cb1r  (Revvity)

Journal: Frontiers in Pharmacology

Article Title: Pharmacological evaluation of enantiomerically separated positive allosteric modulators of cannabinoid 1 receptor, GAT591 and GAT593

doi: 10.3389/fphar.2022.919605

Figure Lengend Snippet: Assessment of GAT1664, GAT1665. GAT1666, and GAT1667 signaling. hCB1R CHO-K1 were treated with 0.10 nM–10 µM GAT compounds ±100 nM CP55,940. (A) cAMP inhibition of GAT compounds as agonists, (B) βarrestin2 recruitment of GAT compounds as agonists, (C) cAMP inhibition of GAT compounds as PAMs in the presence of 100 nM CP55,940, and (D) βarrestin2 recruitment of GAT compounds as PAMs in the presence of 100 nM CP55,940. cAMP inhibition and βarrestin2 recruitment data are expressed as % CP55,940 max. Data were fit to a nonlinear regression (3-parameter model, GraphPad v. 9.0) to determine EC 50 and E max . Data are mean ± SEM ( E max ) or 95% CI (EC 50 ); n = 4 independent experiments performed in triplicate, except for CP55,940 where n = 12 independent experiments performed in triplicate as this compound was included in all assays as a reference compound. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to CP55,940; ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001 compared to GAT1664, # p < 0.05, ### p < 0.001 compared to GAT1666 as determined by one-way ANOVA followed by Tukey’s post-hoc test.

Article Snippet: HitHunter (cAMP) and PathHunter (βarrestin2) Chinese hamster ovary (CHO)-K1 cells stably-expressing human CB1R (hCB1R) from DiscoveRx (Eurofins, Freemont, CA) were maintained at 37°C, 5% CO 2 in DMEM/F-12 containing 10% fetal bovine serum (FBS) and 5 U × 10 4 U penicillin/streptomycin (ThermoFisher, Mississauga, ON).

Techniques: Inhibition

Journal: Frontiers in Pharmacology

Article Title: Pharmacological evaluation of enantiomerically separated positive allosteric modulators of cannabinoid 1 receptor, GAT591 and GAT593

doi: 10.3389/fphar.2022.919605

Figure Lengend Snippet: (A) Radioligand binding of 1 nM [ 3 H]CP55,940 and (B,C) G protein binding of [ 35 S]GTPγS to membranes from hCB1R CHO-K1 cells. hCB1R CHO-K1 cells were treated with 0.10 nM–10 µM compounds in the presence 1 nM [ 3 H]CP55,940 (A) or 1 nM [ 35 S]GTPγS (B) . Data are expressed as % radioligand bound (A) or % stimulation above baseline (i.e. vehicle) levels (B,C) . Data were fit to a nonlinear regression (3-parameter model, GraphPad v. 9.0) to determine EC 50 , and E max . Data are mean ± SEM ( E max ) or 95% CI (EC 50 ); n = 6 independent experiments performed in triplicate, except for CP55,940 where n = 12 independent experiments performed in triplicate as this compound was included in all assays as a reference compound. *** p < 0.001 compared to CP55,940, ^^^ p < 0.001 compared to GAT1664, $$$ p < 0.01 compared to GAT1665, ### p < 0.01 compared to GAT1666, as determined by one-way ANOVA followed by Tukey’s post-hoc test.

Article Snippet: HitHunter (cAMP) and PathHunter (βarrestin2) Chinese hamster ovary (CHO)-K1 cells stably-expressing human CB1R (hCB1R) from DiscoveRx (Eurofins, Freemont, CA) were maintained at 37°C, 5% CO 2 in DMEM/F-12 containing 10% fetal bovine serum (FBS) and 5 U × 10 4 U penicillin/streptomycin (ThermoFisher, Mississauga, ON).

