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Principal Investigator: WILLIAM DEGRADO
Organization: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
Fiscal Year: 2022
Award: $3,846,686
Funding agency: National Institute of Allergy and Infectious Diseases
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.
Terms: <1-Aminoadamantane><2019 novel corona virus><2019 novel coronavirus><2019-nCoV><Adamantylamine><Address><Affinity><Alpha Virus><Alphavirus><Amantadine><Amiloride><Antiviral Agents><Antiviral Drugs><Antivirals><Assay><Attention><Bioassay><Biochemistry><Biologic Assays><Biological Assay><Biological Chemistry><CHIKV><COVID-19 virus><COVID19 virus><Cellular Assay><Chikungunya virus><CoV-2><CoV2><Collaborations><Communicable Diseases><Complex><Coronaviridae><Coronavirus><Cryo-electron Microscopy><Cryoelectron Microscopy><Crystallization><Dose><Drug Design><Drug Targeting><Drugs><Electron Cryomicroscopy><Electrophysiology><Electrophysiology (science)><Envelope Protein><Family><Group A Arboviruses><Hand><Head><High Throughput Assay><Human><In Vitro><Infectious Disease Pathway><Infectious Diseases><Infectious Disorder><Influenza A><Influenza A virus><Influenza Viruses Type A><Influenzavirus A><Ion Channel><Ionic Channels><Letters><Liposomal><Liposomes><Maps><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Medication><Medicinal Chemistry><Membrane><Membrane Channels><Membrane Protein Gene><Membrane Proteins><Membrane-Associated Proteins><Methods><Modeling><Modern Man><Molecular><Neurophysiology / Electrophysiology><Nucleocapsid><Nucleocapsid Proteins><Oral><Orthomyxovirus Type A><Pathogenicity><Pathology><Pharmaceutic Chemistry><Pharmaceutic Preparations><Pharmaceutical Chemistry><Pharmaceutical Preparations><Production><Protein Engineering><Protein Family><Proteins><Proteomics><Resistance><Resolution><Role><Ross river virus><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><SSNMR><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome related corona virus 2><Single Crystal Diffraction><Specificity><Structural Protein><Structure><Surface Proteins><Therapeutic Index><Time><Togaviridae><Togaviruses><Type A Influenza><Vesicle><Viral><Viral Gene Products><Viral Gene Proteins><Viral Proteins><Virion><Virus><Virus Assembly><Virus Particle><Virus-like particle><Work><Wuhan coronavirus><X Ray Crystallographies><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray/Neutron Crystallography><Xray Crystallography><Yeasts><analytical ultracentrifugation><anti-viral agents><anti-viral compound><anti-viral drugs><anti-viral medication><anti-viral therapeutic><anti-virals><antiviral compound><antiviral medication><antiviral therapeutic><base><beta CoV><beta coronavirus><betaCoV><betacoronavirus><cell assay><chikungunya><conformer><corona virus><coronavirus disease 2019 virus><coronavirus disease-19 virus><cross-link><crosslink><cryo-EM><cryoEM><design><designing><drug development><drug discovery><drug/agent><electrophysiological><env Antigens><env Gene Products><env Polyproteins><env Protein><forest><genetic protein engineering><hCoV19><high throughput screening><in vitro Assay><in vivo><inhibitor><lead optimization><member><membrane structure><nCoV2><new drug treatments><new drugs><new therapeutics><new therapy><next generation therapeutics><novel drug treatments><novel drugs><novel therapeutics><novel therapy><pandemic><pandemic disease><particle><programs><protein design><rational design><resistant><response><scaffold><scaffolding><screening><simulation><small molecule><social role><solid state NMR><solid state nuclear magnetic resonance><structural biology><viral assembly><virology><virus protein><virus-like nanoparticles><viruslike particle><β CoV><β coronavirus><βCoV>