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An integrated bacterial system for the discovery of chemical rescuers of disease-associated protein misfolding

A Publisher Correction to this article was published on 02 January 2018

This article has been updated

Abstract

Protein misfolding and aggregation are common pathological features of several human diseases, including Alzheimer’s disease and type 2 diabetes. Here, we report an integrated and generalizable bacterial system for the facile discovery of chemical rescuers of disease-associated protein misfolding. In this system, large combinatorial libraries of macrocyclic molecules are biosynthesized in Escherichia coli cells and simultaneously screened for their ability to rescue pathogenic protein misfolding and aggregation using a flow cytometric assay. We demonstrate the effectiveness of this approach by identifying drug-like, head-to-tail cyclic peptides that modulate the aggregation of the Alzheimer’s disease-associated amyloid β peptide. Biochemical, biophysical and biological assays using isolated amyloid β peptide, primary neurons and various established Alzheimer’s disease nematode models showed that the selected macrocycles potently inhibit the formation of neurotoxic amyloid β peptide aggregates. We also applied the system to the identification of misfolding rescuers of mutant Cu/Zn superoxide dismutase—an enzyme linked with inherited forms of amyotrophic lateral sclerosis. Overall, the system enables the identification of molecules with therapeutic potential for rescuing the misfolding of disease-associated polypeptides.

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Fig. 1: High-throughput genetic screening for the discovery of macrocyclic rescuers of pathogenic Aβ misfolding and aggregation.
Fig. 2: The selected cyclic pentapeptides AβC5-34 and AβC5-116 interfere with the normal Aβ aggregation process.
Fig. 3: The selected cyclic pentapeptides AβC5-34 and AβC5-116 inhibit Aβ-induced neurotoxicity in vitro.
Fig. 4: The selected cyclic pentapeptides AβC5-34 and AβC5-116 inhibit Aβ-induced aggregation and cytotoxicity in vivo.
Fig. 5: Structure–activity analyses of the selected cyclic pentapeptides AβC5-34 and AβC5-116.
Fig. 6: Computational modelling of the mode of AβC5-34 and AβC5-116 binding to Aβ.
Fig. 7: Genetic screening and identification of cyclic oligopeptides that rescue the misfolding and aggregation of mutant SOD1.

Change history

  • 02 January 2018

    In the version of this Article originally published, in Fig. 1c–e, on the x axes, the lines labelled ‘Aβ42’ and ‘Aβ42(F19S;L34P)’ grouped the data incorrectly; the line labelled Aβ42 should have grouped the data for Random 1–2 and Clones 1–10, and the line labelled Aβ42(F19S;L34P) should have only grouped the data for Random 1–2 on the right end of the plots and blots. These figures have now been corrected in all versions of the Article.

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Acknowledgements

The authors thank S. Benkovic (Penn State University) and M. H. Hecht (Princeton University) for the plasmids, D. Walsh (Brigham and Women’s Hospital), L. Stefanis (University of Athens) and M. Paravatou-Petsotas (National Center for Scientific Research 'Demokritos') for the cell lines, C. D. Link (University of Colorado Boulder) for the C. elegans CL2331 strain and P. Mehta (Institute for Basic Research in Developmental Disabilities) for the antibodies. The nematode strains used in this study were provided by the Caenorhabditis Genetics Center, supported by the United States National Institutes of Health National Center for Research Resources. We also thank D. Gialama for technical assistance with the initial expression vector construction and E. Megalou for bacterial sample preparations for the C. elegans feeding assays. We gratefully acknowledge G. Georgiou (University of Texas at Austin) for facilitating the flow cytometric sorting experiments and V. Papadimitriou and A. Xenakis for the dynamic light scattering experiments. This work was funded by the following projects: NEUROTHERAPY in the framework of the research grant 'Aristeia', financed by the Hellenic General Secretariat of Research and Technology and the National Strategic Reference Framework (to G.S.); CYCLIPAD in the framework of the research grant 'Thalis', financed by the Hellenic Ministry of Education, Research and Religious Affairs and the National Strategic Reference Framework (to E.S.G., G.S., F.N.K., M.P., M.M. and S.E.); the John S. Latsis Public Benefit Foundation (to N.C. and G.S.); and the Synthetic Biology research infrastructure OMIC-ENGINE, financed by the Hellenic General Secretariat of Research and Technology and the National Strategic Reference Framework. S.B. and Z.I.L. are recipients of fellowships for post-doctoral research by the Hellenic State Scholarships Foundation "IKY Fellowships of Excellence for Postgraduate Studies in Greece - Siemens Program". The Graphics Processing Unit (GPU)-accelerated molecular dynamics simulations were performed at the LinkSCEEM Cy-Tera GPU cluster, supported by the LinkSCEEM-2 project and funded by the European Union FP7 Capacities Research Infrastructure, INFRA-2010-1.2.3 Virtual Research Communities (grant agreement RI-261600). The molecular mechanics Poisson–Boltzmann surface area calculations were supported by computational time granted by the Greek Research and Technology Network in the National High Performance Computing Facility Advanced Research Information System under project identification pr001017.

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G.S. conceived and coordinated the project. G.S., N.C., M.P., K.D.P. and S.E. designed the research. G.S., E.S.G., M.P., S.E., M.M., K.V., N.C. and F.N.K. attracted funding. I.M., D.C.D., B.M., N.P., S.P., S.B., K.D.P., Z.I.L., A.V.S., N.B., K.V. and G.S. performed the research. I.M., D.C.D., B.M., N.P., S.P., S.B., K.D.P., Z.I.L., K.V., S.E., M.P., N.C. and G.S. analysed the data. G.S., N.C., M.P., Ν.Β., K.V., S.E. and M.G.P. supervised the research. G.S. wrote the paper with contributions from I.M., D.C.D., B.M., N.P., S.P., K.D.P., Z.I.L., S.E., N.C. and M.P. All authors read and approved the final version of the paper.

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Correspondence to Georgios Skretas.

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G.S. is the inventor on patent applications for AβC5-34, AβC5-116, SOD1C5-4 and other Aβ- and SOD1-targeting peptide macrocyclic sequences described in this article.

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Matis, I., Delivoria, D.C., Mavroidi, B. et al. An integrated bacterial system for the discovery of chemical rescuers of disease-associated protein misfolding. Nat Biomed Eng 1, 838–852 (2017). https://doi.org/10.1038/s41551-017-0144-3

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