Journal: Biomolecules

Article Title: 1-(Arylsulfonyl-isoindol-2-yl)piperazines as 5-HT 6 R Antagonists: Mechanochemical Synthesis, In Vitro Pharmacological Properties and Glioprotective Activity

doi: 10.3390/biom13010012

Figure Lengend Snippet: ( A ): Functional dose-response curve of inhibition of cAMP production at 5-HT 6 R for selected compounds 3e , 3f , and 3g in 1321N1 cells. Data were obtained from three independent experiments run in triplicate. ( B ): Impact of compounds 3e , 3f , and 3g and SB-258585 on basal cAMP production in NG108-15 cells transiently expressing 5-HT 6 R. For each compound, six independent transfection experiments were performed, and data were measured in triplicate. Data are given as means ± SEM of the values.

Article Snippet: The ability of compounds 3e , 3f , and 3g to inhibit 5-CT-induced production of cAMP was assessed using 1321N1 cells expressing the human 5-HT 6 R (PerkinElmer) using previously described procedures [ , ].

Techniques: Functional Assay, Inhibition, Expressing, Transfection

Journal: Frontiers in Immunology

Article Title: TLQP-21 is a low potency partial C3aR activator on human primary macrophages

doi: 10.3389/fimmu.2023.1086673

Figure Lengend Snippet: hTLQP-2 and mTLQP-21 activate ERK signalling in CHO-K1 and CHO-C3aR cells. hTLQP-21, mTLQP-21 and plasma-derived human C3a were tested in (A) non-transfected CHO-K1 or (B) CHO cells stably expressing human C3aR. CHO cells were serum-starved overnight and then stimulated with various ligands for 10 min before being lysed. The phospho-ERK1/2 content in the lysate was measured and expressed as fold-baseline before being combined. The maximum relative pERK1/2 activity induced by each ligand is shown in (C) . Data represent mean ± S.E.M. of triplicate measurements from 3-4 independent experiments (n = 3-4). Two-way ANOVA with Dunnett’s post hoc analysis. * P < 0.05, *** P < 0.001, **** P < 0.0001. Ligand treated versus medium treated cells for each cell line.

Article Snippet: Non-transfected CHO-K1 cells or CHO cells stably expressing the human C3aR (CHO-C3aR; Product # ES-730-C, PerkinElmer, Melbourne, Australia) were maintained in Ham’s F12 medium containing 10% foetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin, with additional 400 μg/ml G418 ( In vivo Gen, San Diego, USA) added for CHO-C3aR cell culture.

Techniques: Clinical Proteomics, Derivative Assay, Transfection, Stable Transfection, Expressing, Activity Assay

Journal: Frontiers in Immunology

Article Title: TLQP-21 is a low potency partial C3aR activator on human primary macrophages

doi: 10.3389/fimmu.2023.1086673

Figure Lengend Snippet: Summary of potencies and activities of TLQP-21 tested on CHO-C3aR, HMDM and BMDM.

Article Snippet: Non-transfected CHO-K1 cells or CHO cells stably expressing the human C3aR (CHO-C3aR; Product # ES-730-C, PerkinElmer, Melbourne, Australia) were maintained in Ham’s F12 medium containing 10% foetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin, with additional 400 μg/ml G418 ( In vivo Gen, San Diego, USA) added for CHO-C3aR cell culture.

Techniques: Activity Assay

Journal: Frontiers in Immunology

Article Title: TLQP-21 is a low potency partial C3aR activator on human primary macrophages

doi: 10.3389/fimmu.2023.1086673

Figure Lengend Snippet: TLQP-21 triggers ERK signalling through C3aR in murine bone marrow-derive macrophages. BMDMs (90,000/well) from wildtype mice (A) or with C3aR knockout mice (B) , were serum-starved overnight and then stimulated with respective ligands at the indicated concentrations for 10 min. The phospho-ERK1/2 content in the cell lysate was measured and normalised to the medium only-treated levels before being combined. Data represent mean ± S.E.M. of triplicate measurements using cells from 3 mice (n = 3).

Article Snippet: Non-transfected CHO-K1 cells or CHO cells stably expressing the human C3aR (CHO-C3aR; Product # ES-730-C, PerkinElmer, Melbourne, Australia) were maintained in Ham’s F12 medium containing 10% foetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin, with additional 400 μg/ml G418 ( In vivo Gen, San Diego, USA) added for CHO-C3aR cell culture.

Techniques: Knock-Out

Journal: Drug Design, Development and Therapy

Article Title: Preclinical pharmacodynamic and pharmacokinetic characterization of the major metabolites of cariprazine

doi: 10.2147/DDDT.S188760

Figure Lengend Snippet: In vitro receptor binding affinities of cariprazine (CAR), desmethyl-cariprazine (DCAR), and didesmethyl-cariprazine (DDCAR)

Article Snippet: The ValiScreenTM human 5-HT 2B cell line was obtained from PerkinElmer Inc. (Waltham, MA, USA; ES-314-C, lot 450-442-A).

