Research ArticleSIGNAL TRANSDUCTION

Drug discovery for psychiatric disorders using high-content single-cell screening of signaling network responses ex vivo

See allHide authors and affiliations

Science Advances  08 May 2019:
Vol. 5, no. 5, eaau9093
DOI: 10.1126/sciadv.aau9093
  • Fig. 1 Ex vivo CNS drug discovery pipeline.

    (A) Human primary PBMCs provide an accessible ex vivo model of physiological single-cell phenotypes in health and disease. (B) Time-course exploration of responses to 70 ligands (including CNS ligands and neuropsychiatric treatments) across 78 diverse cell signaling epitopes (5460 responses in total) in T cells from healthy control donors (n = 8) at 1, 5, 15, and 30 min ligand incubation times. (C) Identification of functional drug targets by comparing the T cell signaling response profiles of 56 ligands across 66 cell signaling epitopes (3696 responses) in PBMC samples from three clinical groups: healthy controls (n = 12), antipsychotic drug-naïve patients with SCZ (SCZ; n = 12), and the same patients following 6 weeks of clinical treatment with the atypical antipsychotic olanzapine (SCZ + AP; n = 10). (D) Modeling of disease-associated cellular responses and screening of U.S. Food and Drug Administration (FDA)–approved drugs (repurposing) and experimental neuropsychiatric compounds (n = 946 in total) in T cells from healthy control PBMC donors (n = 6 to 12) and human SH-SY5Y neuronal cells. (E) Validation of the ex vivo cellular model relative to in vivo clinical efficacy in an independent cohort of drug-naïve patients with SCZ (n = 30) treated with two of the study compounds. (F) Experimental design: PBMCs from each donor were distributed across a ligand library (A to O) and stimulated for 1 to 30 min at 37°C. Each ligand or vehicle well was stained with a unique combination of fluorescent barcoding dyes. Ligand wells were pooled and distributed across an array of antibodies (01 to 15) targeting specific phosphorylation or total protein sites on different intracellular signaling proteins. Changes in the activation status of the cell signaling proteins were analyzed at the single-cell level using flow cytometry for each donor individually. Assay plate dimensions are restricted to 15 wells for representation; full dimensions are detailed in brackets.

  • Fig. 2 Kinetic distribution of T cell signaling responses across ligand and epitope categories.

    (A) Composition of the ligand array by class and (B) the epitope array by pathway. Number of ligands and epitopes per class is given in brackets. (C) Composition of the total number of significant responses (“nodes”; permuted P < 0.05, Wilcoxon rank sum test; minimum FC 10%) for each time point by ligand class and (D) by epitope pathway. (E) Significant kinetic responses (permuted P < 0.05, Wilcoxon rank sum test; sustained in the same direction for minimum two consecutive time points) to CNS ligands and (F) neuropsychiatric treatments. Legend shows the mean FC in epitope expression across significant time points, calculated per time point as median MFI of the ligand treatment/median MFI of the vehicle treatment across PBMC donors, with labels distributed evenly across the quantile range for negative and positive FCs separately. For down-regulated epitopes, legend shows −1/FC. Compounds and epitopes in the heat maps are ordered by the number of significant nodes across the time points. Targets of the CNS ligands include phencyclidine (an N-methyl-d-aspartate receptor antagonist/D2 receptor agonist/σ receptor agonist), NECA (5’-N-ethylcarboxamidoadenosine; an adenosine A1/2A/3 receptor agonist), SR 57227 (a 5-HT3 receptor agonist), AS 19 (a 5-HT7 receptor agonist), and 7-OH-DPAT (a D3 receptor agonist). Clinical indications of the neuropsychiatric treatments include desipramine and fluoxetine (antidepressants), lithium and valproic acid (mood stabilizers), and clozapine, risperidone, aripiprazole, and haloperidol (antipsychotics). All data represent eight healthy control PBMC donors. CREB, cyclic adenosine monophosphate response element–binding protein; PDPK1, 3-phosphoinositide-dependent protein kinase-1; MEK1, MAPK kinase 1; PLC-γ2, phospholipase Cγ2; Rb, retinoblastoma protein; NF-κB, nuclear factor κB.

