Structural Landscape of SARS‐CoV‐2 entry & activation of spike glycoprotein & potential interventions

The pandemic COVID19 illness caused by SARS‐CoV‐2, which produces pneumonia and lower respiratory tract infections, is a serious public health concern, with frightening mutations causing over 4.5 million deaths globally. Whilst effective immunisation shows promise globally, several antiviral treatments are being clinically evaluated to fill the “therapeutic gap” in treating infected people. Understanding the entire repertoire of diverse host factors engaged by SARS‐CoV‐2 for entry and pathogenicity is required for long‐lasting potential therapeutics or vaccines. Here, using a structural and molecular approach, we show multistage processing of SARS‐CoV‐2 spike‐protein for virion activation, infection, and how mutations influence it. We solved the structures of spike‐protein in complex with different host cell factors (TMPRSS2, Furin, CD26 (DPP4), and NRP1) with functional activity, and these insights into uncovering how viral spike‐protein engages and primed with these multiple host factors, in addition to ACE2, to hijack host cell entry. Furthermore, our COVID19 patient genome sequencing reveals that allele in TMPRSS2 (V160M) and Furin provided protection from COVID19 infection, and its structural mechanism is further addressed and potential drug clinical trials. Additionally, our large‐scale retrospective cohort studies proved Arbidol and derivatives as potential therapies for COVID19, using structural studies, we demonstrated the mechanism of action of Arbidol in disrupting spike function. These findings cognize the complete mechanism of viral spike‐glycoprotein processing/priming that leads to cascading entry into the host cell, paving the door for future vaccine development and identifying key targets. Our comprehensive, multifaceted research reveals the complexity of the SARS‐CoV‐2 spike‐protein and clinical studies aid in therapies. References: 1. Li, F., et al.,Nature Communications 12, 866 (2021). 2. Vankadari, N. & Jacqline, A., Emeging Microbes & Infections, 9(1), 601‐604 (2020). 3. Vankadari, N. Int. Journal of Antimirobial Agents, 56(2),105998 (2020) 4. Andersen, K.G., et al.,Nature Medicine 26, 450–452 (2020). 5. Qibin Liu et al.,MedRxiv CSH Press (2021), March 22 (2) 6. Ravikanth, V et al., mGENE (2021), 5, e100930. NEJM (Reviewed) 7. Vankadari, N. ACS Journal of Physical Chemistry Letters, 11(16), 6655‐663 (2020).

Structure of Human TMPRSS2 in Complex with SARS-CoV-2 Spike Glycoprotein and Implications for Potential Therapeutics.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 520 million people around the globe resulting in more than 6.2 million as of May 2022. Understanding the cell entry mechanism of SARS-CoV-2 and its entire repertoire is a high priority for developing improved therapeutics. The SARS-CoV-2 spike glycoprotein (S-protein) engages with host receptor ACE2 for adhesion and serine proteases furin and TMPRSS2 for proteolytic activation and subsequent entry. Recent studies have highlighted the molecular details of furin and S-protein interaction. However, the structural and molecular interplay between TMPRSS2 and S-protein remains enigmatic. Here, using biochemical, structural, computational, and molecular dynamics approaches, we investigated how TMPRSS2 recognizes and activates the S-protein to facilitate viral entry. First, we identified three potential TMPRSS2 cleavage sites in the S2 domain of S-protein (S2', T1, and T2) and reported the structure of TMPRSS2 with its individual catalytic triad. By employing computational modeling and structural analyses, we modeled the macromolecular structure of TMPRSS2 in complex with S-protein, which incited the mechanism of S-protein processing or cleavage for a new path of viral entry. On the basis of structure-guided drug screening, we also report the potential TMPRSS2 inhibitors and their structural interaction in blocking TMPRSS2 activity, which could impede the interaction with the spike protein. These findings reveal the role of TMPRSS2 in the activation of SARS-CoV-2 for its entry and insight into possible intervention strategies.

Molecular interplay between SARS-CoV-2 and human proteins for viral activation and entry, potential drugs for combat and scope for new therapeutics

The pandemic Coronavirus Disease 2019 (COVID19) caused by SARS-CoV-2 is a serious public health concern with global morbidity over 85 million. Whilst the vaccine trials is underway, there a several antiviral and antibody treatments being clinically evaluated to fill the ?therapeutic gap? in parallel. The development of potential drugs requires an understanding of SARS-CoV-2 pathogenicity and mechanism of action. Thus, it is essential to understand the full repertoire of viral proteins and their interplay with host factors. Here, we show how the SARS-CoV-2 spike protein undergoes 3 stages of processing to allow virion activation and host cell infection. Our comprehensive structural and computational studies reveal why COVID19 is hypervirulent and incites the possible reason for the failure of several antibody treatments. In addition, our resolved complex structures of spike protein with different host cell receptors shows the complexity of entry. We also demonstrate via experimental, biophysical and molecular dynamics studies that how the host proteins CD26 (DPP4), Furin and TMPRSS2 process the viral spike glycoprotein and assist in the viral entry in addition to ACE2. These results cognise the detailed mechanism of spike glycoprotein for its entry or cascade into host cell and also reveal new avenues for potential therapeutics to block different stages of viral entry and new pathways for vaccine development.

