In isolated peritoneal macrophages, 4 μM SR144528 suppressed cholesterol esterification in both wild-type and CB2-null cells, including under acetylated-LDL stimulation. This endpoint therefore cannot be assigned solely to CB2 antagonism. [3]
Human CB2 versus CB1 receptor binding
Biochemical assay · Membranes from CHO cells expressing human CB2 or human CB1 [1]
SR 144528 displaced [3H]-CP 55,940 with substantially higher affinity at human CB2 than CB1. Saturation binding supported competitive interaction: increased radioligand Kd without a significant change in Bmax.
Literature-Reported Treatment Conditions
- Treatment concentration
- Increasing concentrations in competition assays; 1, 3 and 10 nM in Table1 saturation assay
- Exposure duration
- 1 h at 30°C for binding
- Method
- 0.2 nM [3H]-CP 55,940 for competition; nonspecific binding defined with 1 μM unlabeled CP 55,940
- Vehicle
- DMSO, final concentration no greater than 0.1% (v/v); reported without effect on assays
- Co-treatment
- Radioligand binding experiment; no therapeutic combination
- Reported limits
- Ki uncertainty type is not explicitly specified in the Results; no conversion to a treatment concentration.
Methods and source locations
Compound reported in the paper: SR 144528
Published research on the named compound; no validation of the current Adooq lot, purity or formulation.
pp.645–647, Materials, Binding experiments, Results interaction with peripheral cannabinoid receptors; Figs.1–3 and Table1
Competition with [3H]-CP 55,940 at human CB2: Radioligand competition binding; Results do not explicitly identify the uncertainty statistic for this Ki. — p.646 Results and Fig.2B; p.645 Binding experiments
Competition with [3H]-CP 55,940 at human CB1: Radioligand competition binding; uncertainty statistic not explicitly identified for this Ki. — p.646 Results and Fig.2B; p.645 Binding experiments
Effect of SR 144528 on radioligand saturation binding at human CB2: Table1 reports means ± SEM from three experiments performed in duplicate. Fig.3 caption describes one of two experiments, a replication-count inconsistency retained privately. — pp.646–647 Fig.3, Table1 and associated Results
Inverse agonism in native spleen membranes
Biochemical assay · Mouse, rat and human spleen membrane preparations [2]
SR 144528 reduced basal [35S]GTPγS binding in spleen membranes from all three species, consistent with inverse agonism in native tissue. Potencies are functional assay pEC50 values, not binding affinities.
Literature-Reported Treatment Conditions
- Treatment concentration
- Concentration-response testing, approximately 0.1 nM–1 μM as plotted in Fig.6A–C
- Exposure duration
- 60 min for rodent membranes; 30 min for human membranes, at 30°C
- Method
- [35S]GTPγS binding, 0.1 nM tracer; GDP 30 μM in rodents and 10 μM in human spleen
- Vehicle
- 1 μL DMSO per 500 μL assay; DMSO-only membrane controls
- Co-treatment
- No co-administered agonist for inverse response; response plotted relative to CP 55,940 maximum
- Reported limits
- Table1 n is not a specified count of independent donors. Assay signal was low in native tissue.
Methods and source locations
Compound reported in the paper: SR 144528
Published research on the named compound; no validation of the current Adooq lot, purity or formulation.
Introduction identity paragraph; Materials and methods (spleen tissue, membrane preparation, GTPγS assay, Materials); inverse-agonist Results, Table1 and Fig.6A–C
Functional inhibition of basal [35S]GTPγS binding in mouse spleen membranes: Table1 reports pEC50 ± SEM; n is retained as tabulated and is not treated as a donor or animal count. — Table1, SR 144528 row, Mouse columns; Fig.6A
Functional inhibition of basal [35S]GTPγS binding in rat spleen membranes: Table1 reports pEC50 ± SEM; no conversion to a dose or affinity. — Table1, SR 144528 row, Rat columns; Fig.6B
Functional inhibition of basal [35S]GTPγS binding in human spleen membranes: Table1 reports pEC50 ± SEM. Human spleen was surgical excess tissue from males; n does not establish 12 donors. — Table1, SR 144528 row, Human columns; Fig.6C
Cholesterol esterification in wild-type and CB2-null macrophages
Cell-based study · Resident peritoneal macrophages isolated from CB2+/+ and CB2−/− mice [3]
At 4 μM, SR144528 suppressed cholesterol esterification in macrophages of both genotypes, in the presence and absence of acetylated LDL. This provides evidence for a CB2-independent effect on this endpoint.
Literature-Reported Treatment Conditions
- Treatment concentration
- 4 μM SR144528
- Exposure duration
- 1 h pretreatment, followed by 6 h with tracer ± acLDL
- Method
- Neutral-lipid synthesis assay using [3H]oleate (1 μCi/ml), with or without acLDL (100 μg/ml)
- Vehicle
- Vehicle-matched controls; identity not explicitly assigned in the Neutral Lipid Synthesis subsection
- Co-treatment
- acLDL stimulation where indicated; AM-251 tested separately as comparator
- Reported limits
- No lot, salt, purity, donor-mouse count or separate stereochemical certificate reported. Vehicle identity is not assigned by borrowing the apoptosis-assay stock description.
Methods and source locations
Compound reported in the paper: SR 144528
Published research on the named compound; no validation of the current Adooq lot, purity or formulation.
Materials and methods: Cell lines and reagents, Mice, Neutral Lipid Synthesis; Fig.3B/caption and associated Results
Cholesteryl [3H]oleate formation in wild-type and CB2-null macrophages: [3H]oleate incorporation, TLC and scintillation counting; two independent experiments, each with triplicate samples. Donor-mouse count not specified in the acquired account. No percentages estimated from bars. — Fig.3B/caption; Results paragraph comparing CB2+/+ and CB2−/− cells
Microsomal ACAT inhibition
Biochemical assay · Purchased mouse liver microsomes [3]
SR144528 inhibited cholesteryl ester formation in a cell-free microsomal ACAT assay. The result supports an off-target biochemical activity that limits interpretation of micromolar cellular effects.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0–20 μM per assay Methods and Fig.3C caption; 10 μM endpoint explicitly reported
- Exposure duration
- 30 min preincubation with inhibitor, then 5 min with substrate at 37°C
- Method
- Mouse liver microsomes; cholesteryl [14C]oleate formation from [14C]oleoyl-CoA
- Vehicle
- Inhibitors added in 10 μL methanol solution to a 200 μL assay; no-inhibitor reference
- Co-treatment
- AM-251 and 58–035 tested separately, not combined with SR144528
- Reported limits
- Microsomal ACAT isoform not identified; no purified-enzyme binding study. Fig.3C extends beyond the stated 20 μM range; no additional high-concentration doses inferred.
Methods and source locations
Compound reported in the paper: SR 144528
Published research on the named compound; no validation of the current Adooq lot, purity or formulation.
Materials and methods: In Vitro ACAT Assay; Fig.3C/caption and associated Results
Inhibition of microsomal cholesteryl [14C]oleate formation: [14C]oleoyl-CoA assay with TLC and scintillation counting. Fig.3C describes mean activity ± SD for triplicate samples; Results do not separately define the uncertainty of fitted IC50. — Fig.3C/caption; associated Results paragraph reporting microsomal IC50
Microsomal ACAT activity at 10 μM SR144528: Approximate inhibition explicitly reported in Results, not reconstructed from curve. 58–035 served as an ACAT-inhibitor comparator. — Results paragraph immediately following Fig.3C description