Techniques: Binding Assay, Protein Binding

Journal: Frontiers in Pharmacology

Article Title: Pharmacological evaluation of enantiomerically separated positive allosteric modulators of cannabinoid 1 receptor, GAT591 and GAT593

doi: 10.3389/fphar.2022.919605

Figure Lengend Snippet: hCB1R with three different binding sites shown. In these views of hCB1R the orthosteric site is shown in magenta, an identified PAM site in blue, and another identified putative ago-PAM site in tan. Transmembrane helices are: I red, II orange, III yellow, IV light green, V green, VI cyan, and VII blue. (A) is a visualization of CB1R looking down on the extracellular surface. (B) is a visualization of CB1R looking across at the transmembrane domains.

Article Snippet: HitHunter (cAMP) and PathHunter (βarrestin2) Chinese hamster ovary (CHO)-K1 cells stably-expressing human CB1R (hCB1R) from DiscoveRx (Eurofins, Freemont, CA) were maintained at 37°C, 5% CO 2 in DMEM/F-12 containing 10% fetal bovine serum (FBS) and 5 U × 10 4 U penicillin/streptomycin (ThermoFisher, Mississauga, ON).

Techniques: Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Pharmacological evaluation of enantiomerically separated positive allosteric modulators of cannabinoid 1 receptor, GAT591 and GAT593

doi: 10.3389/fphar.2022.919605

Figure Lengend Snippet: GAT1664 and GAT1666 bind to a putative allosteric agonist site. (A) GAT1664 main interactions and the amino acid residues of the binding pocket displayed in tan in . The conformation of GAT1664 before it binds to hCB1R (B1) and then the conformation of GAT1664.after it binds to hCB1R (B2) . (C) GAT1666 main interactions and the amino acid residues of the binding pocket displayed in tan in . The conformation of GAT1666 before it binds to hCB1R (D1) and then the conformation of GAT1664 after it binds to hCB1R (D2) .

Article Snippet: HitHunter (cAMP) and PathHunter (βarrestin2) Chinese hamster ovary (CHO)-K1 cells stably-expressing human CB1R (hCB1R) from DiscoveRx (Eurofins, Freemont, CA) were maintained at 37°C, 5% CO 2 in DMEM/F-12 containing 10% fetal bovine serum (FBS) and 5 U × 10 4 U penicillin/streptomycin (ThermoFisher, Mississauga, ON).

Techniques: Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Pharmacological evaluation of enantiomerically separated positive allosteric modulators of cannabinoid 1 receptor, GAT591 and GAT593

doi: 10.3389/fphar.2022.919605

Figure Lengend Snippet: GAT1665 and GAT1667 bind to a putative PAM site. ( A ) GAT1665 main interactions and the amino acid residues of the binding pocket displayed in pink in . The conformation of GAT1665 before it binds to hCB1R (B1) and then the conformation of GAT1665 after it binds to hCB1R (B2) . (C) GAT1667 main interactions and the amino acid residues of the binding pocket displayed in pink in . The conformation of GAT1667 before it binds to hCB1R (D1) and then the conformation of GAT1667 after it binds to hCB1R (D2) .

Article Snippet: HitHunter (cAMP) and PathHunter (βarrestin2) Chinese hamster ovary (CHO)-K1 cells stably-expressing human CB1R (hCB1R) from DiscoveRx (Eurofins, Freemont, CA) were maintained at 37°C, 5% CO 2 in DMEM/F-12 containing 10% fetal bovine serum (FBS) and 5 U × 10 4 U penicillin/streptomycin (ThermoFisher, Mississauga, ON).

Techniques: Binding Assay

Journal: IBRO Neuroscience Reports

Article Title: The type 1 cannabinoid receptor positive allosteric modulators GAT591 and GAT593 reduce spike-and-wave discharges in Genetic Absence Epilepsy Rats from Strasbourg

doi: 10.1016/j.ibneur.2022.01.006

Figure Lengend Snippet: CB1R-dependent inhibition of FSK-stimulated cAMP and βarrestin2 recruitment. CHO cells stably-expressing hCB1R were treated with (A,B) 10 µM FSK and 0.10 nM – 10 μM compounds ± 10 nM CP55,940 for 90 min, and inhibition of cAMP was measured; or (C,D) 0.10 nM – 10 μM compounds ± 10 nM CP55,940 for 90 min and βarrestin2 recruitment was measured. Data are expressed as % CP55,940 response. Data were fit to a nonlinear regression (3 parameter model, GraphPad v. 9) (see ). Data are mean ± S.E.M. n ≥ 6 independent experiments performed in triplicate.

