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. 2011 Sep 27;108(39):E771-80.
doi: 10.1073/pnas.1106149108. Epub 2011 Sep 6.

Modulatory profiling identifies mechanisms of small molecule-induced cell death

Adam J Wolpaw  1 , Kenichi Shimada, Rachid Skouta, Matthew E Welsch, Uri David Akavia, Dana Pe'er, Fatima Shaik, J Chloe Bulinski, Brent R Stockwell
Affiliations

Modulatory profiling identifies mechanisms of small molecule-induced cell death

Adam J Wolpaw et al. Proc Natl Acad Sci U S A. .

Abstract

Cell death is a complex process that plays a vital role in development, homeostasis, and disease. Our understanding of and ability to control cell death is impeded by an incomplete characterization of the full range of cell death processes that occur in mammalian systems, especially in response to exogenous perturbations. We present here a general approach to address this problem, which we call modulatory profiling. Modulatory profiles are composed of the changes in potency and efficacy of lethal compounds produced by a second cell death-modulating agent in human cell lines. We show that compounds with the same characterized mechanism of action have similar modulatory profiles. Furthermore, clustering of modulatory profiles revealed relationships not evident when clustering lethal compounds based on gene expression profiles alone. Finally, modulatory profiling of compounds correctly predicted three previously uncharacterized compounds to be microtubule-destabilizing agents, classified numerous compounds that act nonspecifically, and identified compounds that cause cell death through a mechanism that is morphologically and biochemically distinct from previously established ones.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Creating modulatory profiles. (A) Cells with or without modulator were seeded in 384-well plates. Lethal compounds in dilution series were then added, and the plates were incubated for 48 h before the addition of the cell viability dye Alamar blue. Readout of fluorescence after a 14-h incubation with Alamar blue allowed the construction of comparative concentration response curves. (B) Two examples of comparative concentration response curves and an illustration of the two parameters—the change in efficacy and change in potency—extracted from each pairwise combination of modulators and lethal compounds. Both examples use HT-1080 cells. (C) Heat map depicting 32 modulatory profiles of characterized lethal compounds (28 distinct compounds and 4 repeat compounds). Lethal compounds are listed on the y axis, and modulators, cell lines, and parameter types are on the x axis. Missing values are depicted in gray.
Fig. 2.
Fig. 2.
Clustering characterized lethal compounds based on modulatory profiles. (A) Heat map of the similarity matrix showing the Spearman correlation between modulatory profiles of lethal compounds. (B) Dendrogram derived from hierarchical clustering of the similarity matrix in A. (C) TC-7 cells stained for acetylated tubulin after 60-min treatment with vehicle (DMSO), 5 μM rotenone, 1 μM colchicine, or 1 μM staurosporine. Representative images were chosen for each treatment.
Fig. 3.
Fig. 3.
Clustering characterized lethal compounds based on gene expression profiles. (A) Heat map of the similarity matrix showing the Spearman correlation between gene expression profiles of lethal compounds. (B) Dendrogram derived from hierarchical clustering of the similarity matrix in A. *Compound used at sublethal concentration. **Compound used at supralethal concentration.
Fig. 4.
Fig. 4.
Clustering uncharacterized lethal compounds based on modulatory profiles. (A) Potency of uncharacterized lethal compounds in BJ-TERT/LT/ST/RASV12 and HT-1080 cells after 48 h. Values represent the average of three replicates ± SEM. (B) Heat map of the similarity matrix showing the Spearman correlation between modulatory profiles of characterized and uncharacterized lethal compounds. (C) Dendrogram derived from hierarchical clustering of the similarity matrix in B.
Fig. 5.
Fig. 5.