  • Fig. 3 Kinetic exploration of neuropsychiatric treatments and novel inhibitors of the Akt/GSK-3β pathway in T cells.

    (A) Kinetic induction of responses (left) at key Akt/GSK-3β pathway epitopes (right) for positive controls (calyculin A, PMA/ionomycin, staurosporine, and GSK 690693) as compared to neuropsychiatric treatments (fluoxetine, desipramine, risperidone, lithium, and aripiprazole) and specific novel inhibitors (rapamycin, CHIR-99021, and JB1121). Only significant responses (permuted P < 0.05, Wilcoxon rank sum test; n = 8 healthy control PBMC donors) are shown. Black dots at 30-min time points represent replication in an independent PBMC cohort (n = 12). Legend shows FC in epitope expression (calculated as median MFI of the ligand treatment/median MFI of the vehicle treatment across PBMC donors), with labels distributed evenly across the quantile range for negative and positive FCs separately. FC is converted to −1/FC for down-regulated epitopes. Proteins are colored with respect to their cellular function: blue (kinase), red (translation), and green (transcription). The position of mTORC1 is shown for mechanistic interpretation, although no epitopes were measured on this protein. (B and C) Inhibition potency and selectivity across all 70 ligands used in the time course for targets (B) GSK-3β (pS9) and (C) 4EBP1 (pT36/pT45) at 30 min. Potency reflects the percentage of inhibition of phosphorylation at target site calculated as (1 − MFI of the ligand treatment/mean MFI of the vehicle treatment) × 100%, averaged across PBMC donors (n = 8). Selectivity reflects the ratio of the percentage of inhibition of phosphorylation at target site to mean percentage of inhibition across Akt (pS473), Akt (pT308), GSK-3β (pS9), and 4EBP1 (pT36/pT45) sites, averaged across eight donors. Only ligands with >10% potency are shown.

  • Fig. 4 Target identification by functional profiling of cell signaling abnormalities in SCZ T cells.

    The drug target was selected on the basis of the normalization of aberrant cell signaling responses following in vivo pharmacological antipsychotic therapy. Shown are clinically associated T cell signaling nodes (n = 39 of 3762 total nodes screened), defined as individual ligand-epitope combinations, which displayed a significant effect (permuted P < 0.05, ANCOVA) between clinical group status and either ligand response (n = 32) or basal epitope expression (n = 7). All nodes were filtered for a minimum stain index of 2 and, for ligand responses, a significant (permuted P < 0.05, Wilcoxon rank sum test) FC of minimum 10% in at least one of the comparison groups. Nodes altered in the SCZ versus healthy control (n = 12 PBMC donors per group) or SCZ (n = 12) versus SCZ + antipsychotic (n = 10) group comparisons are shown in red and blue, respectively. Nodes altered in both comparisons in opposite directions (i.e., “reversed”) are shown in yellow. The yellow nodes represent putative normalization of SCZ cell signaling alterations following efficacious clinical treatment (fig. S12) and form the basis for drug target selection. Nodes are grouped by signaling pathway (top) and ligand class (left). Refer to tables S6 and S7 and fig. S13 for a detailed group comparison. IRS-1, insulin receptor substrate 1; WIP, Wiskott-Adrich syndrome protein (WASP)-Interacting Protein.

  • Fig. 5 Phenotypic drug repurposing based on cellular response.

