Abstract
Protein-protein interactions (PPIs) are emerging as a promising new class of drug targets. Here, we present a novel high-throughput approach to screen inhibitors of PPIs in cells. We designed a library of 50,000 human peptide-binding motifs and used a pooled lentiviral system to express them intracellularly and screen for their effects on cell proliferation. We thereby identified inhibitors that drastically reduced the viability of a pancreatic cancer line (RWP1) while leaving a control line virtually unaffected. We identified their target interactions computationally, and validated a subset in experiments. We also discovered their potential mechanisms of action, including apoptosis and cell cycle arrest. Finally, we confirmed that synthetic lipopeptide versions of our inhibitors have similarly specific and dosage-dependent effects on cancer cell growth. Our screen reveals new drug targets and peptide drug leads, and it provides a rich data set covering phenotypes for the inhibition of thousands of interactions.
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Acknowledgements
We thank the members of the Moffat laboratory for valuable technical assistance with lentiviral screening technology, usage of reagents and equipment. We thank A. Emili, T. Hughes and M. Garton for helpful comments on the manuscript. We thank A. Musacchio (Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology) for providing us with the INCENP cDNA clone. We thank F. Sicheri (Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto) for use of the ITC machine. P.M.K. acknowledges an Operating Grant from the Canadian Institute of Health Research (CIHR MOP-123526) and an Innovation Grant from the Canadian Cancer Society Research Institute (CCSRI# 702884). J.M. is a Tier 2 Canada Research Chair in Functional Genomics of Cancer. The research was supported in part by the Intramural Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research (N.I.T.).
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P.M.K. designed the project, provided study guidance and wrote the bulk of the manuscript. S.N. performed most experiments and contributed to writing of the manuscript. J.J. performed all bioinformatics analysis and interpreted results as well as assisting in manuscript preparation. C.C.-V. and M.-H.S. performed affinity measurements and helped with other biochemical experiments. Y.I. provided the oligonucleotide library and provided study guidance. N.T. provided synthetic peptides and guidance on their use. J.M. helped design the project and provided guidance on lentiviral screening.
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Supplementary information
Supplementary Text and Figures
Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–11. (PDF 2854 kb)
Supplementary Data Set 1
The list of human peptides for dropout screen and their effect on cell viability. (XLS 12194 kb)
Supplementary Data Set 2
A list of cancer-specific inhibitors. (XLS 217 kb)
Supplementary Data Set 3
Peptide-target interactions (XLS 301 kb)
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Nim, S., Jeon, J., Corbi-Verge, C. et al. Pooled screening for antiproliferative inhibitors of protein-protein interactions. Nat Chem Biol 12, 275–281 (2016). https://doi.org/10.1038/nchembio.2026
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DOI: https://doi.org/10.1038/nchembio.2026
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