Article Snippet: Chinese hamster ovary (CHO)-K1 HitHunter (cAMP assay) or PathHunter (βarrestin2 assay) cells stably expressing human CB1R, were from DiscoveRx (Eurofins, Freemont CA) and cultured as described in earlier studies from our group ( ).

Techniques: Inhibition, Stable Transfection, Expressing

Journal: IBRO Neuroscience Reports

Article Title: The type 1 cannabinoid receptor positive allosteric modulators GAT591 and GAT593 reduce spike-and-wave discharges in Genetic Absence Epilepsy Rats from Strasbourg

doi: 10.1016/j.ibneur.2022.01.006

Figure Lengend Snippet: Modulation of CB1R by GAT591 and GAT593. CB1R-dependent inhibition of FSK-stimulated cAMP and βarrestin2 recruitment. CHO cells stably-expressing hCB1R were treated with 10 µM FSK and 0.10 nM – 10 μM compounds ± 10 nM CP55,940 for 90 min, and inhibition of cAMP was measured; or 0.10 nM – 10 μM compounds ± 10 nM CP55,940 for 90 min and βarrestin2 recruitment was measured. Data are expressed as EC 50 (nM) with 95% confidence interval (CI) or E max (% CP55,940 response) mean ± S.E.M. Data were fit to a nonlinear regression (3 parameter model, GraphPad v. 9). n ≥ 6 independent experiments performed in triplicate. Data presented in Fig. 1 .

Article Snippet: Chinese hamster ovary (CHO)-K1 HitHunter (cAMP assay) or PathHunter (βarrestin2 assay) cells stably expressing human CB1R, were from DiscoveRx (Eurofins, Freemont CA) and cultured as described in earlier studies from our group ( ).

Techniques: Inhibition, Stable Transfection

Journal: bioRxiv

Article Title: A fluorescent sensor for spatiotemporally resolved endocannabinoid dynamics in vitro and in vivo

doi: 10.1101/2020.10.08.329169

Figure Lengend Snippet: a , Schematic diagram depicting the design and principle of the GRAB eCB sensor, consisting of the CB1 receptor and circular-permutated GFP. Ligand binding activates the sensor, inducing a change in fluorescence. b , Screening and optimization steps of GRAB eCB sensors and the normalized fluorescence response to 10 μM 2-AG. eCBmut was generated by introducing the F177 2.64 A mutation in eCB2.0. c , Expression and fluorescence change in response to 100 μM 2-AG and AEA in HEK293T cells expressing eCB2.0. Scale bar, 30 μm. d , Dose-response curves measured in HEK293T cells expressing eCB2.0 or eCBmut, with the corresponding EC 50 values for 2-AG and AEA shown; n = 3 wells each. e , Normalized fluorescence change in response to the indicated compounds (each at 10 μM) measured in cells expressing eCB2.0; n = 3–4 well each. Where indicated, the CB1R inverse agonist AM251 was also added. LPA, lysophosphatidic acid; S1P, sphingosine-1-phosphate; ACh, acetylcholine; DA, dopamine; GABA, gamma-aminobutyric acid; Glu, glutamate; Gly, glycine; NE, norepinephrine; 5-HT, 5-hydroxytryptamine; His, histamine; Epi, epinephrine; Ado, adenosine; Tyr, tyramine. f , Illustration of the localized puffing system using a glass pipette containing 100 μM 2-AG and/or AM251 positioned above an eCB2.0-expressing cell. The dotted black line indicates the region of interest for line scanning. Scale bar, 30 μm. g , Change in eCB2.0 fluorescence was measured in an eCB2.0-expressing cell using line scanning; where indicated, 2-AG and AM251 were puffed on the cell. The graph at the right summarizes the on and off time constants measured upon application of 2-AG and upon application of AM251, respectively; n = 11 ( t on ) and 4 (τoff) cells. h , G protein coupling was measured using a BRET G βγ sensor in cells expressing CB1R, eCB2.0, or eCBmut. i , β-arrestin coupling was measured using the Tango assay in cells expressing CB1R, eCB2.0, or eCBmut. Student’s t tests were performed in e and h : ***p < 0.001; n.s., not significant.