    (A) Identification of functional cellular drug target. The attenuated response to thapsigargin at PLC-γ1 in T cells from drug-naïve patients with SCZ (permuted P = 0.0001, Q = 0.018, two-way ANCOVA), relative to healthy controls (CTRL), was reversed after 6 weeks of clinical treatment with the atypical antipsychotic drug olanzapine in vivo (permuted P = 0.017, Q = 0.340; n = 12 healthy controls, n = 12 drug-naïve patients with SCZ, and n = 10 patients with SCZ after antipsychotic treatment). Box plots show interquartile range with the median (horizontal line) and the minimum and maximum values (whiskers), excluding outliers (dots). (B) Results of primary drug screen. Permuted P values from thapsigargin drug interaction testing (two-way ANOVA; n = 6 to 12 healthy PBMC donors) are shown across the combined FDA-approved (n = 786) and experimental (n = 160) compound libraries. Dashed line represents threshold P value of 0.05. Significant hits are shown in green (n = 102), and compounds, which additionally showed selective potentiation of the thapsigargin/PLC-γ1 response (post hoc one-way ANOVA tests; fig. S18), are shown in magenta (n = 22; table S9). (C) Ranking of best selective potentiation candidates at 10 μM concentration in terms of half maximal effective concentration (EC50) shifts in the thapsigargin/PLC-γ1 dose-response curve. Shown are mean values from six healthy PBMC donors (points) with SEM (vertical bars) and fitted four-parameter logistic curves. The y axis represents the MFI standardized as a proportion of minimum and maximum values. Legend shows the EC50 values with 95% confidence intervals (CI). Methylpred., methylprednisolone. (D) Distribution of drug classes across repurposing stages. Extended FDA-approved library screening (left) refers to the primary compound screen at a single dose (20 μM unless otherwise specified in table S8) of compound (n = 946; B). Validation and selectivity testing (center) refer to dose-response titration (24 nM to 200 μM) and validation of selective potentiation candidates and structural class relatives (n = 24; fig. S20; n = 6 healthy PBMC donors). Potentiation testing (right) refers to the titration of thapsigargin (12.5 pM to 20 μM) in the presence of 10 μM concentration of validated compounds (n = 10; C). In vivo effect of olanzapine (A) was reproduced in vitro throughout.

  • Fig. 6 Efficacy of top SCZ drug candidates in human SH-SY5Y neuronal cells.

    (A) Response to 30-min sub-EC50 (40 nM; fig. S21B) thapsigargin stimulation at PLC-γ1 (yellow) in SH-SY5Y neuronal cells after a 45-min preincubation with the vehicle, top drug candidates (nisoldipine and nicardipine), or olanzapine (positive control) at 10 μM. Blue, 4′,6-diamidino-2-phenylindole (DAPI) (nuclei); magenta, βIII-tubulin (neuronal lineage marker). Scale bars, 100 μm; 60× magnification. Representative of three replicate experiments imaged using confocal microscopy at identical laser power and gain settings. (B) High-throughput wide-field microscopy analysis of (A) in SH-SY5Y cells positive for βIII-tubulin expression (fig. S21A). The y axis shows PLC-γ1 MFI standardized to the median control level in the vehicle condition. Boxes show median and interquartile range, whiskers extend to extreme values within 1.5 × interquartile range, and dots mark data points outside this range. Each box plot represents data from an average of 8126 cells from three replicate experiments. Top 0.5% values lie outside of the plot area and were included in statistical analysis. P values from two-way ANOVA for interaction between the drug condition and the thapsigargin/PLC-γ1 response; FC represents potentiation of the thapsigargin/PLC-γ1 response in drug relative to control conditions and is calculated as (1 − response ratio in drug condition)/(1 − response ratio in control condition). Wide-field images were acquired at the same light source and detector settings at 20× magnification.

  • Fig. 7 Correlation of ex vivo drug-target activity with in vivo efficacy.

    (A) Longitudinal clinical patient assessment of the in vivo efficacy of two antipsychotic medications (aripiprazole and risperidone), identified in the drug-screening phase (Fig. 5), on SCZ symptoms assessed using the SAPS in drug-naïve patients with SCZ (n = 12 and 18 for aripiprazole and risperidone treatments, respectively) between treatment time points at 0 weeks (w) and 12 months (m). Treatments were equally efficacious (P > 0.05, two-way ANOVA). Patients remained on the same treatment at least until the 3-month time point. (B) Correlation between the ex vivo efficacy of the two antipsychotic medications (aripiprazole and risperidone), measured as the shift in EC50 (ΔEC50) of the thapsigargin/PLC-γ1 response induced by the respective drug in PBMCs collected at baseline from individual patients with SCZ, and the in vivo efficacy in ameliorating positive symptoms (ΔSAPS) after 3 weeks of clinical treatment in the same patients. Linear regression model with 95% confidence intervals, adjusted for covariates selected in stepwise procedure using Bayesian information criterion.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/5/eaau9093/DC1

    Fig. S1. Construction of a three-dimensional fluorescent cell barcoding matrix for multiplexing of 80 cellular treatments.

