Original Article | Published:

Drug Discovery

High-throughput screening assay for new ligands at human melatonin receptors

Acta Pharmacologica Sinica volume 29, pages 15151521 (2008) | Download Citation

Project supported in part by the Shanghai Municipality Science and Technology Development Fund (No 06DZ22907 and 07DZ22920), the Ministry of Science and Technology (No 2004CB518902), and Servier Beijing Pharmaceutical Research and Development.

Abstract

Aim:

Melatonin (MT) is a neurohormone produced and secreted primarily by the pineal gland in a circadian manner, and mainly acts through 2 receptor subtypes: MT1 and MT2 in humans. The diversity in their tissue distribution is in favor of different functions for each receptor subtype. Selective modulators are therefore required to determine the physiological roles of these melatonin receptor subtypes and their implications in pathological processes.

Methods:

A homogenous MT1/MT2 receptor binding assay was established for high-throughput screening of new ligands at the hMT1 and/or hMT2 receptors. The functional properties (agonists or antagonists) were assessed by a conventional guanosine-5′[γ-35S] triphosphate (GTP-γS) assay.

Results:

Three hMT1 receptor-selective small molecule antagonists and 1 hMT2 receptor-selective small molecule antagonist with novel structural features were identified following a high-throughput screening campaign of 48 240 synthetic and natural compounds.

Conclusion:

The findings may assist in the expansion of chemical probes to these 2 receptor subtypes.

References

  1. 1.

    . Pineal melatonin: cell biology of its synthesis and of its physiological interactions. Endocr Rev 1991; 12: 151–80.

  2. 2.

    , . Structure of melatonin. J Am Chem Soc 1959; 81: 6084–5.

  3. 3.

    . Melatonin's role in vertebrate circadian rhythms. Chronobiol Int 1998; 15: 457–73.

  4. 4.

    , , , . Melatonin and cardiovascular function. Adv Exp Med Biol 1999; 460: 299–310.

  5. 5.

    . Melatonin and human reproduction. Ann Med 1998; 30: 103–8.

  6. 6.

    , , . Melatonin decreases cell proliferation and transformation in a melatonin receptor-dependent manner. Cancer Lett 2000; 151: 133–43.

  7. 7.

    . Characterization of a retinal melatonin receptor. J Pharmacol Exp Ther 1985; 234: 395–401.

  8. 8.

    , . Use of 2-[125I]iodomelatonin to characterize melatonin binding sites in chicken retina. Proc Natl Acad Sci USA 1987; 84: 3916–20.

  9. 9.

    , , . Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses. Neuron 1994; 13: 1177–85.

  10. 10.

    , , , , , . Molecular characterization of a second melatonin receptor expressed in human retina and brain: the Mel1b melatonin receptor. Proc Natl Acad Sci USA 1995; 92: 8734–8.

  11. 11.

    , , . Activation of melatonin MT2 receptors in rat suprachiasmatic nucleus phase advances the circadian clock. Am J Physiol Cell Physiol 2001; 280: C110–C8.

  12. 12.

    , , , , , et al. Identification of the melatonin binding site MT3 as the quinone reductase 2. J Biol Chem 2000; 257: 31 311–7.

  13. 13.

    , , , , , . Comparative pharmacological studies of melatonin receptors: MT1, MT2 and MT3/QR2. Tissue distribution of MT3/QR2. Biochem Pharmacol 2001; 61: 1369–79.

  14. 14.

    , , , , , et al. The IUPHAR compendium of receptor characterization and classification. IUPHAR Media, London 2000: 270–7.

  15. 15.

    , , . Melatonin receptor subtype expression in human cerebellum. Neuroreport 1998; 9: 4063–8.

  16. 16.

    , , , , , . Molecular and pharmacological evidence for MT1 melatonin receptor subtype in tail artery of juvenile Wistar rats. Br J Pharmacol 1999; 127: 987–95.

  17. 17.

    , , , , , . The melatonin receptor in the human brain: cloning experiments and distribution studies. Brain Res Mol Brain Res 1996; 39: 117–26.

  18. 18.

