Journal: Immunology and Cell Biology

Article Title: The vaccinia‐based Sementis Copenhagen Vector coronavirus disease 2019 vaccine induces broad and durable cellular and humoral immune responses

doi: 10.1111/imcb.12539

Figure Lengend Snippet: Spike‐specific antibody and SARS‐CoV‐2 neutralization responses following SCV‐S vaccination. (a) S1 and S2 subunit endpoint IgM and IgG ELISA titers determined from serum of female C57BL/6J mice ( n = 3) at the indicated times after a single vaccination with 10 7 PFU of SCV‐S, with (b) ratio of S1‐ and S2‐specific IgG2c to IgG1 endpoint ELISA titers determined 28 days after vaccination. (c) S1 IgG ELISA binding titers (left panel) and cPass neutralization titers (right panel) in outbred ARC(s) and inbred C57BL/6J female mice ( n = 5) 21 days after a single vaccination with 10 7 PFU of SCV‐S or vector control, with (d) durability of response in the outbred cohort shown by S1‐specific endpoint IgG ELISA titers (left panel) and neutralization titers (right panel) at the indicated times. (e) S1‐specific IgG ELISA (left panel) and neutralization titers (right panel) 50 days after a single‐dose (day 0) or prime‐boost (day 0 and 28) vaccination of female C57BL/6J mice ( n = 5) with 10 7 PFU SCV‐S or control vector, with (f) neutralizing activity in ACE2 and RBD blocking ELISA, and (g) neutralizing activity (IC 80 titers) against lenti‐SARS‐CoV‐2‐S pseudoviruses bearing spike protein from the Wuhan reference strain, the alpha or beta variant, shown for prime‐boost samples. (h) Neutralization titers in young (6–8 weeks old; n = 5) and middle‐aged (9–10 months old; n = 10) C57BL/6J mice at the indicated times after prime‐boost vaccination with 10 7 PFU SCV‐S. Results shown are representative of four independent experiments (indicated above) with binding and neutralizing antibody levels comparable at similar doses and time points across all experiments. Symbols represent individual mice and bars show the mean ± s.e.m. from independent experiments. Data were log transformed and statistical significance determined using Brown–Forsythe and Welch ANOVA with Dunnett T3 multiple comparison test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ACE2, angiotensin‐converting enzyme‐2; IC 80 , 80% inhibitory concentration; Ig, immunoglobulin; ns, not significant; PFU, plaque‐forming units; RBD, receptor‐binding domain; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; SCV‐S, Sementis Copenhagen Vector spike protein.

Article Snippet: ACE2‐HEK293 recombinant cell line (catalog number 79951; BPS Bioscience, San Diego, CA, USA) was maintained in Dulbecco’s Modified Eagle Medium supplemented (Sigma Aldrich, MO, USA) with 10% fetal bovine serum, 2 mM l ‐glutamine and penicillin–streptomycin.

Techniques: Neutralization, Enzyme-linked Immunosorbent Assay, Binding Assay, Plasmid Preparation, Activity Assay, Blocking Assay, Variant Assay, Transformation Assay, Concentration Assay

Journal: Molecular Pharmacology

Article Title: Identification of Positive Allosteric Modulators of the D 1 Dopamine Receptor That Act at Diverse Binding Sites

doi: 10.1124/mol.118.113175

Figure Lengend Snippet: MLS1082 and MLS6585 potentiate dopamine-induced D1R internalization. Receptor internalization was measured using the DiscoverX GPCR internalization assay as described in Materials and Methods. Cells were treated with the indicated concentrations of dopamine for 3 hours in the absence or presence of 50 μM MLS1082 (+ 50 μM 1082), or 50 μM MLS6585 (+ 50 μM 6585). Both MLS1082 and MLS6585 potentiated DA’s potency for inducing receptor internalization (EC50 [95% CI]: DA = 2.79 μM [1.4–5.6], DA + MLS1082 = 0.53 μM [0.16–1.8], P = 0.004), DA + MLS6585 = 0.46 μM [0.15–1.45], P = 0.04). Further, MLS1082 increased DA’s efficacy for internalization (P = 0.04), but MLS6585 showed no potentiation of efficacy (Emax ± S.E.M.: DA = 97.4% ± 4.7%, DA + MLS1082 = 113.5% ± 2.8%, DA + MLS6585 = 97.2% ± 3.0%). Statistical comparisons via Student’s t test, n = 5.

Article Snippet: Assays were performed on D1R-HEK293 cells stably expressing the human D1R (Codex Biosolutions, Gaithersburg, MD).

