Structural basis of main proteases of HCoV-229E bound to inhibitor PF-07304814 and PF-07321332

PF-07321332 and PF-07304814, inhibitors against SARS-CoV-2 developed by Pfizer, exhibit broad-spectrum inhibitory activity against the main protease (Mpro) from various coronaviruses. Structures of PF-07321332 or PF-07304814 in complex with Mpros of various coronaviruses reveal their inhibitory mechanisms against different Mpros. However, the structural information on the lower pathogenic coronavirus Mpro with PF-07321332 or PF-07304814 is currently scarce, which hinders our comprehensive understanding of the inhibitory mechanisms of these two inhibitors. Meanwhile, given that some immunocompromised individuals are still affected by low pathogenic coronaviruses, we determined the structures of lower pathogenic coronavirus HCoV-229E Mpro with PF-07321332 and PF-07304814, respectively, and analyzed and defined in detail the structural basis for the inhibition of HCoV-229E Mpro by both inhibitors. Further, we compared the crystal structures of multiple coronavirus Mpro complexes with PF-07321332 or PF-07304814 to illustrate the differences in the interaction of Mpros, and found that the inhibition mechanism of lower pathogenic coronavirus Mpro was more similar to that of moderately pathogenic coronaviruses. Our structural studies provide new insights into drug development for low pathogenic coronavirus Mpro, and provide theoretical basis for further optimization of both inhibitors to contain potential future coronaviruses.

Structural basis of main proteases of HCoV-229E bound to inhibitor PF-07304814 and PF-07321332.

PF-07321332 and PF-07304814, inhibitors against SARS-CoV-2 developed by Pfizer, exhibit broad-spectrum inhibitory activity against the main protease (Mpro) from various coronaviruses. Structures of PF-07321332 or PF-07304814 in complex with Mpros of various coronaviruses reveal their inhibitory mechanisms against different Mpros. However, the structural information on the lower pathogenic coronavirus Mpro with PF-07321332 or PF-07304814 is currently scarce, which hinders our comprehensive understanding of the inhibitory mechanisms of these two inhibitors. Meanwhile, given that some immunocompromised individuals are still affected by low pathogenic coronaviruses, we determined the structures of lower pathogenic coronavirus HCoV-229E Mpro with PF-07321332 and PF-07304814, respectively, and analyzed and defined in detail the structural basis for the inhibition of HCoV-229E Mpro by both inhibitors. Further, we compared the crystal structures of multiple coronavirus Mpro complexes with PF-07321332 or PF-07304814 to illustrate the differences in the interaction of Mpros, and found that the inhibition mechanism of lower pathogenic coronavirus Mpro was more similar to that of moderately pathogenic coronaviruses. Our structural studies provide new insights into drug development for low pathogenic coronavirus Mpro, and provide theoretical basis for further optimization of both inhibitors to contain potential future coronaviruses.

Structure-Based Discovery and Structural Basis of a Novel Broad-Spectrum Natural Product against the Main Protease of Coronavirus.

Over the past 20 years, the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2 emerged, causing severe human respiratory diseases throughout the globe. Developing broad-spectrum drugs would be invaluable in responding to new, emerging coronaviruses and to address unmet urgent clinical needs. Main protease (Mpro; also known as 3CLpro) has a major role in the coronavirus life cycle and is one of the most important targets for anti-coronavirus agents. We show that a natural product, noncovalent inhibitor, shikonin, is a pan-main protease inhibitor of SARS-CoV-2, SARS-CoV, MERS-CoV, human coronavirus (HCoV)-HKU1, HCoV-NL63, and HCoV-229E with micromolar half maximal inhibitory concentration (IC50) values. Structures of the main protease of different coronavirus genus, SARS-CoV from the betacoronavirus genus and HCoV-NL63 from the alphacoronavirus genus, were determined by X-ray crystallography and revealed that the inhibitor interacts with key active site residues in a unique mode. The structure of the main protease inhibitor complex presents an opportunity to discover a novel series of broad-spectrum inhibitors. These data provide substantial evidence that shikonin and its derivatives may be effective against most coronaviruses as well as emerging coronaviruses of the future. Given the importance of the main protease for coronavirus therapeutic indication, insights from these studies should accelerate the development and design of safer and more effective antiviral agents. IMPORTANCE The current pandemic has created an urgent need for broad-spectrum inhibitors of SARS-CoV-2. The main protease is relatively conservative compared to the spike protein and, thus, is one of the most promising targets in developing anti-coronavirus agents. We solved the crystal structures of the main protease of SARS-CoV and HCoV-NL63 that bound to shikonin. The structures provide important insights, have broad implications for understanding the structural basis underlying enzyme activity, and can facilitate rational design of broad-spectrum anti-coronavirus ligands as new therapeutic agents.

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.

Mechanism of Microbial Metabolite Leupeptin in the Treatment of COVID-19 by Traditional Chinese Medicine Herbs.

