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High-content single-cell drug screening with phosphospecific flow cytometry

Nature Chemical Biology volume 4, pages 132–142 (2008)Cite this article

Abstract

Drug screening is often limited to cell-free assays involving purified enzymes, but it is arguably best applied against systems that represent disease states or complex physiological cellular networks. Here, we describe a high-content, cell-based drug discovery platform based on phosphospecific flow cytometry, or phosphoflow, that enabled screening for inhibitors against multiple endogenous kinase signaling pathways in heterogeneous primary cell populations at the single-cell level. From a library of small-molecule natural products, we identified pathway-selective inhibitors of Jak-Stat and MAP kinase signaling. Dose-response experiments in primary cells confirmed pathway selectivity, but importantly also revealed differential inhibition of cell types and new druggability trends across multiple compounds. Lead compound selectivity was confirmed in vivo in mice. Phosphoflow therefore provides a unique platform that can be applied throughout the drug discovery process, from early compound screening to in vivo testing and clinical monitoring of drug efficacy.

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Figure 1: Cell-based screening with phosphoflow.
Figure 2: Initial screen reveals Jak-Stat and MAP kinase pathway-selective compounds.
Figure 3: Validation of hits from initial cell line screen.
Figure 4: Screening in heterogeneous primary cell populations.
Figure 5: Identification of pathway-selective compound.
Figure 6: Identification of cell type–selective compound.
Figure 7: Druggable signaling pathways and cell types revealed by selectivity factor clustering.
Figure 8: Preclinical monitoring of drug efficacy in vivo.

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Acknowledgements

We thank E. Danna, K. Schulz and K. Gibbs for critical reading of this manuscript. We thank BD Pharmingen, in particular G. Gao, R. Campos and B. Balderas, for kindly providing antibody reagents and technical expertise. We also thank the NCI DTP and J. Johnson for providing the natural product library used in this study. P.O.K. was supported in part by a Howard Hughes Medical Institute Predoctoral Fellowship. G.P.N. was supported by US National Heart, Lung and Blood Institute contract N01-HV-28183 and US National Institutes of Health grant AI35304.

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  1. Department of Microbiology and Immunology, Baxter Laboratory in Genetic Pharmacology, Stanford University, 269 Campus Drive, Stanford, 94305, California, USA

    Peter O Krutzik, Janelle M Crane, Matthew R Clutter & Garry P Nolan

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  1. Peter O Krutzik
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  2. Janelle M Crane
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Contributions

P.O.K. designed the study, performed experiments, analyzed data and wrote the manuscript. J.M.C. performed experiments and analyzed data. M.R.C. performed experiments and helped write the manuscript. G.P.N. helped to design the study and write the manuscript.

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Correspondence to Garry P Nolan.

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Competing interests

G.P.N. and P.O.K. are each paid consultants to Becton Dickinson, a provider of antibodies and flow cytometry-based reagents. G.P.N. consults occasionally with multiple pharmaceutical and biotechnology concerns about flow-based analysis of signaling systems.

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Krutzik, P., Crane, J., Clutter, M. et al. High-content single-cell drug screening with phosphospecific flow cytometry. Nat Chem Biol 4, 132–142 (2008). https://doi.org/10.1038/nchembio.2007.59

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