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Evaluating biological activity of compounds by transcription factor activity profiling

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Science Advances  26 Sep 2018:
Vol. 4, no. 9, eaar4666
DOI: 10.1126/sciadv.aar4666
  • Fig. 1 The FACTORIAL assay enables profiling TF responses to a chemical.

    (A) Flowchart of the FACTORIAL assay. The assay cells were transiently transfected with a mix of 47 TF-specific RTUs. The RTUs contained a restriction tag (the Hpa I site) placed at different positions within the reporter sequences. The total RNA was amplified by RT-PCR, using a common pair of primers. The PCR products were labeled with a fluorescent label, digested by the Hpa I enzyme, and resolved by CE. The CE fluorescence profile reflected the activity of the TFs. The differential TFAP for a chemical shows changes in TF activity in the chemical-treated versus vehicle-treated cells. By this definition, the basal TFAP (in vehicle-treated cells) is a circle with R = 1.0. (B) TF endpoints of the FACTORIAL assay.

  • Fig. 2 The invariant TFAP signatures for inhibitors of mitochondria function and proteasomal degradation.

    Assay cells (HepG2) were incubated for 24 hours with inhibitors of the mETC or the UPP. Each TFAP signature represents the average data of three independent FACTORIAL assays. The consensus TFAPs of mETC and UPP inhibitors were calculated by clustering the TFAPs of individual perturbagens, as described in Materials and Methods. (A) Representative TFAP signatures of mETC inhibitors. (B) Representative TFAP signatures of UPP inhibitors. Tables A and B show the similarity values r for the TFAPs of the individual perturbagens versus the consensus TFAPs, calculated as a Pearson correlation coefficient. The radial graphs show TFAPs of perturbagens overlaying the consensus TFAPs. Note that the values of TF changes are plotted in a log scale. (C) TFAP endpoints.

  • Fig. 3 The invariant TFAP signatures for HDAC inhibitors and cytoskeleton disruptors.

    Assay cells (HepG2) were incubated with HDAC inhibitors or MTDs for 24 hours. Each TFAP represents the average data of three independent FACTORIAL assays. The consensus TFAPs were calculated by clustering those of individual perturbagens, as described in Materials and Methods. (A) Representative TFAP signatures of HDAC inhibitors. (B) Representative TFAP signatures of MTDs. Tables A and B show the similarity values r for the TFAPs of perturbagens at indicated concentrations versus the consensus TFAPs, calculated as a Pearson correlation coefficient r. Note that the values of TF changes are plotted in a log scale. The radial graphs show TFAPs of perturbagens overlaying the consensus TFAPs. (C) TFAP endpoints.

  • Fig. 4 Low and high doses of DNA-damaging agents produce distinct TFAP signatures.

    Assay cells (HepG2) were irradiated by a UV source or treated with the indicated chemicals and harvested at 24 hours after the treatments. Each TFAP represents the average data of three independent FACTORIAL assays. The clustering of TFAPs revealed two clusters for the treatments inducing weak and strong DNA damage. (A) The table shows the similarity values r for the TFAPs of perturbagens at the indicated concentrations versus the TFAPs of the consensus clusters, calculated as a Pearson correlation coefficient. (B and C) The radial graphs show representative TFAPs of perturbagens overlaying the consensus TFAPs. Note that the values of TF changes are plotted in a log scale. (D) TFAP endpoints.

  • Fig. 5 The invariant TFAP signatures enable the identification of compounds with specified bioactivities.