    Fig. S2. Gating strategies for the functional analysis of 80 barcoded T cell populations.

    Fig. S3. MFIs and CVs across 80 barcoded T cell populations for each functional fluorescence channel.

    Fig. S4. Z factor analyses across 80 barcoded T cell populations for each functional fluorescence channel.

    Fig. S5. Reproducibility across time and independent PBMC cohorts.

    Fig. S6. Stain indices of antibody clones against T cell signaling epitopes used in the KP experiments.

    Fig. S7. Kinetic induction of cell signaling responses across the ligand and epitope array (n = 5460 nodes; i.e., ligand-epitope combinations).

    Fig. S8. Distribution of FCs for T cell signaling responses across time points.

    Fig. S9. Dynamic regulation of JAK/STAT T cell signaling across time course.

    Fig. S10. Gating strategies for the functional analysis of 64 barcoded T cell populations.

    Fig. S11. Gating strategy for cell viability and immunophenotyping.

    Fig. S12. Clinical response to antipsychotic treatment with olanzapine in patients with SCZ at 6 weeks.

    Fig. S13. Altered T cell signaling nodes (ligand-epitope combinations) in pretreatment SCZ versus control and pretreatment versus posttreatment SCZ comparisons.

    Fig. S14. Association between the drug target response to thapsigargin at PLC-γ1 in SCZ and the genome-wide significant SCZ risk SNP rs4766428 in the ATP2A2 gene.

    Fig. S15. Normal regulatory response at PLC-γ1 to calcium release from the endoplasmic reticulum and hypothetical mechanism of action in SCZ, based on the altered response to thapsigargin at PLC-γ1 in T cells from patients with SCZ.

    Fig. S16. Gating strategies for the functional analysis of PLC-γ1 expression in four barcoded T cell populations.

    Fig. S17. Thapsigargin dose response at PLC-γ1.

    Fig. S18. Selective potentiation of PLC-γ1 response in the presence of thapsigargin.

    Fig. S19. Tanimoto structural similarity clustering of calcium channel blocker, antipsychotic, corticosteroid, and antibiotic compounds used in PLC-γ1 dose-response validation and selectivity testing.

    Fig. S20. Validation and selectivity testing of calcium channel blocker, antipsychotic, corticosteroid, antibiotic, and other drug classes at PLC-γ1.

    Fig. S21. Validation of top drug candidates in the SH-SY5Y neuronal cell line.

    Fig. S22. Correlation of ex vivo drug-target activity with in vivo efficacy in the CV study.

    Fig. S23. Potentiation of thapsigargin/PLC-γ1 dose response at 30 min by top drug candidates from the screening phase at 10 μM concentration in PBMCs from drug-naïve patients with SCZ.

    Table S1. Antibodies used to detect intracellular cell signaling epitopes and PBMC subtypes.

    Table S2. Ligands used to stimulate/alter cell signaling dynamics in PBMCs.

    Table S3. Activity of ligands across the time course.

    Table S4. Activity of epitopes across the time course.

    Table S5. Demographic characteristics and matching of PBMC donors used in the TI study.

    Table S6. Altered ligand responses at T cell signaling epitopes in healthy control versus pretreatment SCZ and pretreatment versus posttreatment SCZ comparisons.

    Table S7. Altered basal expression of T cell signaling epitopes in pretreatment versus posttreatment SCZ comparison.

    Table S8. Extended FDA-approved compound library.

    Table S9. Extended FDA-approved library screening of compounds which selectively potentiate the PLC-γ1 response in the presence of 0.5 μM thapsigargin.

    Table S10. Demographic characteristics and matching of PBMC donors used in the CV study.

