Monday, 3 Oct 2022

What is the potential role of sphingosine kinase inhibitors as antiviral agents?

In a recent study posted to the bioRxiv* preprint server, researchers explored sphingosine kinases (SK) inhibitors and characterized the role of SKs in Ebola virus (EBOV) entry.

Study: Sphingosine kinases promote Ebola virus infection and can be targeted to inhibit filoviruses, coronaviruses, and arenaviruses using late endocytic trafficking to enter cells. Image Credit: jaddingt/Shutterstock

Viruses, such as EBOV, that invade host cells through late lysosomes or endosomes are known as late-penetrating viruses. Host factors such as signaling molecules are required by such viruses for efficient fusion to host cell membranes, indicating that signaling molecules could be developed as potential antiviral agents.  

The authors of the present study previously identified several compounds (n=35) that inhibited host invasion by EBOV, of which FTY-720 (fingolimod, 2-amino-2-(2-(4-octylphenyl)-ethyl)-1,3-propanediol) and PF-543 interfered with SK/S1PR (sphingosine-1-phosphate receptor 1) signaling, indicating that SK pathways regulate EBOV entry.

About the study

In the present study, researchers investigated the role of SKs in the host invasion by late penetrating viruses such as EBOV.

The potential regulation of SK/S1PR pathways in facilitating host invasion by EBOV was assessed by characterizing the antiviral activities of SK inhibitors such as FTY-720, SK1-I and PF-543. The antiviral efficacy of the three SK inhibitors was confirmed by using filoviral-like particles (VLPs) produced by co-expressing VP40 (EBOV matrix protein) in fusion with a βlam (β-lactamase) reporter, the glycoprotein (GP) of interest and EBOV nucleoprotein (NP).

SK1 and SK2 activities were assessed in vitro by green fluorescent protein (GFP)-based analysis and cell culture experiments using HT1080 cells (fibrosarcoma cell line) transduced with EBOV GP-harboring murine leukemia virus (MLV) pseudotypes and Vero cells. SK1 and SK2 were targeted using dicer-substrate short interfering ribonucleic acid (DsiRNAs) transfected in HT1080 cells and by using VLPs harboring EBOV GP or vesicular stomatitis virus (VSV)-G to determine the role of SKs in viral entry.

Further, the team explored if SK inhibitors decreased EBOV attachment to the host cells using EBOV VLPs by flow cytometry (FC) analysis. In addition, the potential blockage of viral internalization by SK inhibitors was assessed based on the macropinocytic uptake of EBOV. The researchers explored if SK inhibitors prevented GP-mediated cleavage by cathepsins by evaluating the entry of cleaved and uncleaved VLPs in Ca074 (cathepsin B inhibitor)-treated HT1080 cells.

The team also assessed if SK inhibitors blocked viral endolysosomal trafficking to NCP1 (EBOV receptor) by immunostaining analysis and the capability of SK inhibitors to block enveloped viruses by requiring endosomal traffickings such as Junin virus (JUNV), Marburg virus (MARV), Influenza A virus (IAV), LFV and coronaviruses such as middle eastern respiratory syndrome coronaviruses (MERS), severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and SARS-CoV-2.

Results

Immunoblot analysis showed significantly reduced expression of SK1 and SK2 on cellar transfection with their corresponding DsiRNAs. All the three SK inhibitors (FTY-720, SKI-I and PF-543) and SK1,2 knockdown decreased EBOV GP-harboring VLP entry. Still, they had no impact on VSV G-harboring VLPs, indicating that the SK inhibitors targeted EBOV GP-mediated entry.

Dilated intracellular vesicles were observed among FTY720- and SK1-I-treated cells (but not PF-543-treated cells, indicative of endosomal trafficking defects caused by SK inhibitors. In particular, SK inhibitors blocked EBOV VLP endolysosomal trafficking to Niemann Pick C1 (NPC1) irrespective of S1P signaling through S1P receptors (S1PRs).

SK inhibitors also prevented infection of SARS CoV-2 dependent on cathepsins and decreased host entry of other late penetrating viruses such as SARS-CoV-1, SARS-CoV-2, MERS, MARV, JUNV and Lassa fever virus (LFV). Antiviral compounds that inhibited SK1 demonstrated higher potency, and compounds with poor SK1 inhibition and >40% residual activity at 1 μM inhibitor concentration showed poor antiviral activity.

Significant and complete inhibition of EBOV replication was observed by PF-543 at 5µM and 10µM concentrations, respectively. However, the FC analysis showed that no SK inhibitors reduced VLP fluorescence, indicating no significant inhibition of EBOV VLP attachment by SK inhibitors. Further, FTY720 and SK1-I did not impact EBOV VLP internalization, PF-543 showed only modest effects, and SK inhibitors showed equal potency for inhibiting the entry of uncleaved and cleaved VLPs.

PF-543, slightly reduced internalization of EBOV, indicating that PF-543 slightly reduced internalization of EBOV. All SK inhibitors prevented EBOV from trafficking to late-endosomes and/or lysosomes containing NPC1, indicating that SKs play critical roles in endosomal trafficking. PF-543, an SK inhibitor that is not a sphingosine analogue, does not induce the formation of dilated vesicles; it does induce a similar endocytic trafficking defect, explaining the mechanism of antiviral activity of our SK inhibitors.

Overall, the study findings showed that SKs were required for late endolysosomal EBOV trafficking but not for EBOV attachment and internalization and blocked infection by EBOV and other late-penetrating viruses. The findings highlighted the potential of SK inhibitors to be used as pan-antiviral agents.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Corina M. Stewart et al. (2022). Sphingosine kinases promote Ebola virus infection and can be targeted to inhibit filoviruses, coronaviruses, and arenaviruses using late endocytic trafficking to enter cells. bioRxiv. doi: https://doi.org/10.1101/2022.08.12.503750 https://www.biorxiv.org/content/10.1101/2022.08.12.503750v1

Posted in: Medical Science News | Medical Research News | Disease/Infection News

Tags: Cell, Cell Culture, Cell Line, Coronavirus, Cytometry, Efficacy, Fever, Fibrosarcoma, Fingolimod, Flow Cytometry, Fluorescence, Fluorescent Protein, Glycoprotein, in vitro, Influenza, Intracellular, Kinase, Leukemia, Lysosomes, Marburg Virus, Protein, Receptor, Respiratory, Ribonucleic Acid, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Stomatitis, Syndrome, Transfection, Virus

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Pooja Toshniwal Paharia

Dr. based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.

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