Coronavirus disease 2019 (COVID-19) has caused huge deaths and economic losses worldwide in the current pandemic. The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is thought to be an ideal drug target for treating COVID-19. Leupeptin, a broad-spectrum covalent inhibitor of serine, cysteine, and threonine proteases, showed inhibitory activity against Mpro, with a 50% inhibitory concentration (IC50) value of 127.2 µM in vitro in our study here. In addition, leupeptin can also inhibit SARS-CoV-2 in Vero cells, with 50% effective concentration (EC50) values of 42.34 µM. More importantly, various strains of streptomyces that have a broad symbiotic relationship with medicinal plants can produce leupeptin and leupeptin analogs to regulate autogenous proteases. Fingerprinting and structure elucidation using high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS), respectively, further proved that the Qing-Fei-Pai-Du (QFPD) decoction, a traditional Chinese medicine (TCM) formula for the effective treatment of COVID-19 during the period of the Wuhan outbreak, contains leupeptin. All these results indicate that leupeptin at least contributes to the antiviral activity of the QFPD decoction against SARS-CoV-2. This also reminds us to pay attention to the microbiomes in TCM herbs as streptomyces in the soil might produce leupeptin that will later infiltrate the medicinal plant. We propose that plants, microbiome, and microbial metabolites form an ecosystem for the effective components of TCM herbs. IMPORTANCE A TCM formula has played an important role in the treatment of COVID-19 in China. However, the mechanism of TCM action is still unclear. In this study, we identified leupeptin, a metabolite produced by plant-symbiotic actinomyces (PSA), which showed antiviral activity in both cell culture and enzyme assays. Moreover, leupeptin found in the QFPD decoction was confirmed by both HPLC fingerprinting and HRMS. These results suggest that leupeptin likely contributes to the antiviral activity of the QFPD decoction against SARS-CoV-2. This result gives us important insight into further studies of the PSA metabolite and medicinal plant ecosystem for future TCM modernization research.

Screening of potent neutralizing antibodies against SARS-CoV-2 using convalescent patients-derived phage-display libraries.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten public health worldwide, the development of effective interventions is urgently needed. Neutralizing antibodies (nAbs) have great potential for the prevention and treatment of SARS-CoV-2 infection. In this study, ten nAbs were isolated from two phage-display immune libraries constructed from the pooled PBMCs of eight COVID-19 convalescent patients. Eight of them, consisting of heavy chains encoded by the immunoglobulin heavy-chain gene-variable region (IGHV)3-66 or IGHV3-53 genes, recognized the same epitope on the receptor-binding domain (RBD), while the remaining two bound to different epitopes. Among the ten antibodies, 2B11 exhibited the highest affinity and neutralization potency against the original wild-type (WT) SARS-CoV-2 virus (KD = 4.76 nM for the S1 protein, IC50 = 6 ng/mL for pseudoviruses, and IC50 = 1 ng/mL for authentic viruses), and potent neutralizing ability against B.1.1.7 pseudoviruses. Furthermore, 1E10, targeting a distinct epitope on RBD, exhibited different neutralization efficiency against WT SARS-CoV-2 and its variants B.1.1.7, B.1.351, and P.1. The crystal structure of the 2B11-RBD complexes revealed that the epitope of 2B11 highly overlaps with the ACE2-binding site. The in vivo experiment of 2B11 using AdV5-hACE2-transduced mice showed encouraging therapeutic and prophylactic efficacy against SARS-CoV-2. Taken together, our results suggest that the highly potent SARS-CoV-2-neutralizing antibody, 2B11, could be used against the WT SARS-CoV-2 and B.1.1.7 variant, or in combination with a different epitope-targeted neutralizing antibody, such as 1E10, against SARS-CoV-2 variants.

A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes.

COVID-19 pandemic caused by SARS-CoV-2 constitutes a global public health crisis with enormous economic consequences. Monoclonal antibodies against SARS-CoV-2 can provide an important treatment option to fight COVID-19, especially for the most vulnerable populations. In this work, potent antibodies binding to SARS-CoV-2 Spike protein were identified from COVID-19 convalescent patients. Among them, P4A1 interacts directly with and covers majority of the Receptor Binding Motif of the Spike Receptor-Binding Domain, shown by high-resolution complex structure analysis. We further demonstrate the binding and neutralizing activities of P4A1 against wild type and mutant Spike proteins or pseudoviruses. P4A1 was subsequently engineered to reduce the potential risk for Antibody-Dependent Enhancement of infection and to extend its half-life. The engineered antibody exhibits an optimized pharmacokinetic and safety profile, and it results in complete viral clearance in a rhesus monkey model of COVID-19 following a single injection. These data suggest its potential against SARS-CoV-2 related diseases.

Structural basis for SARS-CoV-2 neutralizing antibodies with novel binding epitopes.

