key: cord-334378-dqtnj3y3
authors: Zhang, Yi; Kutateladze, Tatiana G.
title: Molecular structure analyses suggest strategies to therapeutically target SARS-CoV-2
date: 2020-06-10
journal: Nat Commun
DOI: 10.1038/s41467-020-16779-4
sha: 
doc_id: 334378
cord_uid: dqtnj3y3

Amid the COVID-19 pandemic, scientists around the globe have been working resolutely to find therapies to treat patients and avert the spreading of the SARS-CoV-2 virus. In this commentary, we highlight some of the latest studies that provide atomic-resolution structural details imperative for the development of vaccines and antiviral therapeutics.

conformations, which is also observed in the other coronaviruses 4 . In the closed conformation, all three RBDs are buried at the interface between three protomers, whereas in the open conformation one of RBDs rotates up and therefore is primed for binding to ACE2. In addition, Wrapp et al. demonstrate that the open conformation represents a predominant state of the spike protein trimer 2 .

How does RBD of the SARS-CoV-2 spike protein binds to the human receptor ACE2? The studies by Shang et al., Lan et al., and Yan et al. illuminate the atomic-resolution details of this interaction [5] [6] [7] (Fig. 1c) . Of note, ACE2 normally functions to promote the maturation of angiotensin hormone which controls blood pressure. Aberrant ACE2 levels have been linked to cardiovascular diseases 8 and may play a role in disease severity observed in COVID-19 patients with comorbid conditions, such as heart, blood, and lung diseases and diabetes. Yan et al. describe a cryo-EM structure of the complex of full length human ACE2 with RBD from the SARS-CoV-2 spike protein and suggest that two spike protein trimers can simultaneously bind to an ACE2 dimer 7 .

The precise binding interface of the SARS-CoV-2 spike RBD and ACE2 can be dissected from the crystal structures of the complex determined by Shang et al. and Lan et al 5, 6 (Fig. 1d) . Analyzing the structure, Shang et al. uncovered several hotspots that distinguish binding of SARS-CoV-2 and SARS-CoV 5 . The authors found that overall, the RBD-ACE2 interface in the SARS-CoV-2 complex is larger than the binding interface in the SARS-CoV complex. RBD of SARS-CoV-2 forms more contacts with ACE2, including three additional intermolecular salt bridges, involving Q493 and G496 of RBD and K31, E35, and K353 of ACE2 (dashed lines in Fig. 1d ). The receptor-binding ridge of RBD in the SARS-CoV-2 complex adopts a more compact conformation, allowing an adjacent loop to move closer to ACE2 and F486 of RBD to insert in the hydrophobic pocket of ACE2. These structural differences could account for the 4-to 20-fold increase in binding affinity of the SARS-CoV-2 spike RBD toward ACE2 compared to that of SARS-CoV, which was observed independently by several groups 2,4-6 .

Analysis of the crystal structures of RBD from the SARS-CoV spike protein in complex with established SARS-CoV neutralizing human m396 and 80R antibodies reveals that the antibodies are bound in the same binding site as ACE2 9,10 , implying that their neutralizing mechanism is a direct blockage of receptor binding. Lan et al. estimate that 7 out of 21 epitope residues for m396 and 16 out of 25 epitope residues for 80R are not conserved in RBD sequences of SARS-CoV and SARS-CoV-2, which may explain the lack of appreciable cross-reactivity for these antibodies 6 . Interestingly, a recent structure of RBD from the SARS-CoV-2 spike protein bound to another antibody isolated from a SARS patient, CR3022, determined by Yuan et al. shows that CR3022 targets an epitope distal from the ACE2-binding site, which may provide some cross-reactivity that needs to be thoroughly evaluated 11 .

What are other therapeutic targets beyond the receptorbinding domain of the SARS-CoV-2 spike protein? Proteolytic cleavage of the SARS-CoV-2 spike protein by the human serine protease TMPRSS2 is a critical step in the virus entry through the plasma membrane fusion mechanism (Fig. 2) , as shown by Hoffmann et al. 12 . The authors demonstrate that camostat mesylate, a clinically proven serine protease inhibitor that halts TMPRSS2 activity, blocks SARS-CoV-2 infection of lung cells 12 . Following binding to ACE2 and disassociation of the ACE2bound S 1 subunit, the S 2 subunit of the spike protein-still attached to the virus envelope through its C-terminal transmembrane domain and additionally cleaved at the S 2 ′ siteinserts the cleaved terminus into the host cell plasma membrane and thus physically links both viral and cell membranes. This allows the heptad repeats (HR1 and HR2) of the S 2 subunit to interact and assemble into a six-helix bundle, which is needed to bring the membranes closer to each other for final fusion. Xia et al. now show that EK1C4, a lipopeptide that targets heptad repeats, is a potent inhibitor of SARS-CoV-2 fusion in cellular assays and in a mouse model 13 . By using human clinical-grade recombinant soluble ACE2, Monteil et al. reduced SARS-CoV-2 viral growth by over 10 3fold in Vero cells and suppressed infection in engineered human blood vessel organoids and kidney organoids 14 .

In addition to targeting components of the viral attachment and the membrane fusion machinery, other strategies to eliminate SARS-CoV-2 include impeding virus entry into the host cell through the endosomal pathway and disrupting activities of viral proteins. Ou or cathepsin L by apilimod, tetrandrine, or SID 26681509, respectively, reduces host cell entry 3 . Once inside the host cell, viral RNA uses the host ribosomal machinery to produce important for its own life cycle proteins that also serve as attractive targets for the development of anti-SARS-CoV-2 therapeutics. One of such proteins is the SARS-CoV-2 main protease M pro , which is required for processing the polyproteins translated from the viral RNA. Inhibition of the M pro activity impedes viral replication, and the crystal structure of the SARS-CoV-2 M pro in complex with the α-ketoamide inhibitor provides the mechanistic details necessary to advance in this direction 15 .

The speed by which structural information on the SARS-CoV-2 virus has been collected is astonishing. Structural approaches will likely contribute to drug discoveries and validation and aid in the development of vaccines to reduce and eventually eliminate the ongoing public health emergency. 

A new coronavirus associated with human respiratory disease in China

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

Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein

Structural basis of receptor recognition by SARS-CoV-2

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

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

Role of the ACE2/ Angiotensin 1-7 axis of the renin-angiotensin system in heart failure

Structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody

Structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, 80R

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

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion

Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2

Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors

Research in the Kutateladze laboratory is funded by the NIH. 

The authors declare no competing interests.

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