David Veesler - US grants

Enveloped viruses use specialized proteins present at the virus surface to translocate their genetic material across the host cell membrane during infection. For coronaviruses, homotrimers of the spike glycoprotein promote host cell attachment and fusion of the viral and host membranes. Although coronaviruses have a significant pandemic potential, the lack of high-resolution data for any coronavirus spike trimer limits our mechanistic understanding of infection by this family of viruses. The objective of the proposed work is to obtain high-resolution snapshots corresponding to the various stages of the fusion reaction mediated by coronavirus spikes and to study the structural determinants associated with antibody inhibition of viral infection. In Aim I, we propose to elucidate the architecture of the Mouse Hepatitis Virus (MHV) pre-fusion spike using cryoEM. Aim II will be dedicated to studying the conformational changes associated with the fusion reaction with an emphasis on the first intermediate (extended intermediate) and the post-fusion spike. In Aim III, we will characterize the 3D organization of human coronavirus spikes to understand how these viruses overcome the species barrier and to identify structurally conserved regions that could be potential targets for therapeutic initiatives. The final aim will rely on structure-guided protein design to engineer antibodies targeting human coronavirus spikes with the goal of identifying immunogens for raising broadly-neutralizing antibodies.

Cyclic nucleotide-regulated ion channels are exquisite molecular machines that underlie important physiological functions. Cyclic nucleotide-gated (CNG) channels generate the primary electrical response to light in photoreceptors and to odorant in olfactory receptors. The related hyperpolarization-activated cyclic nucleotide-gated (HCN) channels underlie the pacemaker activity of the heart and many neurons in the brain. These cation selective channels are opened by the direct binding of cyclic nucleotides (cAMP and cGMP) to an intracellular domain of the channel. Our goal is to reveal the molecular mechanism for this allostery in CNG channels. Our approach will be to study bacterial CNG channels as a model system for the eukaryotic channels because of the huge advantages they provide for our biochemical methods . We will leverage the power of four different methodologies to determine the structure, conformational heterogeneity, and dynamics of these channels: 1) cryoelectron microscopy (cryo-EM), 2) double electron-electron resonance (DEER), 3) microfluidic rapid freeze quench (µRFQ) in combination with DEER, and 4) Rosetta-based molecular modeling. The proposal includes four investigators who are pioneers in each of these methods. The use of all four methods on the same ion channel under the same conditions is synergistic and ultimately will lead to a comprehensive structural and energetic model for the allostery of this channel. Ultimately a molecular understanding of these channels would inform not only the physiology and pathophysiology of the heart and brain, but also the general mechanisms for allosteric control of many enzymes.

PROJECT SUMMARY One of the most pressing public health priorities for the COVID-19 pandemic is the development of an effective and inexpensive therapeutic. The long-term goal of this proposal is to develop such COVID-19 treatments, as well as the methods needed to rapidly create such molecules as soon as any new pathogen is identified. The central hypothesis is that computational design can be used to quickly create proteins with potent antiviral activity and others that suppress ?cytokine storms? associated with advanced infection. Such countermeasures, if rapidly developed and deployed, could save millions of lives during an outbreak until vaccines are developed. The specific aims are to: 1) overcome current limitations in the discovery and development of protein therapeutics by creating methods for the de novo design of hyper-stable miniproteins that bind tightly to vulnerable binding sites on the SARS-CoV-2 Spike glycoprotein, including the receptor binding domain (RBD) of the ACE-2 cellular receptor and the fusion peptide region; 2) Enhance the avidity of such anti-Spike minibinders through genetic fusion of multiple copies, or through rational design of higher-order oligomers to create drug compounds that are less prone to viral mutagenic escape; 3) Apply the same minibinder design pipeline to create cytokine receptor antagonists of key cytokines IL-6 and IL-1? likely involved in acute respiratory distress syndrome (ADRS) associated with COVID-19 mortality; 4) Assess the efficacy of antiviral and anti-interleukin minibinders by several routes of delivery (intravenous, intranasal and subcutaneous) in rodent models of COVID-19 and assess immunogenicity in order to identify those designs best suited for further preclinical development. As proof of principle, the first anti-Spike minibinders have already been designed, were found to bind to SARS-CoV-2 Spike RBD, and were found to neutralize live virus with activities rivaling the most potent known antibodies. This proposal is innovative because it seeks to apply powerful emerging methods in the computational design of new protein therapeutics to the COVID-19 pandemic. The proposal is significant because it would be the first example of computational protein design yielding potent and entirely de novo antiviral and anti-inflammatory therapeutics for an active pandemic. Ultimately, rapid minibinder design methods have the potential to generate treatments for future pandemics, as well as for many other common and neglected diseases and conditions.

Cross-species transmission of pathogens is a major threat to public health worldwide and accounts for 75% of emerging human infectious diseases. Bats act as asymptomatic reservoir hosts for numerous zoonotic viruses, that are lethal in humans and for which no vaccines or specific therapeutics exist, indicating that the chiropteran immune system can control these viruses. Although there has been a recent growing interest in the peculiarities of bat innate immunity, their humoral immune response remains unexplored at the molecular level despite its known participation to fighting off pathogens. Previous studies revealed unusually fast bat B cell proliferation, compared to other mammals, and an exceptional immunoglobulin combinatorial diversity, suggesting a possible way these mammals successfully cope with an astounding diversity of viruses. We propose that the long co-evolution of bats with viruses could have led to the presence of highly specific immunoglobulin variable heavy chain segments playing a role in successfully controlling pathogens and that bat antibodies represent an untapped source of viral inhibitors. The proposed project will illuminate the role of bat humoral immunity in controlling pathogen infections, identify novel therapeutics against zoonotic viruses and guide the computational design of next-generation protein inhibitors of viral entry. This work will generate tools to combat emerging and re-emerging zoonotic viruses, including some pathogens that have not yet emerged or been discovered, and will be key to assist pandemic preparedness effort.