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