Most antiviral drugs in the clinic today target virus components related to viral replication. These drugs must be able to penetrate into the cell and be of sufficient potency to stop an already active infection. Recently, the step of virus entry has begun to be exploited for drug discovery. Virus entry is the first and committal step toward establishing a virus infection. Compounds that interfere with entry can affect binding of virus to receptor(s), uptake of virus into endosomes and finally membrane fusion of virus and endosomal membranes. Each of these steps can be targeted by drugs that do not need to permeate the cell membrane as each occurs at the cell surface or within endosomes that contain fluid sampled from outside of the cell. Aside from those against HIV and Influenza A, few other antiviral drugs have been identified that target entry. In this project we aim to identify and characterize compounds that inhibit the entry of filoviruses and arenaviruses. Each family of viruses cause hemorrhagic fever and are classified as NIH category 1 biothreat and emerging infectious agents. We will use qHTS at NCGC to identify compounds that inhibit infection of Marburg (a filovirus related to Ebola virus) and Lassa Fever virus (an arenavirus). For both families little is known about the entry mechanism and no useful drugs are available for therapy. We have developed a robust assay platform for identification of virus entry inhibitors for both. The system is based on virus pseudotypes and so can be used at BSL2. Screens can be safely performed at BSL2 and the assay has been optimized at NCGC to give signal-to-noise ratios >25-fold and Z'of >0.5 in the 1536-well format. Readout is by firefly luciferase and shows good dose-response linearity of signal. We will also perform a set of secondary assays designed to identify the step (binding, uptake and membrane fusion) at which virus entry was blocked. These assays have been previously published by our lab for both virus types. Each compound will also be tested for efficacy against wild type virus. We will then have the opportunity to identify agent- specific or broad-spectrum antiviral compounds. Future work will involve development of the best compounds into potentially useful drugs and use of each as probes to better understand the mechanism of virus entry for each virus and the pathways taken to infect the cell.
No effective drugs are available for treatment of disease caused by Filoviruses or Arenaviruses. This proposal seeks to identify new compounds that inhibit the entry of these viruses into cells. A comprehensive screen of 300,000 compounds will be performed at an NIH screening center and the mechanism of action of each hit will be analyzed.
|Anantpadma, Manu; Kouznetsova, Jennifer; Wang, Hang et al. (2016) Large-Scale Screening and Identification of Novel Ebola Virus and Marburg Virus Entry Inhibitors. Antimicrob Agents Chemother 60:4471-81|