Abstract
We present a strategy for identifying off-target effects and hidden phenotypes of drugs by directly probing biochemical pathways that underlie therapeutic or toxic mechanisms in intact, living cells. High-content protein-fragment complementation assays (PCAs) were constructed with synthetic fragments of a mutant fluorescent protein ('Venus', EYFP or both), allowing us to measure spatial and temporal changes in protein complexes in response to drugs that activate or inhibit particular pathways. One hundred and seven different drugs from six therapeutic areas were screened against 49 different PCA reporters for ten cellular processes. This strategy reproduced known structure-function relationships and also predicted 'hidden,' potent antiproliferative activities for four drugs with novel mechanisms of action, including disruption of mitochondrial membrane potential. A simple algorithm identified a 25-assay panel that was highly predictive of antiproliferative activity, and the predictive power of this approach was confirmed with cross-validation tests. This study suggests a strategy for therapeutic discovery that identifies novel, unpredicted mechanisms of drug action and thereby enhances the productivity of drug-discovery research.
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Acknowledgements
The authors thank E. Smith, M. West and the molecular biology staff at Odyssey Thera for excellent technical support. S.W.M. is the Canada Research Chair in Integrative Genomics. This manuscript is dedicated to the memory of Anthony V. Carrano.
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Supplementary information
Supplementary Fig. 1
Enhanced sensitivity of IFP PCA versus EYFP (enhanced YFP) (PDF 61 kb)
Supplementary Fig. 2
Details of Figure 2 matrix showing compound activity versus PCA at different time points and additional treatments (PDF 166 kb)
Supplementary Fig. 3
Assay response matrix from Figure 5c (PDF 51 kb)
Supplementary Table 1
Assay components and assay conditions (PDF 88 kb)
Supplementary Table 2
Drugs and their screening doses (PDF 54 kb)
Supplementary Table 3
Effects of compounds on proliferation of PC3 Cells (PDF 57 kb)
Supplementary Table 4
Assay statistics and positive predictive values (PPVs) for the top 25 assays, and results of leave-n-out analyses (PDF 78 kb)
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MacDonald, M., Lamerdin, J., Owens, S. et al. Identifying off-target effects and hidden phenotypes of drugs in human cells. Nat Chem Biol 2, 329–337 (2006). https://doi.org/10.1038/nchembio790
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DOI: https://doi.org/10.1038/nchembio790
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