Article Snippet: For the G βγ sensor assay, the human CB1R was cloned into the pCI vector (Promega), and eCB2.0 and eCBmut were cloned into the peGFP-C1 vector (Takara), replacing the eGFP open reading frame.

Techniques: Ligand Binding Assay, Fluorescence, Generated, Mutagenesis, Expressing, Transferring

Journal: bioRxiv

Article Title: A fluorescent sensor for spatiotemporally resolved endocannabinoid dynamics in vitro and in vivo

doi: 10.1101/2020.10.08.329169

Figure Lengend Snippet: a , Schematic diagram depicting the structure of the GRAB eCB2.0 sensor. The IgK leader sequence and the sequence derived from GRAB NE are shown. b , Amino acids sequence of the eCB2.0 sensor. The phenylalanine residue at position 177 2.64 in the CB1 receptor was mutated to an alanine to generate the eCB mutant sensor (indicated by the gray box). Note that the numbering used in the figure corresponds to the start of the IgK leader sequence.

Article Snippet: For the G βγ sensor assay, the human CB1R was cloned into the pCI vector (Promega), and eCB2.0 and eCBmut were cloned into the peGFP-C1 vector (Takara), replacing the eGFP open reading frame.

Techniques: Sequencing, Derivative Assay, Mutagenesis

Journal: Journal of medicinal chemistry

Article Title: Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of CB1 Receptor Positive Allosteric Modulators

doi: 10.1021/acs.jmedchem.9b01142

Figure Lengend Snippet: Structure of representative CB1 allosteric modulators

Article Snippet: Cell culture: HitHunter® (cAMP) and PathHunter® (βarrestin2) CHO-K1 cells stably-expressing human CB1R (hCB1R) from DiscoveRx® (Eurofins, Fremont, CA) were maintained between passage 5–35 at 37°C, 5% CO 2 in F-12 DMEM (Corning Cellgro, Manassas VA) containing 10% FBS and 1% penicillin-streptomycin with 800 μg/mL geneticin (HitHunter®) or 800 μg/mL geneticin and 300 μg/mL hygromycin B (PathHunter®).

Techniques:

Journal: Journal of medicinal chemistry

Article Title: Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of CB1 Receptor Positive Allosteric Modulators

doi: 10.1021/acs.jmedchem.9b01142

Figure Lengend Snippet: Effect of 6r, 9j, and 6s on the binding of a) 1 nM [3H]SR141716A or b) [3H]CP55,940 to membranes obtained from CHO cells stably-expressing human CB1R; or the binding of [35S]GTPγS to G protein in membranes obtained from CHO cells stably-expressing human CB1R and treated with c) CP55,940 and 1 μM GAT compound, or d) GAT compound alone. Ki (nM) and EC50 (nM) are mean with 95% CI. EMax data are mean ± SEM. Data were fit to a variable slope (4 parameter) nonlinear regression model, mean ± SEM, n = 4–6 independent experiments performed in duplicate. ***P < 0.001 Student’s one sample t-test.

Article Snippet: Cell culture: HitHunter® (cAMP) and PathHunter® (βarrestin2) CHO-K1 cells stably-expressing human CB1R (hCB1R) from DiscoveRx® (Eurofins, Fremont, CA) were maintained between passage 5–35 at 37°C, 5% CO 2 in F-12 DMEM (Corning Cellgro, Manassas VA) containing 10% FBS and 1% penicillin-streptomycin with 800 μg/mL geneticin (HitHunter®) or 800 μg/mL geneticin and 300 μg/mL hygromycin B (PathHunter®).

