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Research ArticleArticle

Pyrimidinyl Biphenylureas Act as Allosteric Modulators to Activate Cannabinoid Receptor 1 and Initiate β-Arrestin–Dependent Responses

Caitlin A. D. Jagla, Caitlin E. Scott, Yaliang Tang, Changjiang Qiao, Gabriel E. Mateo-Semidey, Guillermo A. Yudowski, Dai Lu and Debra A. Kendall
Molecular Pharmacology January 2019, 95 (1) 1-10; DOI: https://doi.org/10.1124/mol.118.112854
Caitlin A. D. Jagla
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut (C.A.D.J., C.E.S., Y.T., D.A.K.); Department of Anatomy and Neurobiology (G.E.M.-S., G.A.Y.) and Institute of Neurobiology (G.E.M.-S., G.A.Y.), University of Puerto Rico, San Juan, Puerto Rico; and Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, Texas (C.Q., D.L.)
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Caitlin E. Scott
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut (C.A.D.J., C.E.S., Y.T., D.A.K.); Department of Anatomy and Neurobiology (G.E.M.-S., G.A.Y.) and Institute of Neurobiology (G.E.M.-S., G.A.Y.), University of Puerto Rico, San Juan, Puerto Rico; and Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, Texas (C.Q., D.L.)
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Yaliang Tang
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut (C.A.D.J., C.E.S., Y.T., D.A.K.); Department of Anatomy and Neurobiology (G.E.M.-S., G.A.Y.) and Institute of Neurobiology (G.E.M.-S., G.A.Y.), University of Puerto Rico, San Juan, Puerto Rico; and Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, Texas (C.Q., D.L.)
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Changjiang Qiao
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut (C.A.D.J., C.E.S., Y.T., D.A.K.); Department of Anatomy and Neurobiology (G.E.M.-S., G.A.Y.) and Institute of Neurobiology (G.E.M.-S., G.A.Y.), University of Puerto Rico, San Juan, Puerto Rico; and Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, Texas (C.Q., D.L.)
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Gabriel E. Mateo-Semidey
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut (C.A.D.J., C.E.S., Y.T., D.A.K.); Department of Anatomy and Neurobiology (G.E.M.-S., G.A.Y.) and Institute of Neurobiology (G.E.M.-S., G.A.Y.), University of Puerto Rico, San Juan, Puerto Rico; and Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, Texas (C.Q., D.L.)
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Guillermo A. Yudowski
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut (C.A.D.J., C.E.S., Y.T., D.A.K.); Department of Anatomy and Neurobiology (G.E.M.-S., G.A.Y.) and Institute of Neurobiology (G.E.M.-S., G.A.Y.), University of Puerto Rico, San Juan, Puerto Rico; and Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, Texas (C.Q., D.L.)
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Dai Lu
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut (C.A.D.J., C.E.S., Y.T., D.A.K.); Department of Anatomy and Neurobiology (G.E.M.-S., G.A.Y.) and Institute of Neurobiology (G.E.M.-S., G.A.Y.), University of Puerto Rico, San Juan, Puerto Rico; and Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, Texas (C.Q., D.L.)
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Debra A. Kendall
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut (C.A.D.J., C.E.S., Y.T., D.A.K.); Department of Anatomy and Neurobiology (G.E.M.-S., G.A.Y.) and Institute of Neurobiology (G.E.M.-S., G.A.Y.), University of Puerto Rico, San Juan, Puerto Rico; and Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, Texas (C.Q., D.L.)
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  • Fig. 1.
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    Fig. 1.

    Structures of allosteric modulators of CB1: PSNCBAM1, 1-(4-chlorophenyl)-3-(3-(6-(pyrrolidin-1-yl)pyridin-2-yl)phenyl)urea; LDK1285, 1-(4-cyanophenyl)-3-(3-(2-(pyrrolidin-1-yl)pyrimidine-4-yl)-phenyl)urea; LDK1288, 1-(4-cyanophenyl)-3-(3-(4-(pyrrolidin-1-yl)pyrimidine-2-yl)-phenyl)urea; and LDK1305, 1-(4-cyanophenyl)-3-(3-(6-(pyrrolidin-1-yl)pyridin-2-yl)phenyl)urea.

