key: cord-0814825-j3bmbqfi
authors: Cox, Robert M; Plemper, Richard K
title: The impact of high-resolution structural data on stemming the COVID-19 pandemic
date: 2021-06-03
journal: Curr Opin Virol
DOI: 10.1016/j.coviro.2021.05.005
sha: c6021b980b61c9be638a0bbaa747e813cf4cbcac
doc_id: 814825
cord_uid: j3bmbqfi

The coronavirus disease 2019 (COVID-19) pandemic has had a catastrophic impact on human health and the world economy. The response of the scientific community was unparalleled, and a combined global effort has resulted in the creation of vaccines in a shorter time frame than previously unimaginable. Reflecting this concerted effort, the structural analysis of the etiological agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has progressed with an unprecedented pace. Since the onset of the pandemic, over 1,000 high-resolution structures of a broad range of SARS-CoV-2 proteins have been solved and made publicly available. These structures have aided in the identification of numerous potential druggable targets and have contributed to the design of different vaccine candidates. This opinion article will discuss the impact of high-resolution structures in understanding SARS-CoV-2 biology and explore their role in the development of vaccines and antivirals.

The coronavirus disease 2019 (COVID- 19) pandemic has resulted in a global crisis with devastating effects on public health and the global economy. The scientific community has invested tremendous efforts into characterizing and understanding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19. An unprecedented volume of data has been produced, providing the scientific world with a plethora of information on SARS-CoV-2 biology. Shortly after publishing the SARS-CoV-2 genome sequence ( Figure 1a to develop broad-spectrum coronavirus protease inhibitors, since many of the previously identified SARS-CoV/MERS-CoV Mpro inhibitors were also active against SARS-CoV-2.

There are currently over 250 high-resolution structures deposited for SARS-CoV-2 Mpro, including co-crystal structures with different protease inhibitors and fragment compounds [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] . These crystal structures have revealed that the substrate binding pocket is well defined, independent of whether a substrate was bound or absent ( Figure   2c ) [4] [5] [6] [7] [8] [9] 11, 12] , supporting feasibility of a structure-guided inhibitor design approach.

Investigations into Mpro blockers greatly benefited from the previously developed inhibitors of SARS-CoV Mpro that showed cross-inhibitory activity against SARS-CoV-2.

Of these compounds, compound 11a and PF-07304814 are two of the most advanced Mpro inhibitors [7, 13] . Both were originally developed using structure-guided approaches [7, 13] , and both are peptidomimetics. Co-crystal structures of each inhibitor bound to SARS-CoV-2 Mpro have been solved (Figure 2c) [7, 13] . Compound 11a was originally designed based on the substrate-binding pocket of SARS-CoV Mpro. A recent report showed compound 11a to be active in cell culture against SARS-CoV-2, revealing encouraging pharmacokinetics properties in three animal models (mice, SD rats, and beagle dogs) when administered intravenously [13] . However, some acute toxicity was observed in SD rats at high doses, and antiviral efficacy has not been confirmed in any SARS-CoV-2 animal model [13] . Whereas these initial results are overall promising, compound 11a has yet to proceed to clinical trials. PF-00835231 was also developed using structure-based approaches to target SARS-CoV Mpro, and is J o u r n a l P r e -p r o o f capable of inhibiting SARS-CoV-2 in cell culture [7, 14] . Although likewise untested in animal models, initial pharmacokinetics results for PF-07304814, a phosphate prodrug of PF-00835231, were favorable and the compound is currently in a phase Ib clinical trial in hospitalized COVID-19 patients (NCT04535167). Although no SARS-CoV-2 Mpro inhibitor has succeeded in the clinic yet, data gleaned from current and previous SARS-CoV and MERS-CoV studies have provided a valuable template for how to successfully engage the Mpro active site using peptidomimetics [4-9,11-14].

