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State-based discovery: a multidimensional screen for small-molecule modulators of EGF signaling

Abstract

As an alternative to conventional, target-oriented drug discovery, we report a strategy that identifies compounds on the basis of the state that they induce in a signaling network. Immortalized human cells are grown in microtiter plates and treated with compounds from a small-molecule library. The target network is then activated and lysates derived from each sample are arrayed onto glass-supported nitrocellulose pads. By probing these microarrays with antibodies that report on the abundance or phosphorylation state of selected proteins, a global picture of the target network is obtained. As proof of concept, we screened 84 kinase and phosphatase inhibitors for their ability to induce different states in the ErbB signaling network. We observed functional connections between proteins that match our understanding of ErbB signaling, indicating that state-based screens can be used to define the topology of signaling networks. Additionally, compounds sort according to the multidimensional phenotypes they induce, suggesting that state-based screens may inform efforts to identify the targets of biologically active small molecules.

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Figure 1: State-based screening using lysate microarrays.
Figure 2: Evaluation of antibodies for use in lysate microarrays.
Figure 3: Lysate microarrays resulting from a state-based screen of 84 kinase and phosphatase inhibitors.
Figure 4: Clustering of data from a state-based screen.
Figure 5: Dose-response curves determined with lysate microarrays.

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Acknowledgements

We thank the Bauer Center for Genomics Research at Harvard University for support with instrumentation and automation. This work was supported by awards from the Arnold and Mabel Beckman Foundation and the W.M. Keck Foundation. M.S. is the recipient of an Alfred and Isabel Bader fellowship and a Jacques-Émile Dubois fellowship.

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Correspondence to Gavin MacBeath.

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Supplementary information

Supplementary Fig. 1

Evaluation of antibodies for lysate microarrays. (PDF 300 kb)

Supplementary Fig. 2

Verification of compound activities using traditional western blotting. (PDF 55 kb)

Supplementary Table 1

Antibodies used in the state-based screen. (PDF 58 kb)

Supplementary Table 2

Identity of compounds in the model library. (PDF 12 kb)

Supplementary Methods

Reagents, cell culture, immunoblotting, and determination of dose-response behavior. (PDF 68 kb)

Supplementary Note

Data processing steps used in the state-based screen and how these steps affect uncertainty in the data. (PDF 97 kb)

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Sevecka, M., MacBeath, G. State-based discovery: a multidimensional screen for small-molecule modulators of EGF signaling. Nat Methods 3, 825–831 (2006). https://doi.org/10.1038/nmeth931

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