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

Journal: Nature Communications

Article Title: Immune cell topography predicts response to PD-1 blockade in cutaneous T cell lymphoma

doi: 10.1038/s41467-021-26974-6

Figure Lengend Snippet: a SpatialScore schematic. The SpatialScore is calculated by taking the ratio of the physical distance between each CD4 + T cell and its nearest tumor cell (distance “right”) relative to its nearest Treg (distance “left”). [1] A lower SpatialScore (i.e., CD4 + T cells closer to tumor cells than Tregs) suggests increased T cell effector activity. [2] A higher spatial score (i.e., CD4 + T cells closer to Tregs than tumor cells) suggests increased T cell suppression. b – c SpatialScore calculated per cell for all CD4 + T cells ( b ) and PD-1 + CD4 + T cells ( c ) across patient groups (mean ± s.e.m.). P values calculated with a linear mixed-effect model taking a patient identifier as a random effect. d GZMB protein expression on PD-1 + CD4 + T cells by CODEX per tissue microarray spot (mean fluorescence intensity (arbitrary units, a.u.), red bar). P values calculated with a linear mixed-effect model with Bonferroni’s corrections for multiple comparisons. e CODEX images showing contact between a tumor cell (cross) and GZMB-expressing PD-1 + CD4 + T cell (arrow) in responder patient 13 post-treatment. Scale bars, 10 µm. f Cytotoxicity gene scores, computed on bulk RNA-seq data, per tissue microarray spot. Boxes, median ± upper and lower quartiles; whiskers, 1.5x IQR. P values calculated with a linear mixed-effect model with Bonferroni’s corrections for multiple comparisons. g – h Pre- to post-treatment changes in tumor therapy resistance gene scores, computed on bulk RNA-seq data, per patient in responders ( g ) and nonresponders ( h ). Boxes, median ± upper and lower quartiles; whiskers, 1.5x IQR. P values were calculated by two-sided Wilcoxon’s signed-rank tests. i Ki-67 + tumor cell frequencies per tissue microarray spot (mean, red bar). P values calculated with a linear mixed-effect model with Bonferroni’s corrections for multiple comparisons. j SpatialScore calculated from Vectra mIHC data per cell for all PD-1 + CD4 + T cells (mean ± s.e.m.). P values calculated with a linear mixed-effect model taking a patient identifier as a random effect. k Vectra mIHC images (left panels) and corresponding spatial plots (right panels)from responder patient 13 (R) and nonresponder patient 14 (NR) pretreatment. Scale bars, 20 µm. l SpatialScore calculated from Vectra mIHC data per patient in responders and nonresponders pretreatment(mean, red bar). P value calculated by a two-sided Wilcoxon’s rank-sum test, with no adjustments for multiple hypotheses. Source data are provided as a Source Data file.

Article Snippet: Collectively, these results show that findings identified by highly multiplexed imaging platforms like CODEX can be translated to more clinically accessible platforms like Vectra.

Techniques: Activity Assay, Expressing, Microarray, Fluorescence, RNA Sequencing

Journal: Nature Communications

Article Title: Immune cell topography predicts response to PD-1 blockade in cutaneous T cell lymphoma

doi: 10.1038/s41467-021-26974-6

Figure Lengend Snippet: a Seven genes from bulk RNAseq data predictive of the SpatialScore . b Normalized bulk CXCL13 gene expression per tissue microarray spot. Boxes, median ± upper and lower quartiles; whiskers, 1.5x IQR. P values calculated with a linear mixed-effect model with Bonferroni’s corrections for multiple comparisons. c CXCL13 protein expression by IHC per tissue microarray spot (mean, red bar). P values calculated with a linear mixed-effect model with Bonferroni’s corrections for multiple comparisons. d Representative CXCL13 IHC images from responder patient 9 (left panels) and nonresponder patient 14 (right panels). Scale bars, 20 µm. e – f CXCL13 expression in single-cell transcriptomes from CTCL skin tumors (Gaydosik et al.) . e Normalized expression of CXCL13 in single cells; excluded cells with CXCL13 log1p normalized read counts < 0.5. Boxes, median ± upper and lower quartiles; whiskers, 1.5x IQR. f Proportion of CXCL13-expressing cells per cell type. g CIBERSORTx workflow schematic. A CSx deconvolution signature matrix was generated from single-cell transcriptomes (Gaydosik et al.) (left) and applied to CTCL bulk transcriptomes obtained with laser-capture microdissection (LCM) and Smart-3Seq (right) to enumerate cell type fractions and resolve gene expression profiles. h Heatmap correlation of CSx-resolved and CODEX-identified cell type frequencies; Spearman coefficients are on the diagonal. i – j Differential expression of CSx-resolved tumor cell genes in responders ( j ) and nonresponders ( k ) pre- and post-treatment. P values calculated with a linear mixed-effect model with Benjamini-Hochberg correction (significantly different genes ( p < 0.1), red; CXCL13 highlighted yellow). k Vectra mIHC image (top left), corresponding tumor cell depiction (top right), corresponding CXCL13 IHC image (bottom left), and corresponding overlay image of CXCL13 staining and tumor cells (bottom right) in responder patient 9 post-treatment. Scale bars, 20 µm. l Normalized CSx-resolved CXCL13 expression in tumor cells per tissue microarray spot. Boxes, median ± upper and lower quartiles; whiskers, 1.5x IQR. P values calculated with a linear mixed-effect model with Bonferroni’s corrections for multiple comparisons. m – n Pre- to post-treatment changes in normalized CXCL13 gene expression from CSx-resolved tumor genes per patient in responders ( m ) and nonresponders ( n ). Boxes, median ± upper and lower quartiles; whiskers, 1.5x IQR. P values calculated by two-sided Wilcoxon’s signed-rank tests. o Correlation of CSx-resolved tumor cell CXCL13 expression and bulk CXCR5 expression per tissue microarray spot. Data evaluated with two-sided Spearman test. Source data are provided as a Source Data file.

Article Snippet: Collectively, these results show that findings identified by highly multiplexed imaging platforms like CODEX can be translated to more clinically accessible platforms like Vectra.

Techniques: Gene Expression, Microarray, Expressing, Generated, Laser Capture Microdissection, Quantitative Proteomics, Staining