Journal: eLife

Article Title: Coalescing beneficial host and deleterious antiparasitic actions as an antischistosomal strategy

doi: 10.7554/eLife.35755

Figure Lengend Snippet: ( A ) Confocal images showing cell surface expression in mammalian HEK293 cells of 5-HT receptors cloned from S. mansoni ( Sm .5HTR L ), S. haematobium ( Sh. 5HTR) and S. japonicum ( Sj .5HTR) localized by COOH-terminally tagged eGFP. Scalebar, 50 µm. ( B ) Schematic of luminescent cAMP sensor bioassay. cAMP generated by schistosome 5-HTRs (blue) binds the engineered GloSensor luciferase, switching the sensor to a more active conformation resulting in enhanced luminescence output. ( C ) Kinetics of signal following the addition of 5-HT (1 µM) to HEK293 cells co-transfected with luminescent cAMP sensor and individual schistosome 5-HT receptors. Open circles, cells not transfected with 5-HT receptor, colored circles represent measurements in cells transfected with Sm .5HTR L (black), ( Sh. 5HTR (blue) and Sj .5HTR (red). ( D ) Serotonin dose-response curves for each of the three receptors (colored circles) and HEK293 cells expressing the cAMP sensor alone (open circles). Data reflect mean ± standard error of at least 3 biological replicates.

Article Snippet: Commercial assay or kit , GloSensor 22F cAMP sensor , Company , Promega; E2301 , .

Techniques: Expressing, Clone Assay, Bioassay, Generated, Luciferase, Transfection

Journal: eLife

Article Title: Coalescing beneficial host and deleterious antiparasitic actions as an antischistosomal strategy

doi: 10.7554/eLife.35755

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , GloSensor 22F cAMP sensor , Company , Promega; E2301 , .

Techniques: Recombinant, Software

Journal: Molecular Pharmacology

Article Title: Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics

doi: 10.1124/mol.116.105551

Figure Lengend Snippet: Summary data for A2A agonist pharmacology, physicochemical properties, and antagonist reversal rate pEC 50 is derived from concentration-response curves using the GloSensor cAMP assay (Sample curves in Supplemental Fig. 2 ) measured at 20°C for 100 minutes. LogD 7.4 and log K IAM are experimentally measured physicochemical properties used as indicators of, respectively, lipophilicity and membrane partitioning. Antagonist reversal t 1/2 to estimated agonist off-rate was derived from normalized antagonist reversal curves (one-phase exponential decay) shown in Fig. 3 . Curves were constrained to give a minimal response of “0” as incomplete reversal observed over the time-course. Accurate reversal t 1/2 values were difficult to derive for THESE data, but we can say with confidence that t 1/2 > 100 minutes. pEC 50 values and reversal half-life estimates are an average of 3 or 4 experiments performed in triplicate.

Article Snippet: GloSensor 22F (Promega) was used to monitor real-time cAMP responses.

Techniques: Derivative Assay, Concentration Assay, cAMP Assay

Journal: Molecular Pharmacology

Article Title: Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics

doi: 10.1124/mol.116.105551

Figure Lengend Snippet: Sustained wash-resistant A2A agonist response measured by physically isolating agonist pre-treated cells from the original treatment vessel. (A) Cells were pre-treated with 20× EC 90 concentrations of each agonist for 2 hours before washout and incubation in drug-free buffer for 1 hour (37°C) and measurement of cAMP capture by subsequent inclusion of rolipram. Sustained cAMP responses are normalized to acute responses (20× EC 90 of 3cd) measured in parallel in the same population of pre-treated cells. * P < 0.05, ** P < 0.01; *** P < 0.001 determined by ANOVA with Dunett’s post-test compared with vehicle control pretreated cells. (B) Concentration-responses analysis of postwash residual effects of 3cd (blue circles) and 3ch (red squares). Cells were pretreated with the indicated concentrations of agonist before residual cAMP response measured after washout. Responses are normalized to acute responses by direct application of 3cd. For direct comparison, the acute pharmacological responses of 3cd (light blue triangles) and 3ch (light red triangles) in the GloSensor cAMP assay are shown alongside. (C and D) Time course of sustained responses after pretreatment with EC 90 concentrations of 3cd (C) or 3ch (D) after washout, with (red circles) or without (blue triangles) subsequent inclusion of antagonist (100 nM ZM-241385) for the indicated time. Responses are normalized to responses measured at 1 hour postwash. Data represent an average ± S.E.M. of 3–5 experiments performed in triplicate.

Article Snippet: GloSensor 22F (Promega) was used to monitor real-time cAMP responses.