Besides the common challenges of traditional drug development (e.g. PK-ADME, efficacy, stability, formulation, route of administration), structure-based approaches face the added demand of time required to solve high-resolution structures. Extensive efforts must first be made to determine protein structures that lay the groundwork for structure- 

To date, no allosteric SARS-CoV-2 RdRP inhibitors have been developed and advanced to clinical testing. For NA inhibitors, the SARS-CoV-2 exonuclease (nsp14) poses a potential problem, due to proofreading and excision of NAs such as ribavirin and 5-flurouracil, from the viral RNA [36] [37] [38] [39] [40] . The ability of remdesivir to overcome SARS-CoV-2 exonuclease activity is likely due to its delayed chain termination mechanism of action, which blocks polymerase translocation three nucleotides after incorporation [27, 28, 41] . Since several nucleotides would need to be removed to excise the incorporated form of remdesivir (GS-441524), the drug may partially avoid removal by the SARS-CoV-2 exonuclease. In support of this hypothesis, it has been observed J o u r n a l P r e -p r o o f that the rate of incorporation of GS-441524 is greater than the rate of excision by the exonuclease [42] . Identification of nucleoside analogs that can avoid detection by the SARS-CoV-2 exonuclease might boost the success of NA therapies. Favipiravir [43, 44] and molnupiravir [45] both utilize an alternate mechanism, induction of viral error catastrophe, to inhibit viral replication, albeit favipiravir is reportedly thought to act as a chain terminator also in some cases [46] . These two NAs are thought to avoid excision by the SARS-CoV-2 exonuclease, since both are rapidly incorporated into newly synthesized viral RNA and have been shown to induce lethal mutagenesis [43] [44] [45] [47] [48] [49] [50] [51] . Interestingly, sequencing of SARS-CoV-2 infected hamsters treated with favipiravir did reveal mutations in the SARS-CoV-2 exonuclease, which could represent the development of resistant virus populations [52] .

Two high-resolution cryo-EM structures of the SARS-CoV-2 RdRP in complex with favipiravir were recently solved [33, 34] . In these structures, favipiravir forms noncanonical base-pair interactions, providing an explanation for how it can be incorporated as either adenosine or guanosine. Of note, favipiravir was bound in a non-productive binding mode, which could account for the low potency observed in vitro (EC50 = 118-207 µM) and in vivo (1,000 mg/kg b.i.d. in Syrian hamsters) [33, 34, 43, 53] . Results from clinical studies are ambiguous but tend to suggest that treatment with favipiravir may offer some benefit against COVID-19 [54] [55] [56] [57] [58] [59] . However, study design and validity of the results of at least one of these trials are a subject of debate [60, 61] . Molnupiravir has demonstrated oral efficacy against SARS-CoV-2 in multiple animal models [62] [63] [64] [65] , was found to be safe for human use in phase I clinical trial [66] , and is currently in advanced To date, there are also no high-resolution structures for the SARS-CoV-2 exonuclease alone or in complex with other components of the RTC. Therefore, a key piece of information is missing for the structure-guided development of NA therapies against SARS-CoV-2. Until it is understood how the SARS-CoV-2 exonuclease recognizes incorporated NAs in newly synthesized RNA, it will be difficult to use structure-guided approaches to proactively design next generation analogs that are resistant to detection and excision.

The SARS-CoV-2 spike (S) protein is a homo-trimeric envelope glycoprotein that is responsible for receptor binding and fusion of the viral envelope with host membranes [67] . The S protein is a type I fusion protein and exists in a metastable prefusion state, which can refold into an energetically far lower stable postfusion conformation [67] . A hallmark of type I fusion proteins is synthesis as an inactive precursor protein that must be proteolytically matured into two major subunits. In the case of SARS-CoV-2 maturation occurs predominantly by furin or host TMPRSS2 cleavage into S1/S2 and S2', respectively (Figure 4a) , to gain membrane fusion activity [67] . Of the proteolysis products, S1 is entirely extracellular and mediates receptor binding, whereas transmembrane S2 induces membrane merger [67] . The predominant cognate receptor of SARS-CoV-2 is human angiotensin-converting enzyme 2 (hACE2), which is recognized by the S protein receptor-binding domain (RBD) located in S1 [68] [69] [70] [71] . Viral attachment leads to endocytotic uptake of the virion and ultimately activation of the J o u r n a l P r e -p r o o f fusogenic S2 subunit, which undergoes deep-seated structural changes that result in shedding of the S1 subunit and insertion of the fusion peptide into the host cell membrane, followed by S protein refolding into a hairpin-like structure and opening of a fusion pore [72] [73] [74] .