    , . The Mel1a melatonin receptor gene is expressed in human suprachiasmatic nuclei. Neuroreport 1996; 8: 109–12.

  19. 19.

    , , , , . Studies of the renal action of melatonin: evidence that the effects are mediated by 37 kDa receptors of the Mel1a subtype localized primarily to the basolateral membrane of the proximal tubule. FASEB J 1997; 11: 93–100.

  20. 20.

    , . Melatonin, its receptors and relationships with biological rhythms disorders. Clin Neuropharmacol 1997; 20: 482–510.

  21. 21.

    , , . Melatonin receptor ligands and their potential clinical applications. Annu Rep Med Chem 1997: 31–40.

  22. 22.

    , , . Development of a complex scintillation proximity assay for high-throughput screening of PPARγ modulators. Acta Pharmacol Sin 2005; 26: 339–44.

  23. 23.

    , . The [35S]GTPγS binding assay: approaches and applications in pharmacology. Life Sci 2003; 74: 489–508.

  24. 24.

    , , , , , et al. New selective ligands of human cloned melatonin MT1 and MT2 receptors. Naunyn Schmiedeberg's Arch Pharmacol 2003; 367: 553–61.

  25. 25.

    . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248–54.

  26. 26.

    , . Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol 1973; 22: 3099–108.

  27. 27.

    , , . The effects of saponin on the binding and functional properties of the human adenosine A1 receptor. Br J Pharmacol 1996; 117: 1521–9.

  28. 28.

    , , . A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999; 4: 67–73.

  29. 29.

    , , , . Melatonin receptor antagonists that differentiate between the human Mel1a and Mel1b recombinant subtypes are used to assess the pharmacological profile of the rabbit retina ML1 presynaptic heteroreceptor. Naunyn-Schmiedeberg's Arch Pharmacol 1997; 355: 365–75.

  30. 30.

    , , , , , et al. Analysis of structure-activity relationships for MT2 selective antagonists by melatonin MT1 and MT2 receptor models. J Med Chem 2005; 48: 4049–60.

  31. 31.

    , , , . Circadian uses of melatonin in humans. Chronobiol Int 2006; 23: 403–12.

  32. 32.

    , , , , , et al. Melatonin in mood disorders. World J Biol Psychiatry 2006; 7: 138–51.

  33. 33.

    . Melatonin, aging, and age-related diseases: perspectives for prevention, intervention, and therapy. Endocrine 2005; 27: 201–12.

  34. 34.

    , , , , , . Melatonin, immune function and aging. Immun Ageing 2005; 2: 17.

  35. 35.

    , , , , , et al. Pineal and cortical melatonin receptors MT1 and MT2 are decreased in Alzheimer's disease. Eur J Histochem 2006; 50: 311–6.

  36. 36.

    , , . Melatonin and anesthesia: a clinical perspective. J Pineal Res 2007; 42: 12–21.

  37. 37.

    . Agomelatin: the first “melatoninergic” antidepressant. Neuropsychopharmacol Hung 2006; 8: 105–12.

  38. 38.

    , , , . Intake of melatonin is associated with amelioration of physiological changes, both metabolic and morphological pathologies associated with obesity: an animal model. Int J Exp Pathol 2007; 88: 19–29.

  39. 39.

    , . Therapeutic potential of melatonin ligands. Chronobiol Int 2006; 23: 413–8.

Download references

Author information

Affiliations

  1. The National Center for Drug Screening and the State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

    • Jian-hua Yan
    • , Hao-ran Su
    •  & Ming-wei Wang
  2. Les Laboratoires Servier, 22 rue Garnier, 92200 Neuilly-sur-Seine, France

    • Jean A Boutin
    •  & M Pierre Renard

Authors

  1. Search for Jian-hua Yan in:

  2. Search for Hao-ran Su in:

  3. Search for Jean A Boutin in:

  4. Search for M Pierre Renard in:

  5. Search for Ming-wei Wang in:

Corresponding author

Correspondence to Ming-wei Wang.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1111/j.1745-7254.2008.00903.x

Further reading