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Small molecule inhibitors reveal Niemann–Pick C1 is essential for Ebola virus infection

Abstract

Ebola virus (EboV) is a highly pathogenic enveloped virus that causes outbreaks of zoonotic infection in Africa. The clinical symptoms are manifestations of the massive production of pro-inflammatory cytokines in response to infection1 and in many outbreaks, mortality exceeds 75%. The unpredictable onset, ease of transmission, rapid progression of disease, high mortality and lack of effective vaccine or therapy have created a high level of public concern about EboV2. Here we report the identification of a novel benzylpiperazine adamantane diamide-derived compound that inhibits EboV infection. Using mutant cell lines and informative derivatives of the lead compound, we show that the target of the inhibitor is the endosomal membrane protein Niemann–Pick C1 (NPC1). We find that NPC1 is essential for infection, that it binds to the virus glycoprotein (GP), and that antiviral compounds interfere with GP binding to NPC1. Combined with the results of previous studies of GP structure and function, our findings support a model of EboV infection in which cleavage of the GP1 subunit by endosomal cathepsin proteases removes heavily glycosylated domains to expose the amino-terminal domain3,4,5,6,7, which is a ligand for NPC1 and regulates membrane fusion by the GP2 subunit8. Thus, NPC1 is essential for EboV entry and a target for antiviral therapy.

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Figure 1: Structure and function of Ebola virus entry inhibitors.
Figure 2: NPC1 is essential for Ebola virus infection.
Figure 3: Protease-cleaved EboV GP binds to NPC1.
Figure 4: NPC1 is a target of the small molecule inhibitors.

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Acknowledgements

We thank B. Considine, A. Nilsson and S. Wilkes for assistance, S. Chiang for critical reading of the manuscript, G. Beltz, N. Gray, S. Grinstein, Y. Iannou, R. Infante, J. Kornhuber, F. Sharom and S. Whelan for discussion. This work was supported by grants from U54 AI057159, R01 CA104266 to J.C., PIDS-Sanofi-Pasteur Fellowship, K12-HD052896 and 5K08AI079381 to J.M., 5-T32- HL007623 to A.B., and fellowship from Fonds de la Recherche en Santé du Québec to M.C.; C.M.F. was supported by the Postgraduate Research Participation Program at the US Army Medical Research and Material Command administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and USAMRMC.

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Contributions

M.C., J.M., T.R. and A.B. equally contributed to this work. K.C. and T.R. performed the inhibitor screen. K.L. synthesized and purified 3.0 analogues and T.R. tested them. T.R., A.B., J.M., Q.L. and M.C. carried out infection assays with pseudotyped viruses. A.B. performed microscopy. J.M. purified recombinant glycoprotein. M.C. and J.M. designed and performed binding assays. M.C. performed immunoprecipitation. D.O. provided NPC1 constructs, antibodies and CHO cell lines. Ebola virus infections were performed in the lab of L.H. by C.M.F.; J.C. supervised the project and wrote the manuscript. All authors reviewed the manuscript.

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Correspondence to James Cunningham.

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The authors declare no competing financial interests.

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This file contains Supplementary Figures 1-8 with legends and Supplementary Text and Data detailing the synthesis and characterization of the novel chemicals used in the manuscript. (PDF 4560 kb)

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Côté, M., Misasi, J., Ren, T. et al. Small molecule inhibitors reveal Niemann–Pick C1 is essential for Ebola virus infection. Nature 477, 344–348 (2011). https://doi.org/10.1038/nature10380

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