Techniques: Binding Assay, Stable Transfection, Expressing

Journal: Journal of medicinal chemistry

Article Title: Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of CB1 Receptor Positive Allosteric Modulators

doi: 10.1021/acs.jmedchem.9b01142

Figure Lengend Snippet: (a-c) CHO cells stably-expressing hCB1R were treated with 0.10 nM – 10 μM GAT compounds alone for 90 min and cAMP inhibition (a) or βarrestin2 recruitment was measured (b). Compound agonist bias between cAMP inhibition and βarrestin2 recruitment (ΔΔLogR (cAMP - βarr2)] is shown in c. (d-f) CHO cells stably-expressing hCB1R were treated with 100 nM CP55,940 + 0.10 nM – 10 μM GAT compounds for 90 min and cAMP inhibition (d) or βarrestin2 recruitment was measured (e). Compound PAM bias between cAMP inhibition and βarrestin2 recruitment (ΔΔLogR (cAMP - βarr2)] is shown in f. cAMP data are expressed as % Forskolin response within compound treatment. βarrestin2 recruitment data are expressed as % CP55,940 response. Data were fit to a nonlinear regression (4 parameter model, GraphPad v. 7) for statistics in Table 5; or fit to the operational model (eq. 1) to calculate bias (c,f). Data are mean ± S.E.M. or 95% CI (c,f), n ≥ 6 independent experiments performed in triplicate.

Article Snippet: Cell culture: HitHunter® (cAMP) and PathHunter® (βarrestin2) CHO-K1 cells stably-expressing human CB1R (hCB1R) from DiscoveRx® (Eurofins, Fremont, CA) were maintained between passage 5–35 at 37°C, 5% CO 2 in F-12 DMEM (Corning Cellgro, Manassas VA) containing 10% FBS and 1% penicillin-streptomycin with 800 μg/mL geneticin (HitHunter®) or 800 μg/mL geneticin and 300 μg/mL hygromycin B (PathHunter®).

Techniques: Stable Transfection, Expressing, Inhibition

Journal: Journal of medicinal chemistry

Article Title: Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of CB1 Receptor Positive Allosteric Modulators

doi: 10.1021/acs.jmedchem.9b01142

Figure Lengend Snippet: The CB1 positive allosteric modulators 6r and 6s suppress CFA-Induced mechanical allodynia. (A, B) Both 6r (0.1, 0.3, 1, 3, 10 and 30 mg/kg i.p.) and 6s (0.1, 0.3, 1, 3, 10 and 30 mg/kg i.p.) increase mechanical paw withdrawal thresholds in the paw ipsilateral (A) but not contralateral (B) to CFA injection. (C, D) Both 6r (30 mg/kg i.p.) and 6s (30 mg/kg i.p.) elevated mechanical paw withdrawal thresholds in the paw ipsilateral (C) but not contralateral (D) to CFA injection with efficacy observed over at least 2.5 h post injection. (E, F) Neither 6r nor 6s altered mechanical paw withdrawal thresholds in CB1 KO mice in the paw ipsilateral (E) or contralateral (F) to CFA injection.

Article Snippet: Cell culture: HitHunter® (cAMP) and PathHunter® (βarrestin2) CHO-K1 cells stably-expressing human CB1R (hCB1R) from DiscoveRx® (Eurofins, Fremont, CA) were maintained between passage 5–35 at 37°C, 5% CO 2 in F-12 DMEM (Corning Cellgro, Manassas VA) containing 10% FBS and 1% penicillin-streptomycin with 800 μg/mL geneticin (HitHunter®) or 800 μg/mL geneticin and 300 μg/mL hygromycin B (PathHunter®).

Techniques: Injection

Journal: Journal of medicinal chemistry

Article Title: Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of CB1 Receptor Positive Allosteric Modulators

doi: 10.1021/acs.jmedchem.9b01142

Figure Lengend Snippet: Effect of key CB1R ago-PAMs on catalepsy, hypothermia and tail flick test. Male C57BL/6 mice treated with 0.1 – 10 mg/kg compound or vehicle (1:1:18 ethanol:cremaphor:saline) and physiological assessments of catalepsy (5 min, % MPE 60 sec) (a), body temperature relative to vehicle (15 min, 100%) (b), and nociception in the tail flick assay (52°C) (20 min, % MPE 20 sec) (c). n = 5–6, data are mean ± SEM. *P < 0.05, **P < 0.01 6r compared to vehicle; ^P < 0.05, ^^^P < 0.001 6s compared to vehicle as determined via two-way ANOVA followed by Tukey’s post-hoc test.