Previously uncharacterized compounds destabilize microtubules. (A) Structures of the well-characterized microtubule destabilizers vinblastine, podophyllotoxin, and colchicine, the previously reported destabilizer rotenone, and the three compounds predicted to destabilize microtubules based on their modulatory profiles. (B) Heat map of the Spearman correlations between the modulatory profiles of the uncharacterized compounds NPC4, NPC7, and NPC25 and the characterized compounds colchicine, vinblastine, carmustine, trichostatin A, MG132, and doxorubicin. (C) Time course of TC-7 cells stained for acetylated tubulin after treatment with 1 μM colchicine, 28 μM NPC4, 28 μM NPC7, or 14 μM NPC25. Staining for total tubulin and acetylated tubulin after a 60-min treatment with vehicle (DMSO) is shown at the top right. Representative images were chosen from each time point.
Fig. 6.
Fig. 6.
Two clusters of previously uncharacterized lethal compounds act nonspecifically. (A) Distribution of reactive compounds and amines throughout the clusters from Fig. 4C. Reactivity was calculated by adding activated chloroarenes to filters described previously (43). (B) Fraction nonpolar van der Waals surface area vs. pKa of the most basic residue. Fraction nonpolar surface area was calculated with the built-in parameter in the software MOE. The most basic pKa was calculated with the web-based software SPARC (SI Appendix, SI Methods). Using a one-way ANOVA and Tukey's multiple comparison test, compounds in cluster C are significantly different in their fraction nonpolar surface area from those compounds in cluster D (P < 0.01) and significantly more basic than those compounds in cluster B or D (P < 0.01). (C) Average potency of uncharacterized compounds. The average of the potency in HT-1080 and BJ-TERT/LT/ST/RASV12 cells is shown. Using a one-way ANOVA and Tukey's multiple comparison test, the compounds in clusters B and C are significantly less potent than the compounds in cluster D, and the compounds in cluster C are significantly less potent than the compounds in cluster E (P < 0.05). (D) Average modulatability of compounds. Modulatability was calculated by taking the mean of the absolute value of all of the normalized changes in a compound's modulatory profile. Lines represent cluster averages ± SEM. Using a one-way ANOVA and Tukey's multiple comparison test, compounds in clusters A, B, and C each are significantly less modulatable than those compounds in clusters D and E (P < 0.05).
Fig. 7.
Fig. 7.
Previously uncharacterized compounds induce BAX-/BAK-independent mitochondrial cell death. (A) Structures of erastin, RSL3, and NPC26, which are the three compounds in cluster E. (B) Caspase activity in HT-1080 cells treated for 12–15 h with erastin, RSL3, NPC26, and staurosporine. Caspase activity was measured by the cleavage of a fluorogenic caspase substrate and is shown on the left y axis (points represent the mean of three replicates ± SEM). Cell viability is shown by the shaded region and on the right y axis (points represent the mean of three replicates ± SEM). (C) Lethality of erastin, RSL3, NPC26, and camptothecin in WT or Bax−/−Bak−/− MEFs after 48 h of treatment. Data represent the mean of three replicates ± SEM. (D) EM images taken of BJ-TERT/LT/ST/RASV12 cells after a 3-h treatment with either DMSO or 9 μM NPC26 and EM images taken of HT-1080 cells after a 6-h treatment with either DMSO or 9 μM NPC26. Arrows show the nuclei, and arrowheads show mitochondria. Boxes show the portion of the image shown at higher magnification in the next image to the right. (E) Fluorescence images of HT-1080 cells expressing a mitochondrially targeted dsRed construct infected with either shRNA targeting DRP1 or a control nontargeting shRNA treated with either DMSO or 9 μM NPC26 for 6 h. (F) Heat map depicting the ability of various kinase inhibitors to alter death induced by NPC26 and other lethal compounds. Protection is depicted in yellow, and sensitization is depicted in blue. Experiments were performed in HT-1080 cells.
Fig. P1.
Fig. P1.
Schematic representation of modulatory profiling. (A) Concentration response curves for a lethal compound in the presence and absence of a modulator known to modulate cell death in a particular manner. The quantified changes are shown with red arrows. (B) A heat map illustrating the construction of modulatory profiles by assembling the changes produced by different modulators. Yellow indicates inhibition, and blue indicates sensitization. (C) Dendrogram derived from a hierarchical clustering of the lethal compounds based on their modulatory profiles. The mechanism of action of the compounds in each cluster is labeled.
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