Techniques: Incubation, Concentration Assay, cAMP Assay

Journal: Molecular Pharmacology

Article Title: Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics

doi: 10.1124/mol.116.105551

Figure Lengend Snippet: Explanation and demonstration of the GloSensor reversal assay used to estimate agonist dissociation rate. (A) Schematic cartoon describing the system. Cells are first incubated with agonist (blue circles) to permit assumed equilibrium binding to receptors (red circles), which generates a relatively stable cAMP response. Excess antagonist (green circles) is then directly added to the cells. Upon dissociation of the agonist from the receptor, antagonist then occupies the binding site and prevents further activation by the agonist. This causes cessation of cAMP responses, at a rate that is dependent upon agonist dissociation kinetics. (B and C) Sample GloSensor reversal traces for 1aa (B) and 3cd (C) demonstrate that reversal rate is agonist dependent. Upon incubation of agonist (EC 90 ) and generation of relatively stable cAMP responses for 100 minutes, addition of vehicle control (blue triangles) has little effect on responses for a further 200 minutes. However, addition of antagonist at 100 minutes (red circles; 100 nM ZM-241385) causes a complete reversal of cAMP response to basal levels. 1aa reversal is faster, consistent with a more rapid off-rate compared with 3cd. Data represent an average of three experiments performed in triplicate.

Article Snippet: GloSensor 22F (Promega) was used to monitor real-time cAMP responses.

Techniques: Incubation, Binding Assay, Activation Assay

Journal: Molecular Pharmacology

Article Title: Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics

doi: 10.1124/mol.116.105551

Figure Lengend Snippet: Normalized GloSensor reversal traces for a sample of eighteen A2A agonists at 20°C. (A–R) Stable responses were generated to each agonist applied at an EC 90 concentration (100 minutes, 20°C) before antagonist (100 nM ZM-241385) or vehicle was applied for 200 minutes (20°C). Data points represent antagonist-treated responses expressed as a percentage of vehicle control for each time point to display reversal rate traces over the postantagonist period. Best-fit lines represent nonlinear regression one-phase exponential decay from the average ± S.E.M. of three experiments performed in triplicate. For example, plots (C) and (D) represent normalized data from and , respectively.

Article Snippet: GloSensor 22F (Promega) was used to monitor real-time cAMP responses.

Techniques: Generated, Concentration Assay

Journal: Molecular Pharmacology

Article Title: Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics

doi: 10.1124/mol.116.105551

Figure Lengend Snippet: Correlation of sustained wash-resistant agonist responses and the reversal rates of agonists in the GloSensor assay. Linear regression plot of log converted reversal t 1/2 values (from ) against sustained responses (from above). Compound 3ce is represented as a red circle, to highlight that is an outlier. Data from is plotted on the x -axis and data from is plotted on the y - axis.

Article Snippet: GloSensor 22F (Promega) was used to monitor real-time cAMP responses.

Techniques:

Journal: International Journal of Neuropsychopharmacology

Article Title: In Vitro Comparison of Ulotaront (SEP-363856) and Ralmitaront (RO6889450): Two TAAR1 Agonist Candidate Antipsychotics

doi: 10.1093/ijnp/pyad049

Figure Lengend Snippet: Functional evaluation of ralmitaront at trace amine-associated receptor-1 (TAAR1). (A) Chemical structures of the TAAR1 agonists ulotaront and ralmitaront. (B) Comparison of the recruitment of LgBiT-miniGα s to 3xHA-9β2-TAAR1-SmBiT by p-tyramine, ulotaront, and ralmitaront in the NanoBiT assay. Each data point represents mean ± SEM from 3 separate experiments, each using 2 replicate wells for each agonist concentration. Data shown are from 15 minutes after agonist addition. (C) Time dependence of pEC 50 s in the TAAR1 NanoBiT assay. (D) Stimulation of cAMP production measured as light intensity change in HEK293T cells expressing 3xHA-9β2-TAAR1 and GloSensor 22F at different time points following agonist addition. Each data point represents mean ± SEM from 3 separate experiments, each using 8 replicate wells for each agonist concentration. (E) Time dependence of pEC 50 s in the TAAR1 cAMP assay. (F) Activation of G protein–coupled inward-rectifying potassium channels upon ulotaront and ralmitaront application to Xenopus oocytes expressing G protein-coupled inward rectifier potassium (GIRK) 1/4 channels, 3xHA-9β2-TAAR1, and G s . Current amplitudes at 1 minute following application of each agonist concentration were normalized, within cells, to the response to 100 µM p-tyramine. Each data point represents mean ± SEM from 4 to 8 separate oocytes. (G) Activation rates of TAAR1-mediated GIRK responses elicited by ralmitaront (slope 0.006 ± 0.001 s −1 µM −1 ; y-intercept 0.019 ± 0.007 s −1 , n = 3–7 oocytes, concentrations tested: 1 µM, 3 µM, 6.5 µM, 10 µM) and ulotaront (slope 0.163 ± 0.022 s −1 µM −1 ; y-intercept 0.035 ± 0.008 s −1 , n = 3–6 oocytes, concentrations tested: 100 nM, 300 nM, 650 nM). (H) Response decay time courses following washout of 3 µM ralmitaront (n = 4) or 300 nM of ulotaront (n = 5). The TAAR1 agonists were applied for 15 seconds, and the washout duration was 260 seconds. Data are presented as mean ± SEM.