The ability of the host to generate a robust antibody response against the SARS-CoV-2 S protein, in particular against S1, is critical for virus neutralization and effective immunoprotection [67, 71, [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] . Therapeutic antibodies targeting the SARS-CoV-2 S protein have been granted emergency use authorization by the FDA for the treatment of COVID-19 patients (Table 1) . Structural studies mapping SARS-CoV-2 neutralizing epitopes identified two major sites for neutralizing antibodies (nAbs) derived from convalescent patient sera; the S1 N-terminal region [79, 85, 86] and the RBD [67, 70, 80, 81, 87] (Figure 4a-b) . Of particular importance are antibodies targeting the RBD, based on their potential for broad-spectrum activity against other betacoronaviruses, including MERS-CoV and SARS-CoV [67, [87] [88] [89] [90] [91] [92] . A detailed structural understanding of the conserved epitopes or binding motifs recognized by these broad-spectrum antibodies could potentially be utilized to ultimately design a universal vaccine capable of providing protection against multiple coronaviruses.

Efforts to stabilize the pre-fusion state are critical for effective vaccine design, since important neutralizing motifs are present in the S1 subunit. Historically, stabilization of type-1 fusion proteins has been implemented for numerous viral species using a wide array of techniques, such as modifying cleavage sites, introducing disulfide bonds, and stabilizing flexible regions important for the conformational change between the pre-and post-fusion states [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] . Structure-guided strategies have been applied to some of the current SARS-CoV-2 vaccines to stabilize S in a pre-fusion state and enhance the vaccine induced antibody response against the RBD and neutralizing epitopes in S1 [104, 105] . Previous structure-based studies on the fusion proteins of HIV-1 and respiratory syncytial virus identified that proline substitutions in specific regions of the S2 subunit can hinder the conformational change to a post-fusion state [93] [94] [95] . South Africa [110] [111] [112] [113] [114] [115] [116] .

Since the initial design of SARS-CoV-2 vaccine candidates utilizing S-2P, several studies have identified and structurally characterized additional substitutions in S that led to higher levels of expression and greater thermostability compared to S-2P [117] [118] [119] [120] . However, these new substitutions have not advanced to any candidates in clinical trials. pandemic is that a rapid first-line response is critical to limit virus spread. With current technology, this challenge can only be met when it is possible to build on a rich trough of pre-existing data generated for related viral pathogens. Even so, structure-guided drug design is unlikely to deliver a clinical candidate with the turn-around time required to impact the spread of a pandemic. Accordingly, no structure-guided antiviral specifically targeting SARS-CoV-2 has advanced to clinical use. However, available structural data have greatly shortened the timeline to vaccine development and approval, providing a tangible example of how proactively establishing a solid scientific foundation can prepare against an unexpected pandemic threat. 

Structure of M(pro) from SARS-CoV-2 and discovery of its inhibitors

Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication

Structural basis of SARS-CoV-2 main protease inhibition by a broadspectrum anti-coronaviral drug

Structural basis for the inhibition of SARS-CoV-2 main protease by antineoplastic drug carmofur

Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease

* Initial development of compound 11a, a potent inhibitor of the SARS-CoV-2 main protease. Designed based on the structure of the SARS-CoV Mpro substrate binding pocket

Discovery of a Novel Inhibitor of Coronavirus 3CL Protease as a Clinical Candidate for the Potential Treatment of COVID-19

* The authors performed extensive pharmacokinetic profiling of PF-00835231, the phosphate prodrug of PF-00835231. In vitro antiviral activity against SARS-CoV-2 was assessed as well as potential synergistic effects of PF-00835231 in combination with remdesivir

