Inhibition of SARS-CoV-2 nucleocapsid protein-RNA interaction by guanosine oligomeric RNA.

The interaction of the ß-coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid (N) protein with genomic RNA is initiated by specific RNA regions and subsequently induces the formation of a continuous polymer with characteristic structural units for viral formation. We hypothesized that oligomeric RNAs, whose sequences are absent in the 29.9-kb genome sequence of SARS-CoV-2, might affect RNA-N protein interactions. We identified two such hexameric RNAs, In-1 (CCGGCG) and G6 (GGGGGG), and investigated their effects on the small filamentous/droplet-like structures (< a few µm) of N protein-genomic RNA formed by liquid-liquid phase separation. The small N protein structures were sequence-specifically enhanced by In-1, whereas G6 caused them to coalesce into large droplets. Moreover, we found that a guanosine 12-mer (G12, GGGGGGGGGGGG) expelled preexisting genomic RNA from the small N protein structures. The presence of G12 with the genomic RNA suppressed the formation of the small N protein structures, and alternatively apparently altered phase separation to induce the formation of large droplets with unclear phase boundaries. We showed that the N-terminal RNA-binding domain is required for the stability of the small N protein structures. Our results suggest that G12 may be a strong inhibitor of the RNA-N protein interaction.

Glycyrrhizin as a promising kryptonite against SARS-CoV-2: Clinical, experimental, and theoretical evidences.

COVID-19 is the most devastating disease in recent times affecting most people globally. The higher rate of transmissibility and mutations of SARS-CoV-2 along with the lack of potential therapeutics has made it a global crisis. Potential molecules from natural sources could be a fruitful remedy to combat COVID-19. This systematic review highlights the detailed therapeutic implication of naturally occurring glycyrrhizin and its related derivatives against COVID-19. Glycyrrhizin has already been established for blocking different biomolecular targets related to the SARS-CoV-2 replication cycle. In this article, several experimental and theoretical evidences of glycyrrhizin and related derivatives have been discussed in detail to evaluate their potential as a promising therapeutic strategy against COVID-19. Moreover, the implication of glycyrrhizin in traditional Chinese medicines for alleviating the symptoms of COVID-19 has been reviewed. The potential role of glycyrrhizin and related compounds in affecting various stages of the SARS-CoV-2 life cycle has also been discussed in detail. Derivatization of glycyrrhizin for designing potential lead compounds along with combination therapy with other anti-SARS-CoV-2 agents followed by extensive evaluation may assist in the formulation of novel anti-coronaviral therapy for better treatment to combat COVID-19.

Advances in the development of therapeutic strategies against COVID-19 and perspectives in the drug design for emerging SARS-CoV-2 variants.

Since Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was identified in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has challenged public health around the world. Currently, there is an urgent need to explore antiviral therapeutic targets and effective clinical drugs. In this study, we systematically summarized two main therapeutic strategies against COVID-19, namely drugs targeting the SARS-CoV-2 life cycle and SARS-CoV-2-induced inflammation in host cells. The development of above two strategies is implemented by repurposing drugs and exploring potential targets. A comprehensive summary of promising drugs, especially cytokine inhibitors, and traditional Chinese medicine (TCM), provides recommendations for clinicians as evidence-based medicine in the actual clinical COVID-19 treatment. Considering the emerging SARS-CoV-2 variants greatly impact the effectiveness of drugs and vaccines, we reviewed the appearance and details of SARS-CoV-2 variants for further perspectives in drug design, which brings updating clues to develop therapeutical agents against the variants. Based on this, the development of broadly antiviral drugs, combined with immunomodulatory, or holistic therapy in the host, is prior to being considered for therapeutic interventions on mutant strains of SARS-CoV-2. Therefore, it is highly acclaimed the requirements of the concerted efforts from multi-disciplinary basic studies and clinical trials, which improves the accurate treatment of COVID-19 and optimizes the contingency measures to emerging SARS-CoV-2 variants.

The status and analysis of common mutations found in the SARS-CoV-2 whole genome sequences from Bangladesh.