Article Snippet: Twenty-four hours after transfection, cells were lifted off, pelleted, and resuspended in Hank’s buffered salt solution supplemented with 300 µM 3-isobutyl-1-methylxanthine (Sigma-Aldrich) and GloSensor reagent (Promega).

Techniques: Functional Assay, Comparison, Concentration Assay, Expressing, cAMP Assay, Activation Assay

Journal: International Journal of Neuropsychopharmacology

Article Title: In Vitro Comparison of Ulotaront (SEP-363856) and Ralmitaront (RO6889450): Two TAAR1 Agonist Candidate Antipsychotics

doi: 10.1093/ijnp/pyad049

Figure Lengend Snippet: Ralmitaront lacks detectable activity at the serotonin 5-HT 1A receptor (5-HT 1A R) and the dopamine D 2 receptor (D 2 R). (A) Comparison of the recruitment of LgBiT-miniGα i to 5-HT 1A R-SmBiT by 5-HT, ulotaront, and ralmitaront in the NanoBiT assay. Each data point represents mean ± SEM from 3 separate experiments, each using 2 replicate wells for each agonist concentration. (B) Inhibition of cAMP production, measured as light intensity change, in HEK293T cells expressing the 5-HT 1A R and GloSensor 22F at 40 minutes following agonist addition. Each data point represents mean ± SEM from 3 separate experiments, each using 8 replicate wells for each agonist concentration. (C) Average traces representing mean ± SEM showing responses to application of 30 µM ulotaront (n = 5), 30 µM ralmitaront (n = 6), and 100 nM 5-HT (n = 11) in Xenopus oocytes coexpressing the 5-HT 1A R with G protein-coupled inward rectifier potassium (GIRK) 1/4 channels. (D) Comparison of the recruitment of LgBiT-miniGα i to D 2 R-SmBiT by quinpirole, ulotaront, and ralmitaront in the NanoBiT assay. Each data point represents mean ± SEM from 3 separate experiments, each using 2 replicate wells for each agonist concentration. (E) Inhibition of cAMP production, measured as light intensity change, in HEK293T cells expressing the D 2 R and GloSensor 22F at 40 minutes following agonist addition. Each data point represents mean ± SEM of data from 3 independent experiments, each using 8 replicate wells for each agonist concentration. (F) Average traces representing mean ± SEM showing responses to applications of 30 µM ulotaront (n = 4), 30 µM ralmitaront (n = 6), and 1 µM dopamine (n = 10), respectively, to Xenopus oocytes co-expressing the D 2 R with GIRK1/4 channels.

Article Snippet: Twenty-four hours after transfection, cells were lifted off, pelleted, and resuspended in Hank’s buffered salt solution supplemented with 300 µM 3-isobutyl-1-methylxanthine (Sigma-Aldrich) and GloSensor reagent (Promega).

Techniques: Activity Assay, Comparison, Concentration Assay, Inhibition, Expressing

Journal: ACS Chemical Biology

Article Title: Conformation Guides Molecular Efficacy in Docking Screens of Activated β-2 Adrenergic G Protein Coupled Receptor

doi: 10.1021/cb400103f

Figure Lengend Snippet: Functional assays for β2AR agonists. Six compounds considerably increased cAMP formation and β-arrestin recruitment, consistent with agonism (compounds 1, 4, 10, 12, 14, and 22 as indicated colors). (A) Dose–respone curves measuring G-protein activation through cAMP formation using the GloSensor assay. (B) Known β2AR agonists used as controls in the GloSensor assay: isoproterenol (ISO, black), epinephrine (Epi, green), hydroxybenzylisoproterenol (HBI, blue), and BI-167107 (BI, red). (C) Dose–response curves measuring β-arrestin recruitment using the β2 V2R Tango assay. For compound 4, a connected line of each data point is presented instead of its dose–response curve since its fitting was not converged. (D) The control β2AR agonists in the Tango assay as described for panel B. Each data point represents mean ± SE obtained from three independent experiments done in duplicates. Dose–response curves for each compound were obtained using the nonlinear iterative curve-fitting computer program Prism.

Article Snippet: Bright-Glo and Glosensor reagents were obtained from Promega (Madison, WI).

Techniques: Functional Assay, Activation Assay, Control

Journal: ACS Chemical Biology

Article Title: Conformation Guides Molecular Efficacy in Docking Screens of Activated β-2 Adrenergic G Protein Coupled Receptor

doi: 10.1021/cb400103f

Figure Lengend Snippet: Functional assays for DRD2 agonists and inverse-agonists. (A) Two compounds (29 and 33, green and blue squares, respectively) activated Gi in GloSensor assays, consistent with partial agonism. QUI is the known agonist quinpirole (black circle). (B) The GloSensor assay was run in inverse-agonist mode with addition of 100 nM quinpirole. One compound (38, red square) inhibited Gi activation. HAL is the known inhibitor haloperidol (black diamond).

Article Snippet: Bright-Glo and Glosensor reagents were obtained from Promega (Madison, WI).

Techniques: Functional Assay, Activation Assay