinion on Drug Discovery, 2, 1669–1681.

    Article  CAS  Google Scholar 

  36. Liu, Y., Sun, Y., Zhu, T., Xie, Y., Yu, J., Sun, W. L., Ding, G. X., & Hu, G. (2007). 11 beta-Hydroxysteroid dehydrogenase type 1 promotes differentiation of 3T3-L1 preadipocyte. Acta Pharmacologica Sinica, 28, 1198–1204.

    Article  PubMed  CAS  Google Scholar 

  37. Atanasov, A. G., Nashev, L. G., Schweizer, R. A. S., Frick, C., & Odermatt, A. (2004). Hexose-6-phosphate dehydrogenase determines the reaction direction of 11 beta-hydroxysteroid dehydrogenase type 1 as an oxoreductase. FEBS Letters, 571, 129–133.

    Article  PubMed  CAS  Google Scholar 

  38. Zhang, J. H., Chung, T. D., & Oldenburg, K. R. (1999). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. Journal of Biomolecular Screening, 4, 67–73.

    Article  PubMed  Google Scholar 

  39. Su, X., Vicker, N., Lawrence, H., Smith, A., Purohit, A., Reed, M. J., & Potter, B. V. (2007). Inhibition of human and rat 11beta-hydroxysteroid dehydrogenase type 1 by 18beta-glycyrrhetinic acid derivatives. The Journal of Steroid Biochemistry and Molecular Biology, 104, 312–320.

    Article  PubMed  CAS  Google Scholar 

  40. Monder, C., Stewart, P. M., Lakshmi, V., Valentino, R., Burt, D., & Edwards, C. R. (1989). Licorice inhibits corticosteroid 11 beta-dehydrogenase of rat kidney and liver: In vivo and in vitro studies. Endocrinology, 125, 1046–1053.

    Article  PubMed  CAS  Google Scholar 

  41. Buhler, H., Perschel, F. H., & Hierholzer, K. (1991). Inhibition of rat renal 11 beta-hydroxysteroid dehydrogenase by steroidal compounds and triterpenoids; structure/function relationship. Biochimica et Biophysica Acta, 1075, 206–212.

    PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We wish to thank Gordon McIntyre and Thomas Meek for their support in working on the program.

Author information

Authors and Affiliations

  1. Biological Reagents and Assay Development, GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA, 19426, USA

    Da-Yuan Wang, Quinn Lu, Lisa Payne & Hu Li

  2. Discovery Technology Group, GlaxoSmithKline, Collegeville, PA, USA

    Stacey L. Walsh

  3. Biological Reagents and Assay Development, GlaxoSmithKline, Gunnels Wood Road, Stevenage, SG1 2NY, UK

    Sundip S. Modha

  4. Discovery Technology Group, GlaxoSmithKline, Gunnels Wood Road, Stevenage, SG1 2NY, UK

    Martin J. Scott

  5. Biological Reagents and Assay Development, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA, 19406, USA

    Thomas D. Sweitzer & Robert S. Ames

  6. Cardiovascular and Urology – Center of Excellence for Drug Discovery, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA, 19406, USA

    Daniel J. Krosky

Authors
  1. Da-Yuan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Quinn Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stacey L. Walsh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lisa Payne
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sundip S. Modha
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Martin J. Scott
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Thomas D. Sweitzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Robert S. Ames
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Daniel J. Krosky
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Hu Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hu Li.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, DY., Lu, Q., Walsh, S.L. et al. Development of a High-Throughput Cell-Based Assay for 11β-Hydroxysteroid Dehydrogenase Type 1 Using BacMam Technology. Mol Biotechnol 39, 127–134 (2008). https://doi.org/10.1007/s12033-008-9050-y

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-008-9050-y

Keywords

Search

Navigation