Multiple Cationic Amphiphiles Induce a Niemann-Pick C Phenotype and Inhibit Ebola Virus Entry and Infection

Abstract

Ebola virus (EBOV) is an enveloped RNA virus that causes hemorrhagic fever in humans and non-human primates. Infection requires internalization from the cell surface and trafficking to a late endocytic compartment, where viral fusion occurs, providing a conduit for the viral genome to enter the cytoplasm and initiate replication. In a concurrent study, we identified clomiphene as a potent inhibitor of EBOV entry. Here, we screened eleven inhibitors that target the same biosynthetic pathway as clomiphene. From this screen we identified six compounds, including U18666A, that block EBOV infection (IC50 1.6 to 8.0 µM) at a late stage of entry. Intriguingly, all six are cationic amphiphiles that share additional chemical features. U18666A induces phenotypes, including cholesterol accumulation in endosomes, associated with defects in Niemann–Pick C1 protein (NPC1), a late endosomal and lysosomal protein required for EBOV entry. We tested and found that all six EBOV entry inhibitors from our screen induced cholesterol accumulation. We further showed that higher concentrations of cationic amphiphiles are required to inhibit EBOV entry into cells that overexpress NPC1 than parental cells, supporting the contention that they inhibit EBOV entry in an NPC1-dependent manner. A previously reported inhibitor, compound 3.47, inhibits EBOV entry by blocking binding of the EBOV glycoprotein to NPC1. None of the cationic amphiphiles tested had this effect. Hence, multiple cationic amphiphiles (including several FDA approved agents) inhibit EBOV entry in an NPC1-dependent fashion, but by a mechanism distinct from that of compound 3.47. Our findings suggest that there are minimally two ways of perturbing NPC1-dependent pathways that can block EBOV entry, increasing the attractiveness of NPC1 as an anti-filoviral therapeutic target.

Cholesterol Flux Is Required for Endosomal Progression of African Swine Fever Virions during the Initial Establishment of Infection

“…These chemical compounds have the ability to accumulate cholesterol in late endosomes and inhibit EBOV entry through an NPC1-dependent pathway (53). Filovirus entry seems to require the interaction of GP with NPC1 protein as an intracellular receptor (44,56).…”

Characterization of Human and Murine T-Cell Immunoglobulin Mucin Domain 4 (TIM-4) IgV Domain Residues Critical for Ebola Virus Entry

“…Prior to transduction, the hTIM-4-expresssing HEK 293 stable cell line was incubated at 37°C for 30 min with increasing concentrations of arginyl-glycylaspartic acid (RGD) peptide (Abbiotec) or 3.47 inhibitor (a kind gift from Kartik Chandran, Albert Einstein College of Medicine). Concentrations were based on amounts that have been previously demonstrated to inhibit integrin binding (RGD peptide) or EBOV entry (3.47) (38)(39)(40). Cells in the presence of the inhibitor were transduced with FL EBOV VSV-GFP pseudovirions (MOI of 0.7 [titer determined on Vero cells]).…”

“…Initially, we verified that commercially purchased RGD peptide inhibited fibronectin-Vero cell interactions in a dose-dependent manner, as previously reported (46, 47) (data not shown). Upon verification, HEK 293 cells that stably express hTIM-4 were incubated with increasing concentrations of RGD peptide or, as a control, a well-established EBOV entry inhibitor, 3.47, that blocks late endosomal events required for EBOV entry (39,40). The cells were transduced with EBOV pseudovirions and analyzed 24 h later for GFP expression.…”

The transition from a rural to an urban environment alters expression of the human Ebola virus receptor Neiman-Pick C1: implications for the current epidemic in West Africa

“…More recent studies have confirmed the recognition of NPC1 by Ebola virus (Miller and Chandran 2012;Krishnan et al 2012;Ng et al 2014). Finally, cationic amphiphiles that target NPC1 were also found to inhibit Ebola virus cell entry and infection (Shoemaker et al 2013).…”