Discovery of a beta-d-2'-deoxy-2'-alpha-fluoro-2'-beta-C-methyluridine nucleotide prodrug (PSI-7977) for the treatment of hepatitis C virus

In vitro characterization of baloxavir acid, a first-in-class cap-dependent endonuclease inhibitor of the influenza virus polymerase PA subunit

Nucleotide Reverse Transcriptase Inhibitors: A Thorough Review, Present Status and Future Perspective as HIV Therapeutics

Structural analysis of the putative SARS-CoV-2 primase complex

Crystal Structure of the SARS-CoV-2 Non-structural Protein 9

Crystal Structure of Non-Structural Protein 10 from Severe Acute Respiratory Syndrome Coronavirus-2

High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants

Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA

Coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics

Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency

Remdesivir Is Effective in Combating COVID-19 because It Is a Better Substrate than ATP for the Viral RNA-Dependent RNA Polymerase

Rapid incorporation of Favipiravir by the fast and permissive viral RNA polymerase complex results in SARS-CoV-2 lethal mutagenesis

T-705 (favipiravir) induces lethal mutagenesis in influenza A H1N1 viruses in vitro

Orally Efficacious Broad-Spectrum Ribonucleoside Analog Inhibitor of Influenza and Respiratory Syncytial Viruses

Favipiravir, an anti-influenza drug against life-threatening RNA virus infections

Mutations in the chikungunya virus non-structural proteins cause resistance to favipiravir (T -705), a broad-spectrum antiviral

The mechanism of resistance to favipiravir in influenza

(4)-Hydroxycytidine Is a Potent Anti-alphavirus Compound That Induces a High Level of Mutations in the Viral Genome

Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia

Small-Molecule Antiviral beta-d-N (4)-Hydroxycytidine Inhibits a Proofreading-Intact Coronavirus with a High Genetic Barrier to Resistance

Favipiravir and severe acute respiratory syndrome coronavirus 2 in hamster model

Favipiravir at high doses has potent antiviral activity in SARS-CoV-2-infected hamsters, whereas hydroxychloroquine lacks activity

A Prospective, Randomized, Open-Label Trial of Early versus Late Favipiravir Therapy in Hospitalized Patients with COVID-19

Outcome of Non-Critical COVID-19 Patients with Early Hospitalization and Early Antiviral Treatment Outside the ICU

AVIFAVIR for Treatment of Patients with Moderate COVID-19: Interim Results of a Phase II/III Multicenter Randomized Clinical Trial

Clinical Outcomes and Plasma Concentrations of Baloxavir Marboxil and Favipiravir in COVID-19 Patients: An Exploratory Randomized, Controlled Trial

Efficacy and safety of favipiravir, an oral RNAdependent RNA polymerase inhibitor, in mild-to-moderate COVID-19: A randomized, comparative, open-label, multicenter, phase 3 clinical trial

Experimental Treatment with Favipiravir for COVID-19: An Open-Label Control Study. Engineering (Beijing) 2020

Favipiravir, an antiviral for COVID-19?

Comment on: Favipiravir, an antiviral for COVID-19?

Acute SARS-CoV-2 Infection is Highly Cytopathic, Elicits a Robust Innate Immune Response and is Efficiently Prevented by EIDD-2801

Therapeutically administered ribonucleoside analogue MK-4482/EIDD-2801 blocks SARS-CoV-2 transmission in ferrets

Orally delivered MK-4482 inhibits SARS-CoV-2 replication in the Syrian hamster model

Molnupiravir (EIDD-2801) inhibits SARS-CoV2 replication in Syrian hamsters model

Human Safety, Tolerability, and Pharmacokinetics of a Novel Broad-Spectrum Oral Antiviral Compound, Molnupiravir, with Activity Against SARS-CoV-2

Veesler D: Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2

Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Publication of the prefusion structure of the SARS-CoV-2 spike protein. Remarkably, the structure was released less than two months after the publication of the SARS-CoV-2 genome. The prefusion conformation was stabilized using a SARS-CoV-2 spike protein possessing the S-2P substitution

Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2

Cryo-EM Structures of SARS-CoV-2 Spike without and with ACE2 Reveal a pH-Dependent Switch to Mediate Endosomal Positioning of Receptor-Binding Domains

The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex

Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion

Structures of J o u r n a l P r e -p r o o f Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies

Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients' B Cells

Human monoclonal antibodies block the binding of SARS-CoV-2 spike protein to angiotensin converting enzyme 2 receptor

Human neutralizing antibodies elicited by SARS-CoV-2 infection

A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2

Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike

Convergent antibody responses to SARS-CoV-2 in convalescent individuals

Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model

Analysis of a SARS-CoV-2-Infected Individual Reveals Development of Potent Neutralizing Antibodies with Limited Somatic Mutation

Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein

Potent SARS-CoV-2 Neutralizing Antibodies Directed Against Spike N-Terminal Domain Target a Single Supersite

A novel neutralizing monoclonal antibody targeting the N-terminal domain of the MERS-CoV spike protein

Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody

A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV

Neutralization of SARS-CoV-2 by Destruction of the Prefusion Spike

A human monoclonal antibody blocking SARS-CoV-2 infection

Identification of SARS-CoV RBD-targeting monoclonal antibodies with cross-reactive or neutralizing activity against SARS-CoV-2

Receptor-binding domain-specific human neutralizing monoclonal antibodies against SARS-CoV and SARS-CoV-2

Stabilization of the soluble, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1

A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism

Uncleaved prefusion-optimized gp140 trimers derived from analysis of HIV-1 envelope metastability

Structure of the uncleaved human H1 hemagglutinin from the extinct 1918 influenza virus

Timing is everything: Fine-tuned molecular machines orchestrate paramyxovirus entry

A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure

Specific single or double proline substitutions in the "spring-loaded" coiled-coil region of the influenza hemagglutinin impair or abolish membrane fusion activity

Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains

Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus

*Initial discovery that proline substitutions in a hinge region of the S2 subunit can create a pre-fusion stabilized glycoprotein for respiratory syncytial virus. The first author continued working on proline substitutions to stabilize the prefusion conformations of type I fusion proteins, leading to the S-2P subsitution

A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus

Structural basis for antibody-mediated neutralization of Lassa virus

Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen

*Publication of the S-2P substitutions used by many of the current SARS-CoV-2 vaccines. The authors show that the MERS-CoV spike proteins possessing S-2P modifications were capable of eliciting high neutralizing antibody titers

Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2

Phase 1-2 Trial of a SARS-CoV-2 Recombinant Spike Protein Nanoparticle Vaccine

RNA-Based COVID-19 Vaccine BNT162b2 Selected for a Pivotal Efficacy Study

An mRNA Vaccine against SARS-CoV-2 -Preliminary Report

Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates

SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma

SARS-CoV-2 escape in vitro from a highly neutralizing COVID-19 convalescent plasma

Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants

evidence that people who receive the Moderna mRNA COVID-19 vaccine (mRNA1273) mount antibody responses capable of neutralizing SARS-CoV-2 variants possessing the K417N, E484K and N501Y mutations. Additionally, sera from non-human primates vaccinated with the Moderna mRNA vaccine were also capable of effectively neutralizing the variant SARS-CoV-2 viruses

SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies

mRNA-1273 vaccine induces neutralizing antibodies against spike mutants from global SARS-CoV-2 variants

Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K, and N501Y variants by BNT162b2 vaccine-elicited sera

This study provides experimental evidence that people who receive the Pfizer mRNA COVID-19 vaccine (BNT162b2) mount antibody responses capable of neutralizing the newly emerged United Kingdom and South African SARS-CoV-2 variants

Stabilizing the closed SARS-CoV-2 spike trimer

A thermostable, closed SARS-CoV-2 spike protein trimer

Structure-based design of prefusionstabilized SARS-CoV-2 spikes

Structure-guided covalent stabilization of coronavirus spike glycoprotein trimers in the closed conformation

Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys