Project 1: Small Molecule Entry Inhibitors of Pandemic Viruses

Project 1 - Small Molecule Entry Inhibitors of Pandemic Viruses ABSTRACT Coronaviruses (CoVs), arenaviruses (Arv), flaviviruses (FLAVs) and filoviruses (FiVs) are enveloped viruses. During virus entry, receptor binding and refolding of the fusion protein, followed by lipid mixing, are three essential steps to release the viral genome. Inhibitors of any one of the three steps may be developed as effective antiviral drugs. Aim 1. Screen. (A) DEC-Tec screen using the purified ectodomain of the SARS2 S protein, glycoprotein of Machupo virus (MACV) and envelope (E) protein of Zika virus (ZIKV). (B) HTS will be carried out using fluorescence assays by targeting the six-helix bundle (6HB) of SARS2 and by competition with compound ALD-1.2 that binds ZIKV E protein. (C) Virtual screens by AutoDock or other computation methods will also be conducted since 3D structures of all target proteins are available. Aim 2. Optimization. Mechanism of action by hit compounds. (SARS2) Hit compounds from aim 1A will be validated by time of addition inhibition assays based on cell culture infection of SARS2 pseudotype. Validated entry inhibitors will be screened for inhibition of receptor binding by the S protein, and formation of 6HB. The receptor binding assay is by ELISA using an ACE2-Fc protein. The 6HB assay is set up by binding a fluorescently labeled HR2 peptide to 5HB. Entry inhibitors of other viruses will follow the same study approach. Inhibitors of lipid mixing. In preliminary efforts, we have identified three inhibitors that have EC50 values as low as 190 nM for SARS2 infection of cell culture. Mechanistic studies confirm that these inhibitors interact with the transmembrane domain of the fusion protein and block membrane fusion during virus entry. The experimental design for optimization is presented. Ebola virus entry inhibitors. These inhibitors are at an advanced stage and serve as a proof-of-concept example for our strategy. Med Chem Optimization. Structure and QSAR-based optimization of the inhibitor compounds will be carried out in collaboration with Cores C and D. Candidates that meet the criteria for further evaluation will be advanced to DMPK/toxicity studies (Core C). Escape mutants. The state-of-the-art approach is developed to evaluate mutants that escape the antiviral activities of the inhibitors, to aid inhibitor optimization. Aim 3. In vivo efficacy. For SARS2, lead candidates, especially prodrugs, will be evaluated for broad antiviral activities against multiple SARS2 and SARS isolates. Potent candidates will be evaluated in hamster and mouse models. For Arv, Stat1-/- and Ifnar1/Ifnar2 double knockout mice will be used as infection models. For Ebola virus, we have identified a series of small molecule inhibitors targeting the Ebola GP with a novel mechanism. We will evaluate and adapt these for the broad-spectrum activity against other significant filoviruses and evaluate their in vivo efficacy in the animal model operating at ABSL4. Top ZIKV inhibitors will be tested in animal models against multiple ZIKV infections. All technical work is carried out in Core E.