RNAi-mediated siRNA sequences to combat the COVID-19 pandemic with the inhibition of SARS-CoV2.

The outbreak of the COVID-19 pandemic has cost five million lives to date, and was caused by a positive-sense RNA virus named SARS-CoV2. The lack of drugs specific to SARS-CoV2, leads us to search for an effective and specific therapeutic approach. Small interfering RNA (siRNA) is able to activate the RNA interference (RNAi) pathway to silence the specific targeted gene and inhibit the viral replication, and it has not yet attracted enough attention as a SARS-CoV2 antiviral agent. It could be a potential weapon to combat this pandemic until the completion of full scale, effective mass vaccination. For this study, specific siRNAs were designed using a web-based bioinformatics tool (siDirect2.0) against 14 target sequences. These might have a high probability of silencing the essential proteins of SARS-CoV2. such as: 3CLpro/Mpro/nsp5, nsp7, Rd-Rp/nsp12, ZD, NTPase/HEL or nsp13, PLpro/nsp3, envelope protein (E), spike glycoprotein (S), nucleocapsid phosphoprotein (N), membrane glycoprotein (M), ORF8, ORF3a, nsp2, and its respective 5' and 3'-UTR. Among these potential drug targets, the majority of them contain highly conserved sequences; the rest are chosen on the basis of their role in viral replication and survival. The traditional vaccine development technology using SARS-CoV2 protein takes 6-8 months; meanwhile the virus undergoes several mutations in the candidate protein chosen for vaccine development. By the time the protein-based vaccine reaches the market, the virus would have undergone several mutations, such that the antibodies against the viral sequence may not be effective in restricting the newly mutated viruses. However, siRNA technology can make sequences based on real time viral mutation status. This has the potential for suppressing SARS-CoV2 viral replication, through RNAi technology.

Antiviral therapeutics directed against RNA dependent RNA polymerases from positive-sense viruses.

Viruses with positive-sense single stranded RNA (+ssRNA) genomes are responsible for different diseases and represent a global health problem. In addition to developing new vaccines that protect against severe illness on infection, it is imperative to identify new antiviral molecules to treat infected patients. The genome of these RNA viruses generally codes for an enzyme with RNA dependent RNA polymerase (RdRP) activity. This molecule is centrally involved in the duplication of the RNA genome. Inhibition of this enzyme by small molecules will prevent duplication of the RNA genome and thus reduce the viral titer. An overview of the different therapeutic strategies used to inhibit RdRPs from +ssRNA viruses is provided, along with an analysis of these enzymes to highlight new binding sites for inhibitors.

Identification of recurrent mutations in exonuclease (nsp14); a potential drug target in SARS-CoV-2.

CONTEXT: The rapid outbreak of SARS-CoV-2 has become a significant global health concern, highlighting the dire need for antiviral therapeutic agents. RNA-dependent RNA polymerase (RdRp) of coronavirus plays crucial roles in RNA synthesis, and hence remains the druggable target for the treatment of this disease. The most potent broad-spectrum inhibitors of viral RdRp are members of nucleoside analogs (NAs). However, SARS-CoV-2 proved to be a challenging one for the novel NA drug designing strategy because coronavirus possesses an exonuclease (ExoN) domain that is capable of excising NAs, thus showing resistance to existing antiviral drugs. AIM: The objective of our study was to compare the SARS-CoV-2 exonuclease (nsp14) protein sequence of Wuhan-type virus with those of Indian SARS-Cov-2 isolates and to study the effect of multiple mutations on the secondary structure alterations of proteins. SUBJECTS AND METHODS: Multiple-sequence alignment of exonuclease amino-acid sequences followed by phylogenetic analysis and prediction of its secondary structure of the protein was performed. RESULTS: Altogether, seven mutations were detected in the nsp14 of Indian SARS-CoV-2 isolates. Subsequently, prediction of their secondary structures revealed that mutations altered the structural stability of exonuclease proteins. CONCLUSIONS: Present findings, therefore, further suggest that evolvability of SARS-CoV-2 is primarily associated with the onset of multiple novel mutations that rapidly spread at several new locations of the viral genome and also provides important insight to develop specific control strategies to fight against COVID-19 infections.

Can remdesivir and its parent nucleoside GS-441524 be potential oral drugs? An in vitro and in vivo DMPK assessment.

Remdesivir (RDV) is the only US Food and Drug Administration (FDA)-approved drug for treating COVID-19. However, RDV can only be given by intravenous route, and there is a pressing medical need for oral antivirals. Significant evidence suggests that the role of the parent nucleoside GS-441524 in the clinical outcomes of RDV could be largely underestimated. We performed an in vitro and in vivo drug metabolism and pharmacokinetics (DMPK) assessment to examine the potential of RDV, and particularly GS-441524, as oral drugs. In our in vitro assessments, RDV exhibited prohibitively low stability in human liver microsomes (HLMs, t 1/2 = âˆ¼1 min), with the primary CYP-mediated metabolism being the mono-oxidation likely on the phosphoramidate moiety. This observation is poorly aligned with any potential oral use of RDV, though in the presence of cobicistat, the microsomal stability was drastically boosted to the level observed without enzyme cofactor NADPH. Conversely, GS-441524 showed excellent metabolic stability in human plasma and HLMs. In further in vivo studies in CD-1 mice, GS-441524 displayed a favorable oral bioavailability of 57%. Importantly, GS-441524 produced adequate drug exposure in the mice plasma and lung, and was effectively converted to the active triphosphate, suggesting that it could be a promising oral antiviral drug for treating COVID-19.

Identification of novel mutations in RNA-dependent RNA polymerases of SARS-CoV-2 and their implications on its protein structure.

The rapid development of the SARS-CoV-2 mediated COVID-19 pandemic has been the cause of significant health concern, highlighting the immediate need for effective antivirals. SARS-CoV-2 is an RNA virus that has an inherently high mutation rate. These mutations drive viral evolution and genome variability, thereby facilitating viruses to have rapid antigenic shifting to evade host immunity and to develop drug resistance. Viral RNA-dependent RNA polymerases (RdRp) perform viral genome duplication and RNA synthesis. Therefore, we compared the available RdRp sequences of SARS-CoV-2 from Indian isolates and the 'Wuhan wet sea food market virus' sequence to identify, if any, variation between them. Our data revealed the occurrence of seven mutations in Indian isolates of SARS-CoV-2. The secondary structure prediction analysis of these seven mutations shows that three of them cause alteration in the structure of RdRp. Furthermore, we did protein modelling studies to show that these mutations can potentially alter the stability of the RdRp protein. Therefore, we propose that RdRp mutations in Indian SARS-CoV-2 isolates might have functional consequences that can interfere with RdRp targeting pharmacological agents.