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Directed molecular evolution of DREADDs: a generic approach to creating next-generation RASSLs

Abstract

G protein–coupled receptors (GPCRs) and their downstream signaling cascades contribute to most physiological processes and a variety of human diseases. Isolating the effects of GPCR activation in an in vivo experimental setting is challenging as exogenous ligands have off-target effects and endogenous ligands constantly modulate the activity of native receptors. Highly specific designer drug–designer receptor complexes are a valuable tool for elucidating the effects of activating particular receptors and signaling pathways within selected cell types in vivo. In this study, we describe a generic protocol for the directed molecular evolution of designer receptors exclusively activated by designer drugs (DREADDs). First, the yeast system is validated with the template receptor. Second, a mutant library is generated by error-prone PCR. Third, the library is screened by drug-dependent yeast growth assays. Mutants exhibiting the desired properties are selected for further rounds of mutagenesis or for characterization in mammalian systems. In total, these steps should take 6–8 weeks of experimentation and should result in the evolution of a receptor to be activated by the chosen ligand. This protocol should help improve the experimental targeting of select cell populations.

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Figure 1: Pheromone signaling pathways of wild-type and engineered yeast Saccharomyces cerevisiae.
Figure 2: Generic scheme for obtaining DREADDs through directed molecular evolution.
Figure 3: Typical pharmacological profiles of 3-d growth of yeast functionally expressing the template GPCR in response to three agonists with differing potency and efficacy.
Figure 4: Typical pharmacological profiles of DREADD candidates.

References

  1. Hopkins, A.L. & Groom, C.R. The druggable genome. Nat. Rev. Drug Discov. 1, 727–730 (2002).

    Article  CAS  PubMed  Google Scholar 

  2. Armbruster, B.N. & Roth, B.L. Mining the receptorome. J. Biol. Chem. 280, 5129–5132 (2005).

    Article  CAS  PubMed  Google Scholar 

  3. Roth, B.L., Sheffler, D.J. & Kroeze, W.K. Magic shotguns versus magic bullets: selectively non-selective drugs for mood disorders and schizophrenia. Nat. Rev. Drug Discov. 3, 353–359 (2004).

    Article  CAS  PubMed  Google Scholar 

  4. Keiser, M.J. et al. Predicting new molecular targets for known drugs. Nature 462, 175–181 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Pei, Y., Rogan, S.C., Yan, F. & Roth, B.L. Engineered GPCRs as tools to modulate signal transduction. Physiology 23, 313–321 (2008).

    Article  CAS  PubMed  Google Scholar 

  6. Conklin, B.R. et al. Engineering GPCR signaling pathways with RASSLs. Nat. Methods 5, 673–678 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Armbruster, B.N. et al. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc. Natl. Acad. Sci. USA 12, 5163–5168 (2007).

    Article  Google Scholar 

  8. Guettier, J-M. et al. Profound metabolic effects following selective activation of distinct G protein signaling pathways in pancreatic β cells in vivo . Proc. Natl. Acad. Sci. USA 106, 19197–19202 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Alexander, G.M. et al. Remote control of neuronal activity in transgenic mice expressing evolved G protein-coupled receptors. Neuron 63, 27–39 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Campbell, J.H., Lengyel, J.A. & Langridge, J. Evolution of a second gene for beta-galactosidase in Escherichia coli . Proc. Natl. Acad. Sci. USA 70, 1841–1845 (1973).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Yuan, L., Kurek, I., English, J. & Keenan, R. Laboratory-directed protein evolution. Microbiol. Mol. Biol. Rev. 69, 373–392 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Bloom, J.D. & Arnold, F.H. In the light of directed evolution: pathways of adaptive protein evolution. Proc. Natl. Acad. Sci. USA 106 (Suppl. 1): 9995–10000 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Erlenbach, I. et al. Functional expression of M(1), M(3) and M(5) muscarinic acetylcholine receptors in yeast. J. Neurochem. 77, 1327–1337 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Pausch, M.H. et al. Heterologous G protein-coupled receptors expressed in Saccharomyces cerevisiae: methods for genetic analysis and ligand identification. In Identification and Expression of G Protein-Coupled Receptors (ed. Lynch, K.R.) 196–212 (Wiley-Liss, New York, 1998).

    Google Scholar 

  15. Wess, J. Muscarinic acetylcholine receptor knockout mice: novel phenotypes and clinical implications. Annu. Rev. Pharmacol. Toxicol. 44, 423–450 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Roth, B.L. Drugs and valvular heart disease. N. Engl. J. Med. 356, 6–9 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Bates, B. et al. Characterization of Gpr101 expression and G-protein coupling selectivity. Brain Res. 1087, 1–14 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Huang, X.P. et al. Parallel functional activity profiling reveals valvulopathogens are potent 5-HT2B receptor agonists: implications for drug safety assessment. Mol. Pharmacol. 76, 710–722 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Pei, Y., Dong, S. & Roth, B.L. Generation of designer receptors exclusively activated by designer drugs (DREADDs) by directed molecular evolution. Curr. Protoc. Neurosci. 50, 4.33.1–4.33.25 (2010).

    Google Scholar 

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Acknowledgements

We thank Blaine N. Armbruster for his work developing this technique. This study was supported by a NARSAD Distinguished Investigator Award and by the following grants from the National Institutes of Health: GM07040 and GM008719 from the National Institute of General Medical Sciences (S.C.R.); and MH087074 (S.C.R.), MH082441 and MH061887 (B.L.R.) from the National Institute of Mental Health.

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S.D., S.C.R. and B.L.R. prepared the paper.

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Correspondence to Bryan L Roth.

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Dong, S., Rogan, S. & Roth, B. Directed molecular evolution of DREADDs: a generic approach to creating next-generation RASSLs. Nat Protoc 5, 561–573 (2010). https://doi.org/10.1038/nprot.2009.239

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