    Table S11. Prediction of in vivo response to treatment from ex vivo treatment activity.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Construction of a three-dimensional fluorescent cell barcoding matrix for multiplexing of 80 cellular treatments.
    • Fig. S2. Gating strategies for the functional analysis of 80 barcoded T cell populations.
    • Fig. S3. MFIs and CVs across 80 barcoded T cell populations for each functional fluorescence channel.
    • Fig. S4. Z factor analyses across 80 barcoded T cell populations for each functional fluorescence channel.
    • Fig. S5. Reproducibility across time and independent PBMC cohorts.
    • Fig. S6. Stain indices of antibody clones against T cell signaling epitopes used in the KP experiments.
    • Fig. S7. Kinetic induction of cell signaling responses across the ligand and epitope array (n = 5460 nodes; i.e., ligand-epitope combinations).
    • Fig. S8. Distribution of FCs for T cell signaling responses across time points.
    • Fig. S9. Dynamic regulation of JAK/STAT T cell signaling across time course.
    • Fig. S10. Gating strategies for the functional analysis of 64 barcoded T cell populations.
    • Fig. S11. Gating strategy for cell viability and immunophenotyping.
    • Fig. S12. Clinical response to antipsychotic treatment with olanzapine in patients with SCZ at 6 weeks.
    • Fig. S13. Altered T cell signaling nodes (ligand-epitope combinations) in pretreatment SCZ versus control and pretreatment versus posttreatment SCZ comparisons.
    • Fig. S14. Association between the drug target response to thapsigargin at PLC-γ1 in SCZ and the genome-wide significant SCZ risk SNP rs4766428 in the ATP2A2 gene.
    • Fig. S15. Normal regulatory response at PLC-γ1 to calcium release from the endoplasmic reticulum and hypothetical mechanism of action in SCZ, based on the altered response to thapsigargin at PLC-γ1 in T cells from patients with SCZ.
    • Fig. S16. Gating strategies for the functional analysis of PLC-γ1 expression in four barcoded T cell populations.
    • Fig. S17. Thapsigargin dose response at PLC-γ1.
    • Fig. S18. Selective potentiation of PLC-γ1 response in the presence of thapsigargin.
    • Fig. S19. Tanimoto structural similarity clustering of calcium channel blocker, antipsychotic, corticosteroid, and antibiotic compounds used in PLC-γ1 dose-response validation and selectivity testing.
    • Fig. S20. Validation and selectivity testing of calcium channel blocker, antipsychotic, corticosteroid, antibiotic, and other drug classes at PLC-γ1.
    • Fig. S21. Validation of top drug candidates in the SH-SY5Y neuronal cell line.
    • Fig. S22. Correlation of ex vivo drug-target activity with in vivo efficacy in the CV study.
    • Fig. S23. Potentiation of thapsigargin/PLC-γ1 dose response at 30 min by top drug candidates from the screening phase at 10 μM concentration in PBMCs from drug-naïve patients with SCZ.
    • Table S1. Antibodies used to detect intracellular cell signaling epitopes and PBMC subtypes.
    • Table S2. Ligands used to stimulate/alter cell signaling dynamics in PBMCs.
    • Table S3. Activity of ligands across the time course.
    • Table S4. Activity of epitopes across the time course.
    • Table S5. Demographic characteristics and matching of PBMC donors used in the TI study.
    • Table S6. Altered ligand responses at T cell signaling epitopes in healthy control versus pretreatment SCZ and pretreatment versus posttreatment SCZ comparisons.
    • Table S7. Altered basal expression of T cell signaling epitopes in pretreatment versus posttreatment SCZ comparison.
    • Table S8. Extended FDA-approved compound library.
    • Table S9. Extended FDA-approved library screening of compounds which selectively potentiate the PLC-γ1 response in the presence of 0.5 μM thapsigargin.
    • Table S10. Demographic characteristics and matching of PBMC donors used in the CV study.
    • Table S11. Prediction of in vivo response to treatment from ex vivo treatment activity.

    Download PDF

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article