Continued Complexity of Mutations in Omicron Sublineages.

The latest SARS-CoV-2 variant of concern (VOC), Omicron (B.1.1.529), has diversified into more than 300 sublineages. With an expanding number of newly emerging sublineages, the mutation profile is also becoming complicated. There exist mutually exclusive and revertant mutations in different sublineages. Omicron sublineages share some common mutations with previous VOCs (Alpha, Beta, Gamma, and Delta), indicating an evolutionary relationship between these VOCs. A diverse mutation profile at the spike-antibody interface, flexibility of the regions harboring mutations, mutation types, and coexisting mutations suggest that SARS-CoV-2's evolution is far from over.

Evolutionary analysis of the Delta and Delta Plus variants of the SARS-CoV-2 viruses.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been rapidly evolving in the form of new variants. At least eleven known variants have been reported. The objective of this study was to delineate the differences in the mutational profile of Delta and Delta Plus variants. High-quality sequences (n = 1756) of Delta (B.1.617.2) and Delta Plus (AY.1 or B.1.617.2.1) variants were used to determine the prevalence of mutations (≥20 %) in the entire SARS-CoV-2 genome, their co-existence, and change in prevalence over a period of time. Structural analysis was conducted to get insights into the impact of mutations on antibody binding. A Sankey diagram was generated using phylogenetic analysis coupled with sequence-acquisition dates to infer the migration of the Delta Plus variant and its presence in the United States. The Delta Plus variant had a significant number of high-prevalence mutations (≥20 %) than in the Delta variant. Signature mutations in Spike (G142D, A222V, and T95I) existed at a more significant percentage in the Delta Plus variant than the Delta variant. Three mutations in Spike (K417N, V70F, and W258L) were exclusively present in the Delta Plus variant. A new mutation was identified in ORF1a (A1146T), which was only present in the Delta Plus variant with ~58 % prevalence. Furthermore, five key mutations (T95I, A222V, G142D, R158G, and K417N) were significantly more prevalent in the Delta Plus than in the Delta variant. Structural analyses revealed that mutations alter the sidechain conformation to weaken the interactions with antibodies. Delta Plus, which first emerged in India, reached the United States through England and Japan, followed by its spread to more than 20 the United States. Based on the results presented here, it is clear that the Delta and Delta Plus variants have unique mutation profiles, and the Delta Plus variant is not just a simple addition of K417N to the Delta variant. Highly correlated mutations may have emerged to keep the structural integrity of the virus.

Discovery and Evaluation of Entry Inhibitors for SARS-CoV-2 and Its Emerging Variants.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 19 (COVID-19) pandemic. Despite unprecedented research and developmental efforts, SARS-CoV-2-specific antivirals are still unavailable for the treatment of COVID-19. In most instances, SARS-CoV-2 infection initiates with the binding of Spike glycoprotein to the host cell ACE2 receptor. Utilizing the crystal structure of the ACE2/Spike receptor-binding domain (S-RBD) complex (PDB file 6M0J) in a computer-aided drug design approach, we identified and validated five potential inhibitors of S-RBD and ACE-2 interaction. Two of the five compounds, MU-UNMC-1 and MU-UNMC-2, blocked the entry of pseudovirus particles expressing SARS-CoV-2 Spike glycoprotein. In live SARS-CoV-2 infection assays, both compounds showed antiviral activity with IC50 values in the micromolar range (MU-UNMC-1: IC50 = 0.67 µM and MU-UNMC-2: IC50 = 1.72 µM) in human bronchial epithelial cells. Furthermore, MU-UNMC-1 and MU-UNMC-2 effectively blocked the replication of rapidly transmitting variants of concern: South African variant B.1.351 (IC50 = 9.27 and 3.00 µM) and Scotland variant B.1.222 (IC50 = 2.64 and 1.39 µM), respectively. Following these assays, we conducted "induced-fit (flexible) docking" to understand the binding mode of MU-UNMC-1/MU-UNMC-2 at the S-RBD/ACE2 interface. Our data showed that mutation N501Y (present in B.1.351 variant) alters the binding mode of MU-UNMC-2 such that it is partially exposed to the solvent and has reduced polar contacts. Finally, MU-UNMC-2 displayed high synergy with remdesivir, the only approved drug for treating hospitalized COVID-19 patients. IMPORTANCE The ongoing coronavirus infectious disease 2019 (COVID-19) pandemic is caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). More than 207 million people have been infected globally, and 4.3 million have died due to this viral outbreak. While a few vaccines have been deployed, a SARS-CoV-2-specific antiviral for the treatment of COVID-19 is yet to be approved. As the interaction of SARS-CoV-2 Spike protein with ACE2 is critical for cellular entry, using a combination of a computer-aided drug design (CADD) approach and cell-based in vitro assays, we report the identification of five potential SARS-CoV-2 entry inhibitors. Out of the five, two compounds (MU-UNMC-1 and MU-UNMC-2) have antiviral activity against ancestral SARS-CoV-2 and emerging variants from South Africa and Scotland. Furthermore, MU-UNMC-2 acts synergistically with remdesivir (RDV), suggesting that RDV and MU-UNMC-2 can be developed as a combination therapy to treat COVID-19 patients.

