Targeting Viroporins and Coronavirus M Protein

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PROJECT 3: TARGETING VIROPORINS AND CORONAVIRUS M PROTEIN SUMMARY Coronaviruses express four structural proteins; spike (S), membrane (M), envelope (E) and nucleocapsid (N), which are essential for efficient viral particle formation. We seek to understand and structurally characterize E and M, and to use this information to design antiviral drugs. M is a molecular scaffold that brings together the structural proteins, and E is a member of the viroporin family of proteins, which act as ion channels to control ionic composition in the host and virus. Previously, Hong and DeGrado solved the structures of the viroporins from influenza A virus, AM2, and used this information to design novel drugs that address the problem of resistance. We now have turned our attention to the E protein of SARS-CoV-2. Furthermore, we will determine structures of viroporins from a variety of other coronaviruses and alphaviruses of the Togaviridae family to enable structure-based protein design. M is essential for virus particle assembly. We will structurally characterize M alone and with its viral interacting partners in vesicles and virus-like particles (VLPs). This work will elucidate their role in stabilizing the virus and generating membrane curvature required for budding. We will use this information plus high-throughput screening (HTS) to discover drugs targeting M. More specifically, in Aim 1, we will structurally characterize the E protein and drug complexes using X-ray crystallography, Cryo-EM and solid-state NMR. We will also determine structures of E proteins from multiple alphacoronavirus and betacoronavirus lineages that infect humans. In Aim 2, we will determine Cryo-EM and crystal structures of M alone and in association with viral protein partners, in liposomes, VLPs and virions. The high-resolution structures determined in this aim will enable structure-based drug design. In parallel, we will use a convenient VLP assay to map residues in M and E that are essential for packaging and entry. The VLP assay will also be configured for HTS. In Aim 3, we will expand our studies to viroporins of alphaviruses of the Togaviridae family, which include the chikungunya (CHIKV), Sindbis (SINV), Semliki Forest (SFV), and Ross River (RRV) viruses. These viruses have viroporins, which are essential for effective replication in vivo. Using methods in Aim 1, we will explore their structures and develop inhibitors to facilitate drug discovery. In Aim 4, we will develop Optimized Lead compounds targeting E and M for transfer to Roche. Current compounds that show weak inhibition of E, which include amantadine and hexamethylene amiloride (HMA), provide starting points for design of higher specificity and affinity compounds. In parallel, we will use HTS, based on VLPs and a yeast screen of channel function to develop starting points for optimization of drug leads. In years 2.5 to 5, we will design small molecules to target M. Overall, we aim to develop orally bioavailable Optimized Leads that target E and M with IC50 < 100 nM in cellular assays and in vivo activity at dose <50 mg/kg.