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A potentiator of orthosteric ligand activity at GLP-1R acts via covalent modification

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Abstract

We report that 4-(3-(benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), which behaves as a positive allosteric modulator at the glucagon-like peptide-1 receptor (GLP-1R), covalently modifies cysteines 347 and 438 in GLP-1R. C347, located in intracellular loop 3 of GLP-1R, is critical to the activity of BETP and a structurally distinct GLP-1R ago-allosteric modulator, N-(tert-butyl)-6,7-dichloro-3-(methylsulfonyl)quinoxalin-2-amine. We further show that substitution of cysteine for phenylalanine 345 in the glucagon receptor is sufficient to confer sensitivity to BETP.

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Figure 1: GLP-1R is covalently modified by BETP.
Figure 2: Mutant receptors alter BETP sensitivity.

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Acknowledgements

We thank X. Chen, Y. Cao and K. Fennell (Worldwide Medicinal Chemistry, Pfizer PharmaTherapeutics Research and Development) for advice, constructs and preliminary experiments.

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Authors and Affiliations

  1. Worldwide Medicinal Chemistry, Pfizer PharmaTherapeutics Research and Development, Cambridge, Massachusetts, USA

    Whitney M Nolte, Benjamin D Stevens, David A Griffith, Roger B Ruggeri, Alan M Mathiowetz, David Hepworth & Philip A Carpino

  2. Cardiovascular and Metabolic Diseases Research Unit, Pfizer PharmaTherapeutics Research and Development, Cambridge, Massachusetts, USA

    Jean-Philippe Fortin

  3. Worldwide Medicinal Chemistry, Pfizer PharmaTherapeutics Research and Development, Groton, Connecticut, USA

    Gary E Aspnes & Chris Limberakis

  4. Structural Biology and Biophysics Group, Center for Chemistry Innovation and Excellence, Worldwide Medicinal Chemistry, Pfizer PharmaTherapeutics Research and Development, Groton, Connecticut, USA

    Lise R Hoth

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  1. Whitney M Nolte
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Contributions

W.M.N. conducted all of the experiments and analyzed the data. W.M.N., B.D.S., G.E.A. and C.L. designed PETP and developed a synthetic route for its preparation. W.M.N., J.-P.F., B.D.S., D.A.G., R.B.R., A.M.M., D.H. and P.A.C. conceived the experiments. W.M.N. and L.R.H. developed MS methods for identifying the site (or sites) at which BETP modifies GLP-1R. W.M.N. and P.A.C. wrote the paper.

Corresponding author

Correspondence to Philip A Carpino.

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Competing interests

Declaration: W.N., J.-P.F., G.A., D.G., L.H., R.R., A.M., C.L., D.H. and P.C. are employees of Pfizer. The research was fully funded by Pfizer.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–14 and Supplementary Table 1. (PDF 1307 kb)

Supplementary Data Set 1

BETP inhibitory activity across 64 proteins (XLSX 12 kb)

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Nolte, W., Fortin, JP., Stevens, B. et al. A potentiator of orthosteric ligand activity at GLP-1R acts via covalent modification. Nat Chem Biol 10, 629–631 (2014). https://doi.org/10.1038/nchembio.1581

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  • DOI: https://doi.org/10.1038/nchembio.1581

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