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    Fig. 2.

    Orthosteric ligand binding to CB1 in the presence of allosteric modulators. Left panels show binding assays using [3H]CP55940 against (A) CP55940 (i.e., orthosteric agonist shown for comparison), (B) LDK1285, (C) LDK1288, (D) LDK1305, and (E) PSNCBAM1. Right panels show binding assays using [3H]SR141716A against (F) SR141716A (i.e., orthosteric inverse agonist shown for comparison), (G) LDK1285, (H) LDK1288, (I) LDK1305, and (J) PSNCBAM1. Each data point represents the mean ± S.E. of three independent experiments performed in duplicate.

  • Fig. 3.
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    Fig. 3.

    Internalization of CB1 upon treatment with LDK1285, LDK1288, LDK1305, or PSNCBAM1. (A) Cells expressing CB1T210A-GFP were incubated with either vehicle alone (0.03% DMSO) or 10 μM of the indicated compound for 1, 2, or 3 hours before fixation. The 0-hour time point indicates untreated cells expressing CB1T210A-GFP. Images are representative of three independent transfections. Scale bar, 15 μm (white bar; see bottom-right image). (B) Internal CB1T210A-GFP was quantified as described in Materials and Methods. Percent internal fluorescence is expressed as the mean ± S.E. fold change over vehicle (n = 3 independent experiments). Statistical significance was assessed using one-way analysis of variance and Dunnett’s multiple comparisons test; *P < 0.05; ***P < 0.001. (C) Membrane fluorescence, derived from the experiment in frame A, is expressed as the mean ± S.E. fold change over vehicle (n = 3 independent experiments). Orange line indicates LDK1285; gray, LDK1288; yellow, LDK1305; blue, PSNCBAM1. Statistical significance was assessed using one-way analysis of variance and Dunnett’s multiple-comparisons test; ††P < 0.01 in comparison with PSNCBAM1. (D) Cells expressing CB1-GFP and β-arrestin 2-mcherry were treated with PSNCBAM1 (5 μM; blue curve) or CP55940 (0.5 μM; gray curve) or PSCNBAM1 and CP55940 (5, 0.5 μM, respectively; black curve). β-arrestin-2 recruitment to CB1 was measured by TIRF microscopy and is displayed as percent change in fluorescence intensity over time. CP55940 t1/2 = 36 seconds; CP55940 plus PSNCBAM1 t1/2 = 42 seconds. (E) Cells expressing CB1-GFP and β-arrestin 2-mcherry were treated with LDK1288 (5 μM; orange curve) or CP55940 (0.5 μM; red curve) or LDK1288 and CP55940 (5, 0.5 μM, respectively; black curve). β-arrestin 2 recruitment to CB1 was measured by TIRF microscopy and is displayed as percent change in fluorescence intensity over time. CP55940 t1/2 = 33 second; CP55940 plus LDK1288 = 36 second.

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    Fig. 4.

    Effect of CP55940, LDK1285, LDK1288, or LDK1305 on ERK1/2 phosphorylation over time. HEK293 cells expressing CB1 were untreated (DMSO only) or treated with (A) CP55,940 (0.5 μM), (B) LDK1285 (10 μM), (C) LDK1288 (10 μM), or (D) LDK1305 (10 μM), for 2, 5, 10, 15, or 20 minutes. (E) Cells expressing CB1 and either β-arrestin 1 siRNA or control siRNA were treated with DMSO vehicle only, LDK1285, LDK1288, or LDK1305 (all at 10 μM) for 5 minutes. Cell lysates were separated by SDS-PAGE and analyzed by Western blots probed for phospho-ERK1/2 (pERK1/2). The total level of ERK1/2 was detected for comparison. Note that the two bands correspond to the predominant isoforms of ERK, ERK1 (p44) and ERK2 (p42). Cells not expressing CB1 and treated with LDK1288 showed no kinase phosphorylation relative to vehicle alone treatment (data not shown). Quantification of at least three independent experiments is displayed in the right-hand column and expressed as mean Embedded Image S.E.-fold increase above the basal level of phosphorylation.