Techniques:

Journal: Molecular Pharmacology

Article Title: Identification of Positive Allosteric Modulators of the D 1 Dopamine Receptor That Act at Diverse Binding Sites

doi: 10.1124/mol.118.113175

Figure Lengend Snippet: MLS1082 and MLS6585 increase the efficacy and potency of the D1R agonist dihydrexidine. β-arrestin recruitment was measured following stimulation by the indicated concentrations of dihydrexidine in the absence or presence of 50 μM MLS1082 (+ 50 μM 1082) or MLS6585 (+ 50 μM 6585). DA was run as a control in every experiment and the data were plotted as the percentage of the maximum DA response observed. Both MLS1082 and MLS6585 increased the efficacy and potency of dihydrexidine: (EC50 [95% CI]) dihydrexidine = 73.3 nM [42.8–125.2], dihydrexidine + MLS1082 = 20.9 nM [9.7–45.5], P < 0.0001, dihydrexidine + MLS6585 = 18.6 nM [11.3–30.6], P < 0.0001; (Emax ± S.E.M.) dihydrexidine = 70.5% ± 2.5%; dihydrexidine + MLS1082 = 95.3% ± 3.8%, P < 0.001; dihydrexidine + MLS6585 = 96.9% ± 3.2%, P < 0.001. Data are displayed as a percentage of the maximum control stimulation seen with dopamine, mean ± S.E.M., and statistical comparisons are via paired two-tailed Student’s t test, n = 5.

Article Snippet: Assays were performed on D1R-HEK293 cells stably expressing the human D1R (Codex Biosolutions, Gaithersburg, MD).

Techniques: Two Tailed Test

Journal: Molecular Pharmacology

Article Title: Identification of Positive Allosteric Modulators of the D 1 Dopamine Receptor That Act at Diverse Binding Sites

doi: 10.1124/mol.118.113175

Figure Lengend Snippet: MLS1082 and MLS6585 potentiate the activity of D1R partial agonists. β-arrestin recruitment or cAMP assays were performed in concentration-response curve format using known partial agonists of the D1R in either the presence or absence of the indicated PAM compounds at 50 μM concentration. DA was run as a control in every experiment and the data were plotted as the percentage of the maximum DA response observed. (A) MLS1082 and MLS6585 increased both the efficacy and potency of the partial agonist fenoldopam. Emax ± S.E.M. (% DA response): fenoldopam = 47.7% ± 1.6%; fenoldopam + MLS1082 = 70.8% ± 2.1%, P < 0.05; fenoldopam + MLS6585 = 87.4% ± 2.7%, P < 0.0001. EC50 [95% CI]: fenoldopam = 37.9 nM [22.5–63.8]; fenoldopam + MLS1082 = 7.5 nM [4.7–11.9], P < 0.0001; fenoldopam ± MLS6585 = 8.6 nM [5.3–13.9], P = 0.0002. (B) MLS1082 and MLS6585 increased both the efficacy and potency of the partial agonist apomorphine. Emax ± S.E.M.: apomorphine = 28.6% ± 2.2%; apomorphine + MLS1082 = 54.3% ± 3.2%, P < 0.01; apomorphine + MLS6585 = 80.8% ± 2.7%, P < 0.0001. EC50 [95% CI]: apomorphine = 0.1 μM [0.04–0.26]; apomorphine + MLS1082 = 0.014 μM [0.007–0.027], P = 0.0006; apomorphine + 6585 = 0.024 μM [0.015–0.036], P = 0.001. (C) The G protein-biased agonist SKF38393 exhibited no measurable agonist activity for β-arrestin recruitment but gained efficacy upon concurrent treatment with the PAM compounds. Emax ± S.E.M.: SKF38393 + MLS1082 = 24.1% ± 1.3%; SKF38393 + MLS6585: 28.7% ± 1.3%. EC50 [95% CI]: SKF38393 + MLS1082 = 0.12 μM [0.05–0.29]; SKF38393 + MLS6585 = 0.14 μM [0.08–0.27]. (D) MLS1082 and MLS6585 potentiated the efficacy of SKF77434-stimulated cAMP accumulation. Emax ± S.E.M.: SKF77434 = 24.7% ± 2.0%; SKF77434 + MLS1082 = 56.8% ± 2.9%, P < 0.0001; SKF7743 + MLS6585 = 48.3% ± 2.6%, P < 0.01. Neither PAM affected SKF77434 potency, however. EC50 [95% CI]: SKF77437 = 0.06 μM [0.01–0.03], SKF77434 + MLS1082 = 0.03 μM [0.01–0.07], SKF77434 + MLS6585 = 0.02 μM [0.01–0.05]. Statistical comparisons were determined for Emax values using one-way ANOVA testing, and Student’s t test for potency values, n = 6–8.

Article Snippet: Assays were performed on D1R-HEK293 cells stably expressing the human D1R (Codex Biosolutions, Gaithersburg, MD).

