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

Abstract

Summary Ebolavirus (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 the anti-viral 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 N-terminal domain3–7, 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 anti-viral therapy.

Genetic mutations you want

“…The gene that encodes it, when mutated, causes a rare version of Niemann Pick disease that is usually fatal in childhood to people with two copies. But in animal studies, individuals with only one mutated copy of the gene resist Ebola infection because the virus needs the working version of the protein to infect host cells (9).…”

The Niemann-Pick C1 Inhibitor NP3.47 Enhances Gene Silencing Potency of Lipid Nanoparticles Containing siRNA

“…NPC1 inhibitor NP3.47 was synthesized following the procedures detailed elsewhere. 9 The structure of NP3.47 was confirmed by NMR and the full details are provided in Supplementary Method 1.…”

“…In a screen of small molecules to identify agents that prevent Ebola virus infection, the compound NP3.47 (Figure 1) was shown to enhance cholesterol accumulation in intracellular vacuoles, 9 presumably by preventing endosomal escape of lipoproteins following endocytosis. In the case of Ebola virus it was shown that NP3.47 inhibited NPC1 and blocked cytoplasmic delivery of the virus.…”

“…In the case of Ebola virus it was shown that NP3.47 inhibited NPC1 and blocked cytoplasmic delivery of the virus. 9 For LNP-siRNA systems, longer residence times in intracellular organelles may be expected to result in an enhanced possibility for lytic interactions of component cationic lipids with late endosome or lysosome membranes, resulting in siRNA escape into the cytosol. To assess the influence of NP3.47 on the processing of LNP-siRNA following uptake, various cell lines were incubated with LNP-siRNA in the presence and absence of NP3.47 to determine effects on intracellular accumulation, extracellular recycling, and gene silencing.…”

Clues to NPC1-mediated cholesterol export from lysosomes

“…Native NPC1 is a 1,254-aa-long, multipass membrane glycoprotein located in the limiting membrane of lysosomes; NPC2 is a smaller, 151-residue protein, present in the lysosome lumen. In addition to its important role in cholesterol transport, NPC1 has also been identified as a key component required for the entry of Ebola virus into the cytoplasm (3,4). In PNAS Li et al (5) present a 3D structural model of NPC1, which provides important new clues to the mechanisms by which cholesterol is exported from lysosomes and the mechanism by which Ebola virus docks on the internal surface of the lysosome-limiting membrane as part of the infection process.…”