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    Fig. 5.

    Coupling partner dependency of MEK phosphorylation induced by LDK1285, LDK1288, or LDK1305. HEK293 cells expressing CB1 and β-arrestin 1 siRNA, CB1 and β-arrestin 2 siRNA, or CB1 and control siRNA, or expressing CB1 alone and pretreated with PTX were either untreated (DMSO only) or treated with (A) LDK1285 (10 μM), (B) LDK1288 (10 μM), or (C) LDK1305 (10 μM) for 5 minutes. Cell lysates were separated by SDS-PAGE and analyzed by Western blots probed for phospho-MEK (pMEK). The total level of MEK was detected for comparison. Quantification of at least three independent experiments is displayed in the right-hand column and expressed as mean Embedded Image S.E.-fold increase above the basal level of phosphorylation.

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    Fig. 6.

    Pathway dependency of Src phosphorylation induced by LDK1285, LDK1288, or LDK1305. HEK293 cells expressing CB1 alone, CB1 and β-arrestin 1 siRNA, CB1 and β-arrestin 2 siRNA, CB1 and control siRNA were either untreated (DMSO only) or treated with (A) LDK1285 (10 μM), (B) LDK1288 (10 μM), or (C) LDK1305 (10 μM) for 5 minutes. Cell lysates were separated by SDS-PAGE and analyzed by Western blots probed for phospho-Src (pSrc). The total level of Src was detected for comparison. Quantification of at least three independent experiments is displayed in the right-hand column and expressed as mean Embedded Image S.E.-fold increase above basal level of phosphorylation.

Tables

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    TABLE 1

    Binding parameters of orthosteric ligands and allosteric modulators

    Ki, KB, and α values are shown with corresponding 95% confidence intervals in parentheses. For the first row, [3H]CP55940 and for the second row [3H]SR141716A are used as the orthosteric tracers. Three independent experiments, each done in duplicate, were used in each case.

    CP55940SR141716ALDK1285LDK1288LDK1305PSNCBAM1
    KiKiKBαKBαKBαKBα
    [3H]CP55940nMnMnM11.5nM5.3nM4.3nM2.7
    1.24ND19862.520.041.4
    (0.62–2.48)(141–277)(7.7–17.2)(41.6–94.1)(3.9–7.2)(13.5–29.5)(3.4–5.6)(18.8–91.1)(2.1–3.5)
    [3H]SR141716AND2.134730.12240.11150.31560.1
    (1.37–3.32)(160–1350)(0.0–0.3)(64.4–722)(0.0–0.3)(29.9–454)(0.1–0.4)(14.2–983)(0.0–0.4)
    • Ki, equilibrium dissociation constant of orthosteric ligand; KB, equilibrium dissociation constant of allosteric modulator; α, cooperativity factor between orthosteric and allosteric ligand binding; ND, not done.

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      β-arrestin 2 knockdown

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Molecular Pharmacology: 95 (1)
Molecular Pharmacology
Vol. 95, Issue 1
1 Jan 2019
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Research ArticleArticle

Pyrimidinyl Biphenylureas as Allosteric Modulators of CB1

Caitlin A. D. Jagla, Caitlin E. Scott, Yaliang Tang, Changjiang Qiao, Gabriel E. Mateo-Semidey, Guillermo A. Yudowski, Dai Lu and Debra A. Kendall
Molecular Pharmacology January 1, 2019, 95 (1) 1-10; DOI: https://doi.org/10.1124/mol.118.112854

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Research ArticleArticle

Pyrimidinyl Biphenylureas as Allosteric Modulators of CB1

Caitlin A. D. Jagla, Caitlin E. Scott, Yaliang Tang, Changjiang Qiao, Gabriel E. Mateo-Semidey, Guillermo A. Yudowski, Dai Lu and Debra A. Kendall
Molecular Pharmacology January 1, 2019, 95 (1) 1-10; DOI: https://doi.org/10.1124/mol.118.112854
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