Article Snippet: Cell culture: HitHunter® (cAMP) and PathHunter® (βarrestin2) CHO-K1 cells stably-expressing human CB1R (hCB1R) from DiscoveRx® (Eurofins, Fremont, CA) were maintained between passage 5–35 at 37°C, 5% CO 2 in F-12 DMEM (Corning Cellgro, Manassas VA) containing 10% FBS and 1% penicillin-streptomycin with 800 μg/mL geneticin (HitHunter®) or 800 μg/mL geneticin and 300 μg/mL hygromycin B (PathHunter®).

Techniques: Tail Flick Test, Saline

Journal: Journal of medicinal chemistry

Article Title: Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of CB1 Receptor Positive Allosteric Modulators

doi: 10.1021/acs.jmedchem.9b01142

Figure Lengend Snippet: Structure of representative CB1 allosteric modulators

Article Snippet: HitHunter® (cAMP) and PathHunter® (βarrestin2) CHO-K1 cells stably-expressing human CB1R (hCB1R) from DiscoveRx® (Eurofins, Fremont, CA) were maintained between passage 5–35 at 37°C, 5% CO 2 in F-12 DMEM (Corning Cellgro, Manassas VA) containing 10% FBS and 1% penicillin-streptomycin with 800 μg/mL geneticin (HitHunter®) or 800 μg/mL geneticin and 300 μg/mL hygromycin B (PathHunter®).

Techniques:

Journal: Journal of medicinal chemistry

Article Title: Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of CB1 Receptor Positive Allosteric Modulators

doi: 10.1021/acs.jmedchem.9b01142

Figure Lengend Snippet: Effect of 6r, 9j, and 6s on the binding of a) 1 nM [3H]SR141716A or b) [3H]CP55,940 to membranes obtained from CHO cells stably-expressing human CB1R; or the binding of [35S]GTPγS to G protein in membranes obtained from CHO cells stably-expressing human CB1R and treated with c) CP55,940 and 1 μM GAT compound, or d) GAT compound alone. Ki (nM) and EC50 (nM) are mean with 95% CI. EMax data are mean ± SEM. Data were fit to a variable slope (4 parameter) nonlinear regression model, mean ± SEM, n = 4–6 independent experiments performed in duplicate. ***P < 0.001 Student’s one sample t-test.

Article Snippet: HitHunter® (cAMP) and PathHunter® (βarrestin2) CHO-K1 cells stably-expressing human CB1R (hCB1R) from DiscoveRx® (Eurofins, Fremont, CA) were maintained between passage 5–35 at 37°C, 5% CO 2 in F-12 DMEM (Corning Cellgro, Manassas VA) containing 10% FBS and 1% penicillin-streptomycin with 800 μg/mL geneticin (HitHunter®) or 800 μg/mL geneticin and 300 μg/mL hygromycin B (PathHunter®).

Techniques: Binding Assay, Stable Transfection, Expressing

Journal: Journal of medicinal chemistry

Article Title: Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of CB1 Receptor Positive Allosteric Modulators

doi: 10.1021/acs.jmedchem.9b01142

Figure Lengend Snippet: (a-c) CHO cells stably-expressing hCB1R were treated with 0.10 nM – 10 μM GAT compounds alone for 90 min and cAMP inhibition (a) or βarrestin2 recruitment was measured (b). Compound agonist bias between cAMP inhibition and βarrestin2 recruitment (ΔΔLogR (cAMP - βarr2)] is shown in c. (d-f) CHO cells stably-expressing hCB1R were treated with 100 nM CP55,940 + 0.10 nM – 10 μM GAT compounds for 90 min and cAMP inhibition (d) or βarrestin2 recruitment was measured (e). Compound PAM bias between cAMP inhibition and βarrestin2 recruitment (ΔΔLogR (cAMP - βarr2)] is shown in f. cAMP data are expressed as % Forskolin response within compound treatment. βarrestin2 recruitment data are expressed as % CP55,940 response. Data were fit to a nonlinear regression (4 parameter model, GraphPad v. 7) for statistics in Table 5; or fit to the operational model (eq. 1) to calculate bias (c,f). Data are mean ± S.E.M. or 95% CI (c,f), n ≥ 6 independent experiments performed in triplicate.