Rapid emergence of covid-19 variants by continuous mutation made the world experience continuous waves of infections and as a result, a huge number of death-toll recorded so far. It is, therefore, very important to investigate the diversity and nature of the mutations in the SARS-CoV-2 genomes. In this study, the common mutations occurred in the whole genome sequences of SARS-CoV-2 variants of Bangladesh in a certain timeline were analyzed to better understand its status. Hence, a total of 78 complete genome sequences available in the NCBI database were obtained, aligned and further analyzed. Scattered Single Nucleotide Polymorphisms (SNPs) were identified throughout the genome of variants and common SNPs such as: 241:C>T in the 5'UTR of Open Reading Frame 1A (ORF1A), 3037: C>T in Non-structural Protein 3 (NSP3), 14,408: C>T in ORF6 and 23,402: A>G, 23,403: A>G in Spike Protein (S) were observed, but all of them were synonymous mutations. About 97% of the studied genomes showed a block of tri-nucleotide alteration (GGG>AAC), the most common non-synonymous mutation in the 28,881-28,883 location of the genome. This block results in two amino acid changes (203-204: RG>KR) in the SR rich motif of the nucleocapsid (N) protein of SARS-CoV-2, introducing a lysine in between serine and arginine. The N protein structure of the mutant was predicted through protein modeling. However, no observable difference was found between the mutant and the reference (Wuhan) protein. Further, the protein stability changes upon mutations were analyzed using the I-Mutant2.0 tool. The alteration of the arginine to lysine at the amino acid position 203, showed reduction of entropy, suggesting a possible impact on the overall stability of the N protein. The estimation of the non-synonymous to synonymous substitution ratio (dN/dS) were analyzed for the common mutations and the results showed that the overall mean distance among the N-protein variants were statistically significant, supporting the non-synonymous nature of the mutations. The phylogenetic analysis of the selected 78 genomes, compared with the most common genomic variants of this virus across the globe showed a distinct cluster for the analyzed Bangladeshi sequences. Further studies are warranted for conferring any plausible association of these mutations with the clinical manifestation.

Characterizing genomic variants and mutations in SARS-CoV-2 proteins from Indian isolates.

SARS-CoV-2 is mutating and creating divergent variants by altering the composition of essential constituent proteins. Pharmacologically, it is crucial to understand the diverse mechanism of mutations for stable vaccine or anti-viral drug design. Our current study concentrates on all the constituent proteins of 469 SARS-CoV-2 genome samples, derived from Indian patients. However, the study may easily be extended to the samples across the globe. We perform clustering analysis towards identifying unique variants in each of the SARS-CoV-2 proteins. A total of 536 mutated positions within the coding regions of SARS-CoV-2 proteins are detected among the identified variants from Indian isolates. We quantify mutations by focusing on the unique variants of each SARS-CoV-2 protein. We report the average number of mutation per variant, percentage of mutated positions, synonymous and non-synonymous mutations, mutations occurring in three codon positions and so on. Our study reveals the most susceptible six (06) proteins, which are ORF1ab, Spike (S), Nucleocapsid (N), ORF3a, ORF7a, and ORF8. Several non-synonymous substitutions are observed to be unique in different SARS-CoV-2 proteins. A total of 57 possible deleterious amino acid substitutions are predicted, which may impact on the protein functions. Several mutations show a large decrease in protein stability and are observed in putative functional domains of the proteins that might have some role in disease pathogenesis. We observe a good number of physicochemical property change during above deleterious substitutions.

Mutational insights into the envelope protein of SARS-CoV-2.

The ongoing mutations in the structural proteins of SARS-CoV-2 are the major impediment for prevention and control of the COVID-19 disease. Presently we focused on evolution of the envelope (E) protein, one of the most enigmatic and less studied protein among the four structural proteins (S, E, M and N) associated with multitude of immunopathological functions of SARS-CoV-2. In the present study, we comprehensively analyzed 81,818 high quality E protein sequences of SARS-CoV-2 globally available in the GISAID database as of 20 August 2020. Compared to Wuhan reference strain, our mutational analysis explored only 1.2 % (982/81818) mutant strains undergoing a total of 115 unique amino acid (aa) substitutions in the E protein, highlighting the fact that most (98.8 %) of the E protein of SARS-CoV-2 strains are highly conserved. Moreover, we found 58.77 % (134 of 228) nucleotides (nt) positions of SARS-CoV-2 E gene encountering a total of 176 unique nt-level mutations globally, which may affect the efficacy of real time RT-PCR-based molecular detection of COVID-19. Importantly, higher aa variations observed in the C-terminal domain (CTD) of the E protein, particularly at Ser55-Phe56, Arg69 and the C-terminal end (DLLV: 72-75) may alter the binding of SARS-CoV-2 Envelope protein to tight junction-associated PALS1 and thus could play a key role in COVID-19 pathogenesis. Furthermore, this study revealed the V25A mutation in the transmembrane domain which is a key factor for the homopentameric conformation of E protein. Our analysis also observed a triple cysteine motif harboring mutation (L39M, A41S, A41V, C43F, C43R, C43S, C44Y, N45R) which may hinder the binding of E protein with spike glycoprotein. These results therefore suggest the continuous monitoring of the structural proteins including the envelope protein of SARS-CoV-2 since the number of genome sequences from across the world are continuously increasing.