Techniques: In Vitro, Binding Assay

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: Schemes of the workflow used in this study. (A) Main steps employed in the screening along with the number of compounds left after each step. (B) A scheme showing the detailed order of utilized techniques, especially docking to CB2 structures from PDB IDs 5ZTY and 6KPC and to the CB2 model based on MD of PDB ID 6PT0 .

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques:

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: CB2–ligand complexes. (A–C) Binding sites with ligands (green) and amino acids (gray) important for ligand binding depicted in stick representation. PDB IDs 5ZTY , 6KPC , and 6PT0 , respectively. (D–F) 2D interaction schemes generated using Schrödinger Maestro. Additionally, we marked with gray, dashed circles the amino acids that are too far away from the ligand to create protein–ligand interactions in deposited structures but probably do so alternately, for limited periods of time in natural, nonstatic complexes.

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Binding Assay, Ligand Binding Assay, Generated

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: (A) Radioligand displacement curves for two screened compounds with the lowest K i values toward human CB2—AS-5 and AS-7. WIN 55,212-2 was issued as the reference compound. Both identified CB2 ligands exhibit desired nanomolar K i and structural distinctiveness compared to the other known compounds with high affinity for CB2. (B) Inhibition of CP-55,940-stimulated [ 35 S]GTPγS at the CB2 receptor by the compounds at 10 μM. Results were expressed as mean percent of basal [ 35 S]GTPγS binding in the presence of 100 nM CP-55,940 as stimulating ligand. AM-630 served as a reference CB2 antagonist. Basal binding was set to 100% and is represented by the dotted line. Data was collected from three separate experiments and analyzed with the two-tailed t test. Statistical significance was depicted as follows: ** p < 0.01; *** p < 0.001.

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Inhibition, Binding Assay, Two Tailed Test

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: Best identified compound—AS-7 (green) docked to CB2 models based on PDB IDs 5ZTY (A), 6KPC (B) and 6PT0 MD-derived structure (C). Yellow dashed line, H-bond; teal dashed line, π–π interaction. (D) CB2–WIN 55,212-2 (magenta) complex from the largest 6PT0 MD cluster with AS-7 (green) docked to this model. The superposition shows, that despite the different chemotypes, the binding modes of both ligands exhibit similarities, mainly in the placement of the morpholine moieties and carbonyl oxygen atoms and to a lesser extent in the location of two AS-7 benzene rings in similar positions to WIN 55,212-2 central tricyclic moiety and naphthyl group.

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Derivative Assay, Binding Assay

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: AS-5 (green) docked to CB2 models based on PDB IDs 5ZTY (A) and 6PT0 MD-derived structure (B). Yellow dashed line, H-bond; teal dashed line, π–π interaction.

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Derivative Assay

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: CB2 Structures Deposited in PDB a

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Activity Assay

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: Selected Results of the K i Determination with [ 3 H]CP-55,940 Displacement Assay

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: Activity Assay

Journal: Journal of Chemical Information and Modeling

Article Title: Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation

doi: 10.1021/acs.jcim.2c01503

Figure Lengend Snippet: Docking and MM–GBSA Results for the Four Most Potent Compounds from the In Vitro Assay and Three Already Known CB2 Ligands for Comparison

Article Snippet: Ten micromolar concentrations of each compound were incubated in triplicate with membrane preparations from CHO-K1 cells expressing the human CB2 receptor (0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA) in the presence of 0.08 nM [ 35 S] guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).

Techniques: In Vitro, Comparison

Journal: Animal Cells and Systems

Article Title: Multifunctional antistress effects of standardized aqueous extracts from Hippophae rhamnoides L.

doi: 10.1080/19768354.2016.1250816

Figure Lengend Snippet: Figure 3. The effects of HR extracts on 5-HT-medicated cAMP formation in human astrocytoma 1321n1 cells, stably expressed with the human serotonin 5-HT6 receptor gene. Test extracts were treated in the presence of 100 µM 5-HT, a receptor agonist. After 15 min of treatment with the extracts, intracellular cAMP levels were measured using a ParameterTM cAMP Assay according to the manufacturer’s instructions. The data express the mean latency ± SD. Different letters above the bar indicate significant differences, as determined by ANOVA (p < .05, upper case: compared in 10 μg/mL conc., lower case: compared in 5 μg/mL conc.).

Article Snippet: All imaging data were collected and analyzed using Universal Imaging Software (West Chester, PA) (Kim et al. 2004). cAMP accumulation assay through 5-HT6 receptor Human astrocytoma 1321N1 cells that stably expressed the human serotonin 5-HT6 receptor gene (cat. ES-316CV, clone C1, Perkin-Elmer, Boston, MA, USA) were grown in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, and 0.4 mg/mL Geneticin (Calbiochem, Darmstadt, Germany) for receptor expression selection at 37°C in a 5% CO2 incubator.

Techniques: Stable Transfection, cAMP Assay