    (A) Comparing the TFAP-based prediction of mitochondria inhibitors with the data by a functional assay. Top: A total of 2793 ToxCast chemicals was assessed by MMP and FACTORIAL assays. Five hundred eighteen chemicals were scored as mitochondria inhibitors by the MMP assay. The data set of TFAP signatures was queried by the consensus mETC TFAP. The middle panel shows the recall of MMP-positive chemicals (circles) and the concordance rates (bars) for the two assays. The concordance rate is the fraction of MMP positives among the retrieved chemicals that had TFAP similarity values r within the indicated intervals (r*1rr*2). The cumulative recall curve shows the percentage of 518 MMP positives that were retrieved at different thresholds (rr*). Bottom: The scaled Venn diagram illustrates the relationship between the two assays. The left area represents 518 MMP-positive chemicals. The right area represents the 199 chemicals with TFAP similarity values r ≥ 0.800 (for the list of retrieved chemicals, see table S1). The intersection area represents the 118 retrieved chemicals that were MMP-positive [concordance of ~59% (118 of 199); recall of ~23% (118 of 518)]. The striped area represents the 25 mETC inhibitors known by the literature that were scored negative by the MMP assay but positive by the FACTORIAL assay (see also fig. S7). (B) Querying the TFAP data set by the consensus TFAPs of mETC, UPP, and HDAC inhibitors resulted in retrieved compounds with corresponding bioactivities. Graphs show the similarity of the retrieved and consensus signatures.

  • Fig. 6 Assessing the on-target and off-target activities of polypharmacological drugs by TF activity profiling.

    The TFAPs for the glitazones in HepG2 cells (a 24-hour treatment). Each TFAP represents an average of three signatures by independent FACTORIAL assays. Representative data of the three experiments are shown. (A) The TFAP signature transition with increased concentration indicates an influence of off-target drug activities. (B) The TFAP signatures enable the identification of the on-target and off-target activities of glitazones. Left: The identical TFAPs for low-concentration glitazones reflect on-target activity (PPAR activation). Middle: Identical secondary TFAPs for pioglitazone and troglitazone at higher concentrations indicate mitochondria malfunction. Right: At the highest concentration (60 μM), the troglitazone TFAP is identical to that of hydrogen peroxide, indicating oxidative stress. (C) Assessing dose response of the PPAR RTU to glitazones to determine the AC50 values for the on-target activity. Each data point is an average of three independent measurements.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/9/eaar4666/DC1

    Fig. S1. The effect of mETC inhibitors on the viability of assay cells (HepG2).

    Fig. S2. The effect of UPP inhibitors on the viability of assay cells and the common TFAP signatures for UPP inhibitors in HEK293 and MCF-7 cells.

    Fig. S3. The common TFAP signatures for HDAC inhibitors in HEK293 and MCF-7 cells and the effect of HDAC inhibitors on the viability of assay cells.

    Fig. S4. The effect of MTDs on the viability of assay cells.

    Fig. S5. The effect of microtubule DNA-damaging agents on the viability of assay cells.

    Fig. S6. An alternative presentation of TF responses to perturbagens as a heatmap.

    Fig. S7. The list of known mitochondria disruptors with a high (r > 0.800) TFAP similarity to the mETC TFAP that were scored negative by the MMP assay.

    Table S1. ToxCast chemicals with a high (r ≥ 0.800) similarity to the invariant mETC TFAP.

    References (4165)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. The effect of mETC inhibitors on the viability of assay cells (HepG2).
    • Fig. S2. The effect of UPP inhibitors on the viability of assay cells and the common TFAP signatures for UPP inhibitors in HEK293 and MCF-7 cells.
    • Fig. S3. The common TFAP signatures for HDAC inhibitors in HEK293 and MCF-7 cells and the effect of HDAC inhibitors on the viability of assay cells.
    • Fig. S4. The effect of MTDs on the viability of assay cells.
    • Fig. S5. The effect of microtubule DNA-damaging agents on the viability of assay cells.
    • Fig. S6. An alternative presentation of TF responses to perturbagens as a heatmap.
    • Fig. S7. The list of known mitochondria disruptors with a high (r > 0.800) TFAP similarity to the mETC TFAP that were scored negative by the MMP assay.
    • Table S1. ToxCast chemicals with a high (r ≥ 0.800) similarity to the invariant mETC TFAP.
    • References (4165)

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