Licorice-saponin A3 is a broad-spectrum inhibitor for COVID-19 by targeting viral spike and anti-inflammation.

Currently, human health due to corona virus disease 2019 (COVID-19) pandemic has been seriously threatened. The coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein plays a crucial role in virus transmission and several S-based therapeutic approaches have been approved for the treatment of COVID-19. However, the efficacy is compromised by the SARS-CoV-2 evolvement and mutation. Here we report the SARS-CoV-2 S protein receptor-binding domain (RBD) inhibitor licorice-saponin A3 (A3) could widely inhibit RBD of SARS-CoV-2 variants, including Beta, Delta, and Omicron BA.1, XBB and BQ1.1. Furthermore, A3 could potently inhibit SARS-CoV-2 Omicron virus in Vero E6 cells, with EC50 of 1.016 µM. The mechanism was related with binding with Y453 of RBD determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis combined with quantum mechanics/molecular mechanics (QM/MM) simulations. Interestingly, phosphoproteomics analysis and multi fluorescent immunohistochemistry (mIHC) respectively indicated that A3 also inhibits host inflammation by directly modulating the JNK and p38 MAPK pathways and rebalancing the corresponding immune dysregulation. This work supports A3 as a promising broad-spectrum small molecule drug candidate for COVID-19.

Cross-species recognition and molecular basis of SARS-CoV-2 and SARS-CoV binding to ACE2s of marine animals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has an extremely broad host range that includes hippopotami, which are phylogenetically closely related to whales. The cellular ACE2 receptor is one of the key determinants of the host range. Here, we found that ACE2s from several marine mammals and hippopotami could efficiently bind to the receptor-binding domain (RBD) of both SARS-CoV and SARS-CoV-2 and facilitate the transduction of SARS-CoV and SARS-CoV-2 pseudoviruses into ACE2-expressing cells. We further resolved the cryo-electron microscopy complex structures of the minke whale ACE2 and sea lion ACE2, respectively, bound to the RBDs, revealing that they have similar binding modes to human ACE2 when it comes to the SARS-CoV-2 RBD and SARS-CoV RBD. Our results indicate that marine mammals could potentially be new victims or virus carriers of SARS-CoV-2, which deserves further careful investigation and study. It will provide an early warning for the prospective monitoring of marine mammals.

In Silico Discovery of Small Molecule Modulators Targeting the Achilles' Heel of SARS-CoV-2 Spike Protein.

The spike protein of SARS-CoV-2 has been a promising target for developing vaccines and therapeutics due to its crucial role in the viral entry process. Previously reported cryogenic electron microscopy (cryo-EM) structures have revealed that free fatty acids (FFA) bind with SARS-CoV-2 spike protein, stabilizing its closed conformation and reducing its interaction with the host cell target in vitro. Inspired by these, we utilized a structure-based virtual screening approach against the conserved FFA-binding pocket to identify small molecule modulators of SARS-CoV-2 spike protein, which helped us identify six hits with micromolar binding affinities. Further evaluation of their commercially available and synthesized analogs enabled us to discover a series of compounds with better binding affinities and solubilities. Notably, our identified compounds exhibited similar binding affinities against the spike proteins of the prototypic SARS-CoV-2 and a currently circulating Omicron BA.4 variant. Furthermore, the cryo-EM structure of the compound SPC-14 bound spike revealed that SPC-14 could shift the conformational equilibrium of the spike protein toward the closed conformation, which is human ACE2 (hACE2) inaccessible. Our identified small molecule modulators targeting the conserved FFA-binding pocket could serve as the starting point for the future development of broad-spectrum COVID-19 intervention treatments.

In Silico Discovery of Small Molecule Modulators Targeting the Achilles' Heel of SARS-CoV-2 Spike Protein

The spike protein of SARS-CoV-2 has been a promising target for developing vaccines and therapeutics due to its crucial role in the viral entry process. Previously reported cryogenic electron microscopy (cryo-EM) structures have revealed that free fatty acids (FFA) bind with SARS-CoV-2 spike protein, stabilizing its closed conformation and reducing its interaction with the host cell target in vitro. Inspired by these, we utilized a structure-based virtual screening approach against the conserved FFA-binding pocket to identify small molecule modulators of SARS-CoV-2 spike protein, which helped us identify six hits with micromolar binding affinities. Further evaluation of their commercially available and synthesized analogs enabled us to discover a series of compounds with better binding affinities and solubilities. Notably, our identified compounds exhibited similar binding affinities against the spike proteins of the prototypic SARS-CoV-2 and a currently circulating Omicron BA.4 variant. Furthermore, the cryo-EM structure of the compound SPC-14 bound spike revealed that SPC-14 could shift the conformational equilibrium of the spike protein toward the closed conformation, which is human ACE2 (hACE2) inaccessible. Our identified small molecule modulators targeting the conserved FFA-binding pocket could serve as the starting point for the future development of broad-spectrum COVID-19 intervention treatments. Computationally identified compound SPC-14 stabilizes the ACE2-inaccessible closed conformation of the SARS-CoV-2 spike protein by targeting a less mutation-prone free fatty acids-binding pocket.

Broader-species receptor binding and structural bases of Omicron SARS-CoV-2 to both mouse and palm-civet ACE2s.

The Omicron variant of SARS-CoV-2 carries multiple unusual mutations, particularly in the receptor-binding domain (RBD) of the spike (S) protein. Moreover, host-adapting mutations, such as residues 493, 498, and 501, were also observed in the Omicron RBD, which indicates that it is necessary to evaluate the interspecies transmission risk of the Omicron variant. Herein, we evaluated the interspecies recognition of the Omicron BA.1 and Delta RBDs by 27 ACE2 orthologs, including humans. We found that Omicron BA.1 expanded its receptor binding spectra to palm-civet, rodents, more bats (least horseshoe bat and greater horseshoe bat) and lesser hedgehog tenrec. Additionally, we determined the cryo-electron microscopy (cryo-EM) structure of the Omicron BA.1 S protein complexed with mouse ACE2 (mACE2) and the crystal structure of Omicron RBD complexed with palm-civet ACE2 (cvACE2). Several key residues for the host range have been identified. These results suggest that surveillance should be enhanced on the Omicron variant for its broader-species receptor binding to prevent spillover and expansion of reservoir hosts for a prolonged pandemic.

Binding and structural basis of equine ACE2 to RBDs from SARS-CoV, SARS-CoV-2 and related coronaviruses.

The origin and host range of SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), are important scientific questions as they might provide insight into understanding of the potential future spillover to infect humans. Here, we tested the binding between equine angiotensin converting enzyme 2 (eqACE2) and the receptor binding domains (RBDs) of SARS-CoV, SARS-CoV-2 prototype (PT) and variant of concerns (VOCs), as well as their close relatives bat-origin coronavirus (CoV) RaTG13 and pangolin-origin CoVs GX/P2V/2017 and GD/1/2019. We also determined the crystal structures of eqACE2/RaTG13-RBD, eqACE2/SARS-CoV-2 PT-RBD and eqACE2/Omicron BA.1-RBD. We identified S494 of SARS-COV-2 PT-RBD as an important residue in the eqACE2/SARS-COV-2 PT-RBD interaction and found that N501Y, the commonly recognized enhancing mutation, attenuated the binding affinity with eqACE2. Our work demonstrates that horses are potential targets for SARS-CoV-2 and highlights the importance of continuous surveillance on SARS-CoV-2 and related CoVs to prevent spillover events.

Structural basis of human ACE2 higher binding affinity to currently circulating Omicron SARS-CoV-2 sub-variants BA.2 and BA.1.1.

The currently circulating Omicron sub-variants are the SARS-CoV-2 strains with the highest number of known mutations. Herein, we found that human angiotensin-converting enzyme 2 (hACE2) binding affinity to the receptor-binding domains (RBDs) of the four early Omicron sub-variants (BA.1, BA.1.1, BA.2, and BA.3) follows the order BA.1.1 > BA.2 > BA.3 ≈ BA.1. The complex structures of hACE2 with RBDs of BA.1.1, BA.2, and BA.3 reveal that the higher hACE2 binding affinity of BA.2 than BA.1 is related to the absence of the G496S mutation in BA.2. The R346K mutation in BA.1.1 majorly affects the interaction network in the BA.1.1 RBD/hACE2 interface through long-range alterations and contributes to the higher hACE2 affinity of the BA.1.1 RBD than the BA.1 RBD. These results reveal the structural basis for the distinct hACE2 binding patterns among BA.1.1, BA.2, and BA.3 RBDs.

Atlas of currently available human neutralizing antibodies against SARS-CoV-2 and escape by Omicron sub-variants BA.1/BA.1.1/BA.2/BA.3.

SARS-CoV-2 Omicron variant has presented significant challenges to current antibodies and vaccines. Herein, we systematically compared the efficacy of 50 human monoclonal antibodies (mAbs), covering the seven identified epitope classes of the SARS-CoV-2 RBD, against Omicron sub-variants BA.1, BA.1.1, BA.2, and BA.3. Binding and pseudovirus-based neutralizing assays revealed that 37 of the 50 mAbs lost neutralizing activities, whereas the others displayed variably decreased activities against the four Omicron sub-variants. BA.2 was found to be more sensitive to RBD-5 antibodies than the other sub-variants. Furthermore, a quaternary complex structure of BA.1 RBD with three mAbs showing different neutralizing potencies against Omicron provided a basis for understanding the immune evasion of Omicron sub-variants and revealed the lack of G446S mutation accounting for the sensitivity of BA.2 to RBD-5 mAbs. Our results may guide the application of the available mAbs and facilitate the development of universal therapeutic antibodies and vaccines against COVID-19.

Receptor binding and complex structures of human ACE2 to spike RBD from omicron and delta SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic continues worldwide with many variants arising, some of which are variants of concern (VOCs). A recent VOC, omicron (B.1.1.529), which obtains a large number of mutations in the receptor-binding domain (RBD) of the spike protein, has risen to intense scientific and public attention. Here, we studied the binding properties between the human receptor ACE2 (hACE2) and the VOC RBDs and resolved the crystal and cryoelectron microscopy structures of the omicron RBD-hACE2 complex as well as the crystal structure of the delta RBD-hACE2 complex. We found that, unlike alpha, beta, and gamma, omicron RBD binds to hACE2 at a similar affinity to that of the prototype RBD, which might be due to compensation of multiple mutations for both immune escape and transmissibility. The complex structures of omicron RBD-hACE2 and delta RBD-hACE2 reveal the structural basis of how RBD-specific mutations bind to hACE2.

Binding and molecular basis of the bat coronavirus RaTG13 virus to ACE2 in humans and other species.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading worldwide, causing a global pandemic. Bat-origin RaTG13 is currently the most phylogenetically related virus. Here we obtained the complex structure of the RaTG13 receptor binding domain (RBD) with human ACE2 (hACE2) and evaluated binding of RaTG13 RBD to 24 additional ACE2 orthologs. By substituting residues in the RaTG13 RBD with their counterparts in the SARS-CoV-2 RBD, we found that residue 501, the major position found in variants of concern (VOCs) 501Y.V1/V2/V3, plays a key role in determining the potential host range of RaTG13. We also found that SARS-CoV-2 could induce strong cross-reactive antibodies to RaTG13 and identified a SARS-CoV-2 monoclonal antibody (mAb), CB6, that could cross-neutralize RaTG13 pseudovirus. These results elucidate the receptor binding and host adaption mechanisms of RaTG13 and emphasize the importance of continuous surveillance of coronaviruses (CoVs) carried by animal reservoirs to prevent another spillover of CoVs.