Distinct Metabolic Profile Associated with a Fatal Outcome in COVID-19 Patients during the Early Epidemic in Italy.

In one year of the coronavirus disease 2019 (COVID-19) pandemic, many studies have described the different metabolic changes occurring in COVID-19 patients, linking these alterations to the disease severity. However, a complete metabolic signature of the most severe cases, especially those with a fatal outcome, is still missing. Our study retrospectively analyzes the metabolome profiles of 75 COVID-19 patients with moderate and severe symptoms admitted to Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico (Lombardy Region, Italy) following SARS-CoV-2 infection between March and April 2020. Italy was the first Western country to experience COVID-19, and the Lombardy Region was the epicenter of the Italian COVID-19 pandemic. This cohort shows a higher mortality rate compared to others; therefore, it represents a unique opportunity to investigate the underlying metabolic profiles of the first COVID-19 patients in Italy and to identify the potential biomarkers related to the disease prognosis and fatal outcome. IMPORTANCE Understanding the metabolic alterations occurring during an infection is a key element for identifying potential indicators of the disease prognosis, which are fundamental for developing efficient diagnostic tools and offering the best therapeutic treatment to the patient. Here, exploiting high-throughput metabolomics data, we identified the first metabolic profile associated with a fatal outcome, not correlated with preexisting clinical conditions or the oxygen demand at the moment of diagnosis. Overall, our results contribute to a better understanding of COVID-19-related metabolic disruption and may represent a useful starting point for the identification of independent prognostic factors to be employed in therapeutic practice.

Evolution, correlation, structural impact and dynamics of emerging SARS-CoV-2 variants.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections remain unmanageable in some parts of the world. As with other RNA viruses, mutations in the SARS-CoV-2 gene have been continuously evolving. Recently, four variants have been identified, B.1.1.7, B.1.351, P.1 and CAL.20C. These variants appear to be more infectious and transmissible than the original Wuhan-Hu-1 virus. Using a combination of bioinformatics and structural analyses, we show that the new SARS-CoV-2 variants emerged in the background of an already known Spike protein mutation D614G together with another mutation P323L in the RNA polymerase of SARS-CoV-2. The phylogenetic analysis showed that the CAL.20C and B.1.351 shared one common ancestor, whereas the B.1.1.7 and P.1 shared a different ancestor. Structural comparisons did not show any significant difference between the wild-type and mutant ACE2/Spike complexes. Structural analysis indicated that the N501Y mutation may increase hydrophobic interactions at the ACE2/Spike interface. However, reported greater binding affinity of N501Y Spike with ACE2 does not seem to be entirely due to increased hydrophobic interactions, given that Spike mutation R417T in P.1 or K417N in B.1.351 results in the loss of a salt-bridge interaction between ACE2 and S-RBD. The calculated change in free energy did not provide a clear trend of S protein stability of mutations in the variants. As expected, we show that the CAL.20C generally migrated from the west coast to the east coast of the USA. Taken together, the analyses suggest that the evolution of variants and their infectivity is complex and may depend upon many factors.

Coronavirus helicases: attractive and unique targets of antiviral drug-development and therapeutic patents.

Introduction: Coronaviruses encode a helicase that is essential for viral replication and represents an excellent antiviral target. However, only a few coronavirus helicase inhibitors have been patented. These patents include drug-like compound SSYA10-001, aryl diketo acids (ADK), and dihydroxychromones. Additionally, adamantane-derived bananins, natural flavonoids, one acrylamide derivative [(E)-3-(furan-2-yl)-N-(4-sulfamoylphenyl)acrylamide], a purine derivative (7-ethyl-8-mercapto-3-methyl-3,7-dihydro-1 H-purine-2,6-dione), and a few bismuth complexes. The IC50 of patented inhibitors ranges between 0.82 µM and 8.95 µM, depending upon the assays used. Considering the urgency of clinical interventions against Coronavirus Disease-19 (COVID-19), it is important to consider developing antiviral portfolios consisting of small molecules.Areas covered: This review examines coronavirus helicases as antiviral targets, and the potential of previously patented and experimental compounds to inhibit the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) helicase.Expert opinion: Small molecule coronavirus helicase inhibitors represent attractive pharmacological modalities for the treatment of coronaviruses such as SARS-CoV and SARS-CoV-2. Rightfully so, the current emphasis is focused upon the development of vaccines. However, vaccines may not work for everyone and broad-based adoption of vaccinations is an increasingly challenging societal endeavor. Therefore, it is important to develop additional pharmacological antivirals against the highly conserved coronavirus helicases to broadly protect against this and subsequent coronavirus epidemics.