Techniques: Activity Assay, Concentration Assay

Journal: Molecular Pharmacology

Article Title: Identification of Positive Allosteric Modulators of the D 1 Dopamine Receptor That Act at Diverse Binding Sites

doi: 10.1124/mol.118.113175

Figure Lengend Snippet: Combination experiments suggest that MLS1082 and Compound B act at the same site on D1R, which is separate from that of MLS6585. β-arrestin recruitment was measured following stimulation with dopamine in the absence (DA) or in the presence of 50 μM MLS1082 (+ 50 μM 1082), 50 μM MLS6585 (+ 50 μM 6585), 100 μM Compound B (+ 100 μM Cmpd B), or a combination of the three compounds. (A) Structure of Compound B. (B) MLS1082 and Compound B both potentiate DA’s potency (EC50 [95% CI]: DA = 4.19 μM [2.4–7.5]; DA + MLS1082 = 0.68 μM [0.36–1.3], P = 0.004; DA + Compound B = 0.56 μM [0.22–1.4], P = 0.01). Addition of MLS1082 and Compound B together caused the same level of potentiation as either compound alone (EC50 [95% CI]: DA + MLS1082 + Compound B = 0.5 μM [0.29–0.86]). (C) MLS6585 and Compound B both potentiated DA’s potency for β-arrestin recruitment (EC50 [95% CI]: DA = 4.19 μM [2.4–7.5]; DA + MLS6585 = 0.39 μM [0.27–0.54], P = 0.0002); DA + Compound B = 0.56 μM [0.22–1.4], P = 0.01). Addition of MLS6585 and Compound B together resulted in a greater potentiation of DA’s potency than either compound alone (EC50 ± S.E.M.: DA + MLS6585 + Compound B = 0.09 μM [0.02–0.46], P = 0.004). Statistical comparisons via paired two-tailed Student’s t test, n = 5.

Article Snippet: Assays were performed on D1R-HEK293 cells stably expressing the human D1R (Codex Biosolutions, Gaithersburg, MD).

Techniques: Two Tailed Test

Journal: Molecular Pharmacology

Article Title: Identification of Positive Allosteric Modulators of the D 1 Dopamine Receptor That Act at Diverse Binding Sites

doi: 10.1124/mol.118.113175

Figure Lengend Snippet: R130Q mutation abolishes MLS1082 but not MLS6585 PAM activity. Dopamine-stimulated β-arrestin recruitment and G protein (Gαs) engagement were measured using BRET assays as described in Materials and Methods. Briefly, cells were transfected with either the wild-type D1R or the R130Q mutant along with the indicated biosensor. Cells were then stimulated with the indicated concentrations of dopamine alone (DA) or in the presence of 50 μM MLS1082 (+ 50 μM 1082) or 50 μM MLS6585 (+ 50 μM 6585). (A) Both MLS1082 and MLS6585 potentiated DA’s potency for β-arrestin recruitment to the wild-type D1R (EC50 [95% CI]: DA = 1.76 μM [0.69–4.5]; DA + MLS1082 = 0.45 μM [0.2–1.0], P < 0.001; DA + MLS6585 = 0.43 μM [0.21–0.86], P < 0.001). Further, both MLS1082 and MLS6585 increased DA’s efficacy (Emax ± S.E.M.: DA = 99.9% ± 1.2%; DA + MLS1082 = 132.7% ± 2.2%; DA + MLS6585 = 114.5% ± 1.6%, P < 0.01). (B) With the mutant R130Q receptor, MLS6585, but not MLS1082, potentiated DA’s potency (EC50 [95% CI]: DA = 1.67 μM [0.79–3.5]; DA + MLS1082 = 1.23 μM [0.44–3.5]; DA + MLS6585 = 0.38 μM [0.19–0.75], P < 0.0001). Further, MLS1082 did not potentiate DA’s efficacy for activating the R130Q mutant (Emax ± S.E.M.: DA = 98.4% ± 5.2%; DA + MLS1082 = 105.4% ± 2.9%); however, MLS6585 did potentiate the Emax (DA + MLS6585 = 111.3 ± 3.1, P < 0.03). (C) With the wild-type receptor, both MLS1082 and MLS6585 enhanced DA’s potency for stimulating D1R-Gs interactions (EC50 [95% CI]: DA = 0.37 μM [0.24–0.57]; DA + MLS1082 = 0.12 μM [0.09–0.16], P = 0.0001; DA + MLS6585 (0.07 μM [0.04–0.12], P = 0.001). MLS1082 also promoted a measurable increase in DA efficacy, whereas MLS6585 did not (Emax ± S.E.M.: DA = 100.3% ± 2.1%; DA+MLS1082 = 109.4% ± 2.2%; DA+MLS6585 = 101.7% ± 3.4%). (D) With the mutant R130Q receptor, MLS6585, but not MLS1082, enhanced DA’s potency (EC50 [95% CI]: DA = 0.39 μM [0.25–0.6]; DA + MLS1082 = 0.23 μM [0.17–0.31]; DA + MLS6585 = 0.069 μM [0.05–0.11], P < 0.0001). Further, neither compound increased DA’s efficacy at the R130Q receptor (Emax ± S.E.M.: DA = 100.3% ± 49%; DA+MLS1082 = 98.2% ± 4.8%; DA+MLS6585 = 107.3% ± 5.3%). Statistical comparisons via paired two-tailed Student’s t test, and one-way ANOVA; n = 5 or 6.

Article Snippet: Assays were performed on D1R-HEK293 cells stably expressing the human D1R (Codex Biosolutions, Gaithersburg, MD).

Techniques: Mutagenesis, Activity Assay, Transfection, Two Tailed Test