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) threatens global public health and economy unprecedentedly, requiring accelerating development of prophylactic and therapeutic interventions. Molecular understanding of neutralizing antibodies (NAbs) would greatly help advance the development of monoclonal antibody (mAb) therapy, as well as the design of next generation recombinant vaccines. Here, we applied H2L2 transgenic mice encoding the human immunoglobulin variable regions, together with a state-of-the-art antibody discovery platform to immunize and isolate NAbs. From a large panel of isolated antibodies, 25 antibodies showed potent neutralizing activities at sub-nanomolar levels by engaging the spike receptor-binding domain (RBD). Importantly, one human NAb, termed PR1077, from the H2L2 platform and 2 humanized NAb, including PR953 and PR961, were further characterized and subjected for subsequent structural analysis. High-resolution X-ray crystallography structures unveiled novel epitopes on the receptor-binding motif (RBM) for PR1077 and PR953, which directly compete with human angiotensin-converting enzyme 2 (hACE2) for binding, and a novel non-blocking epitope on the neighboring site near RBM for PR961. Moreover, we further tested the antiviral efficiency of PR1077 in the Ad5-hACE2 transduction mouse model of COVID-19. A single injection provided potent protection against SARS-CoV-2 infection in either prophylactic or treatment groups. Taken together, these results shed light on the development of mAb-related therapeutic interventions for COVID-19.

Structurally Resolved SARS-CoV-2 Antibody Shows High Efficacy in Severely Infected Hamsters and Provides a Potent Cocktail Pairing Strategy.

Understanding how potent neutralizing antibodies (NAbs) inhibit SARS-CoV-2 is critical for effective therapeutic development. We previously described BD-368-2, a SARS-CoV-2 NAb with high potency; however, its neutralization mechanism is largely unknown. Here, we report the 3.5-Å cryo-EM structure of BD-368-2/trimeric-spike complex, revealing that BD-368-2 fully blocks ACE2 recognition by occupying all three receptor-binding domains (RBDs) simultaneously, regardless of their "up" or "down" conformations. Also, BD-368-2 treats infected adult hamsters at low dosages and at various administering windows, in contrast to placebo hamsters that manifested severe interstitial pneumonia. Moreover, BD-368-2's epitope completely avoids the common binding site of VH3-53/VH3-66 recurrent NAbs, evidenced by tripartite co-crystal structures with RBDs. Pairing BD-368-2 with a potent recurrent NAb neutralizes SARS-CoV-2 pseudovirus at pM level and rescues mutation-induced neutralization escapes. Together, our results rationalized a new RBD epitope that leads to high neutralization potency and demonstrated BD-368-2's therapeutic potential in treating COVID-19.

Both Boceprevir and GC376 efficaciously inhibit SARS-CoV-2 by targeting its main protease.

COVID-19 was declared a pandemic on March 11 by WHO, due to its great threat to global public health. The coronavirus main protease (Mpro, also called 3CLpro) is essential for processing and maturation of the viral polyprotein, therefore recognized as an attractive drug target. Here we show that a clinically approved anti-HCV drug, Boceprevir, and a pre-clinical inhibitor against feline infectious peritonitis (corona) virus (FIPV), GC376, both efficaciously inhibit SARS-CoV-2 in Vero cells by targeting Mpro. Moreover, combined application of GC376 with Remdesivir, a nucleotide analogue that inhibits viral RNA dependent RNA polymerase (RdRp), results in sterilizing additive effect. Further structural analysis reveals binding of both inhibitors to the catalytically active side of SARS-CoV-2 protease Mpro as main mechanism of inhibition. Our findings may provide critical information for the optimization and design of more potent inhibitors against the emerging SARS-CoV-2 virus.

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

The recent emergence of a novel coronavirus (SARS-CoV-2) in China has caused significant public health concerns. Recently, ACE2 was reported as an entry receptor for SARS-CoV-2. In this study, we present the crystal structure of the C-terminal domain of SARS-CoV-2 (SARS-CoV-2-CTD) spike (S) protein in complex with human ACE2 (hACE2), which reveals a hACE2-binding mode similar overall to that observed for SARS-CoV. However, atomic details at the binding interface demonstrate that key residue substitutions in SARS-CoV-2-CTD slightly strengthen the interaction and lead to higher affinity for receptor binding than SARS-RBD. Additionally, a panel of murine monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) against SARS-CoV-S1/receptor-binding domain (RBD) were unable to interact with the SARS-CoV-2 S protein, indicating notable differences in antigenicity between SARS-CoV and SARS-CoV-2. These findings shed light on the viral pathogenesis and provide important structural information regarding development of therapeutic countermeasures against the emerging virus.

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

A new and highly pathogenic coronavirus (severe acute respiratory syndrome coronavirus-2, SARS-CoV-2) caused an outbreak in Wuhan city, Hubei province, China, starting from December 2019 that quickly spread nationwide and to other countries around the world1-3. Here, to better understand the initial step of infection at an atomic level, we determined the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 bound to the cell receptor ACE2. The overall ACE2-binding mode of the SARS-CoV-2 RBD is nearly identical to that of the SARS-CoV RBD, which also uses ACE2 as the cell receptor4. Structural analysis identified residues in the SARS-CoV-2 RBD that are essential for ACE2 binding, the majority of which either are highly conserved or share similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly indicate convergent evolution between the SARS-CoV-2 and SARS-CoV RBDs for improved binding to ACE2, although SARS-CoV-2 does not cluster within SARS and SARS-related coronaviruses1-3,5. The epitopes of two SARS-CoV antibodies that target the RBD are also analysed for binding to the SARS-CoV-2 RBD, providing insights into the future identification of cross-reactive antibodies.