Article Snippet: HitHunter® (cAMP) and PathHunter® (βarrestin2) CHO-K1 cells stably-expressing human CB1R (hCB1R) from DiscoveRx® (Eurofins, Fremont, CA) were maintained between passage 5–35 at 37°C, 5% CO 2 in F-12 DMEM (Corning Cellgro, Manassas VA) containing 10% FBS and 1% penicillin-streptomycin with 800 μg/mL geneticin (HitHunter®) or 800 μg/mL geneticin and 300 μg/mL hygromycin B (PathHunter®).

Techniques: Stable Transfection, Expressing, Inhibition

Journal: Journal of medicinal chemistry

Article Title: Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of CB1 Receptor Positive Allosteric Modulators

doi: 10.1021/acs.jmedchem.9b01142

Figure Lengend Snippet: The CB1 positive allosteric modulators 6r and 6s suppress CFA-Induced mechanical allodynia. (A, B) Both 6r (0.1, 0.3, 1, 3, 10 and 30 mg/kg i.p.) and 6s (0.1, 0.3, 1, 3, 10 and 30 mg/kg i.p.) increase mechanical paw withdrawal thresholds in the paw ipsilateral (A) but not contralateral (B) to CFA injection. (C, D) Both 6r (30 mg/kg i.p.) and 6s (30 mg/kg i.p.) elevated mechanical paw withdrawal thresholds in the paw ipsilateral (C) but not contralateral (D) to CFA injection with efficacy observed over at least 2.5 h post injection. (E, F) Neither 6r nor 6s altered mechanical paw withdrawal thresholds in CB1 KO mice in the paw ipsilateral (E) or contralateral (F) to CFA injection.

Article Snippet: HitHunter® (cAMP) and PathHunter® (βarrestin2) CHO-K1 cells stably-expressing human CB1R (hCB1R) from DiscoveRx® (Eurofins, Fremont, CA) were maintained between passage 5–35 at 37°C, 5% CO 2 in F-12 DMEM (Corning Cellgro, Manassas VA) containing 10% FBS and 1% penicillin-streptomycin with 800 μg/mL geneticin (HitHunter®) or 800 μg/mL geneticin and 300 μg/mL hygromycin B (PathHunter®).

Techniques: Injection

Journal: Journal of medicinal chemistry

Article Title: Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of CB1 Receptor Positive Allosteric Modulators

doi: 10.1021/acs.jmedchem.9b01142

Figure Lengend Snippet: Effect of key CB1R ago-PAMs on catalepsy, hypothermia and tail flick test. Male C57BL/6 mice treated with 0.1 – 10 mg/kg compound or vehicle (1:1:18 ethanol:cremaphor:saline) and physiological assessments of catalepsy (5 min, % MPE 60 sec) (a), body temperature relative to vehicle (15 min, 100%) (b), and nociception in the tail flick assay (52°C) (20 min, % MPE 20 sec) (c). n = 5–6, data are mean ± SEM. *P < 0.05, **P < 0.01 6r compared to vehicle; ^P < 0.05, ^^^P < 0.001 6s compared to vehicle as determined via two-way ANOVA followed by Tukey’s post-hoc test.

Article Snippet: HitHunter® (cAMP) and PathHunter® (βarrestin2) CHO-K1 cells stably-expressing human CB1R (hCB1R) from DiscoveRx® (Eurofins, Fremont, CA) were maintained between passage 5–35 at 37°C, 5% CO 2 in F-12 DMEM (Corning Cellgro, Manassas VA) containing 10% FBS and 1% penicillin-streptomycin with 800 μg/mL geneticin (HitHunter®) or 800 μg/mL geneticin and 300 μg/mL hygromycin B (PathHunter®).

Techniques: Tail Flick Test