Structure of the SARS-CoV-2 Nsp1/5'-Untranslated Region Complex and Implications for Potential Therapeutic Targets, a Vaccine, and Virulence.

SARS-CoV-2 is the cause of the ongoing Coronavirus disease 19 (COVID-19) pandemic around the world causing pneumonia and lower respiratory tract infections. In understanding the SARS-CoV-2 pathogenicity and mechanism of action, it is essential to depict the full repertoire of expressed viral proteins. The recent biological studies have highlighted the leader protein Nsp1 of SARS-CoV-2 importance in shutting down the host protein production. Besides, it still enigmatic how Nsp1 regulates for translation. Here we report the novel structure of Nsp1 from SARS-CoV-2 in complex with the SL1 region of 5'UTR of SARS-CoV-2, and its factual interaction is corroborated with enzyme kinetics and experimental binding affinity studies. The studies also address how leader protein Nsp1 of SARS-CoV-2 recognizes its self RNA toward translational regulation by further recruitment of the 40S ribosome. With the aid of molecular dynamics and simulations, we also demonstrated the real-time stability and functional dynamics of the Nsp1/SL1 complex. The studies also report the potential inhibitors and their mode of action to block viral protein/RNA complex formation. This enhance our understanding of the mechanism of the first viral protein Nsp1 synthesized in the human cell to regulate the translation of self and host. Understanding the structure and mechanism of SARS-CoV-2 Nsp1 and its interplay with the viral RNA and ribosome will open the arena for exploring the development of live attenuated vaccines and effective therapeutic targets for this disease.

Emerging WuHan (COVID-19) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human CD26.

The recent outbreak of pneumonia-causing COVID-19 in China is an urgent global public health issue with an increase in mortality and morbidity. Here we report our modelled homo-trimer structure of COVID-19 spike glycoprotein in both closed (ligand-free) and open (ligand-bound) conformation, which is involved in host cell adhesion. We also predict the unique N- and O-linked glycosylation sites of spike glycoprotein that distinguish it from the SARS and underlines shielding and camouflage of COVID-19 from the host the defence system. Furthermore, our study also highlights the key finding that the S1 domain of COVID-19 spike glycoprotein potentially interacts with the human CD26, a key immunoregulatory factor for hijacking and virulence. These findings accentuate the unique features of COVID-19 and assist in the development of new therapeutics.

Structure of Furin Protease Binding to SARS-CoV-2 Spike Glycoprotein and Implications for Potential Targets and Virulence.

The COVID-19 pandemic is an urgent global health emergency, and the presence of Furin site in the SARS-CoV-2 spike glycoprotein alters virulence and warrants further molecular, structural, and biophysical studies. Here we report the structure of Furin in complex with SARS-CoV-2 spike glycoprotein, demonstrating how Furin binds to the S1/S2 region of spike glycoprotein and eventually cleaves the viral protein using experimental functional studies, molecular dynamics, and docking. The structural studies underline the mechanism and mode of action of Furin, which is a key process in host cell entry and a hallmark of enhanced virulence. Our whole-exome sequencing analysis shows the genetic variants/alleles in Furin were found to alter the binding affinity for viral spike glycoprotein and could vary in infectivity in humans. Unravelling the mechanisms of Furin action, binding dynamics, and the genetic variants opens the growing arena of bona fide antibodies and development of potential therapeutics targeting the blockage of Furin cleavage.

Overwhelming mutations or SNPs of SARS-CoV-2: A point of caution.

The morbidity of SARS-CoV-2 (COVID-19) is reaching 3 Million landmark causing and a serious public health concern globally and it is enigmatic how several antiviral and antibody treatments were not effective in the different period across the globe. With the drastic increasing number of positive cases around the world WHO raised the importance in the assessment of the risk of spread and understanding genetic modifications that could have occurred in the SARS-CoV-2. Using all available deep sequencing data of complete genome from all over the world (NCBI repository), we identified several hundreds of point mutations or SNPs in SARS-CoV-2 all across the genome. This could be the cause for the constant change and differed virulence with an increase in mortality and morbidity. Among the 12 different countries (one sequence from each country) with complete genome sequencing data, we noted the 47 key point mutations or SNPs located along the entire genome that might have impact in the virulence and response to different antivirals against SARS-CoV-2. In this regard, key viral proteins of spike glycoprotein, Nsp1, RdRp and the ORF8 region got heavily mutated within these 3 months via person-to-person passage. We also discuss what could be the possible cause of this rapid mutation in the SARS-CoV-2.

Arbidol: A potential antiviral drug for the treatment of SARS-CoV-2 by blocking trimerization of the spike glycoprotein.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic is a global public health emergency, and new therapeutics are needed. This article reports the potential drug target and mechanism of action of Arbidol (umifenovir) to treat coronavirus disease 2019 (COVID-19). Molecular dynamics and structural analysis were used to show how Arbidol targets the SARS-CoV-2 spike glycoprotein and impedes its trimerization, which is key for host cell adhesion and hijacking, indicating the potential of Arbidol to treat COVID-19. It is hoped that knowledge of the potential drug target and mechanism of action of Arbidol will help in the development of new therapeutics for SARS-CoV-2.