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1.
EMBO Mol Med ; 14(4): e15811, 2022 04 07.
Article in English | MEDLINE | ID: covidwho-1743028

ABSTRACT

There is an urgent need to bring new antivirals to SARS-CoV-2 to the market. Indeed, in the last 3 months, we have seen at least two new antivirals approved, molnupiravir and paxlovid. Both are older established antivirals that show some efficacy against SARS-CoV-2. The work by Chang et al (2022) in the current issue of EMBO Molecular Medicine explores the use of short interfering RNAs to directly target SARS-CoV-2 and shows that RNAi is an effective approach to reducing, or even eliminating viral replication, depending on the experimental setting. This antiviral effect results in significant prevention of infection-related pathology in animals. The key feature of this approach, besides its simplicity as naked siRNAs, is that all current variants are covered by this treatment.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/therapy , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology , RNA, Small Interfering/therapeutic use , SARS-CoV-2/genetics , Virus Replication
2.
Chem Biol Drug Des ; 99(2): 233-246, 2022 02.
Article in English | MEDLINE | ID: covidwho-1488186

ABSTRACT

Coronavirus (SARS-CoV-2) as a global pandemic has attracted the attention of many scientific centers to find the right treatment. We expressed and purified the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein, and specific RBD aptamers were designed using SELEX method. RNAi targeting nucleocapsid phosphoprotein was synthesized and human lung cells were inoculated with aptamer-functionalized lipid nanoparticles (LNPs) containing RNAi. The results demonstrated that RBD aptamer having KD values of 0.290 nm possessed good affinity. Based on molecular docking and efficacy prediction analysis, siRNA molecule was showed the best action. LNPs were appropriately functionalized by aptamer and contained RNAi molecules. Antiviral assay using q-PCR and ELISA demonstrated that LNP functionalized with 35 µm Apt and containing 30 nm RNAi/ml of cell culture had the best antiviral activity compared to other concentrations. Applied aptamer in the nanocarrier has two important functions. First, it can deliver the drug (RNAi) to the surface of epithelial cells. Second, by binding to the SARS-CoV-2 spike protein, it inhibits the virus entrance into cells. Our data reveal an interaction between the aptamer and the virus, and RNAi targeted the virus RNA. CT scan and the clinical laboratory tests in a clinical case study, a 36-year old man who presented with severe SARS-CoV-2, demonstrated that inhalation of 10 mg Apt-LNPs-RNAi nebulized/day for six days resulted in an improvement in consolidation and ground-glass opacity in lungs on the sixth day of treatment. Our findings suggest the treatment of SARS-CoV-2 infection through inhalation of Aptamer-LNPs-RNAi.


Subject(s)
Antiviral Agents/administration & dosage , Aptamers, Nucleotide/chemistry , COVID-19/drug therapy , Liposomes/chemistry , Nanoparticles/chemistry , RNA, Small Interfering/administration & dosage , Spike Glycoprotein, Coronavirus/metabolism , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Administration, Inhalation , Adult , Alanine/analogs & derivatives , Alanine/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , COVID-19/pathology , COVID-19/virology , Cell Line , Humans , Lung/diagnostic imaging , Lung/pathology , Male , Protein Domains/genetics , RNA Interference , RNA, Small Interfering/chemistry , RNA, Small Interfering/metabolism , RNA, Small Interfering/pharmacology , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , SELEX Aptamer Technique , Severity of Illness Index , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/genetics , Viral Load/drug effects
3.
Viruses ; 13(10)2021 10 08.
Article in English | MEDLINE | ID: covidwho-1463841

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected almost 200 million people worldwide and led to approximately 4 million deaths as of August 2021. Despite successful vaccine development, treatment options are limited. A promising strategy to specifically target viral infections is to suppress viral replication through RNA interference (RNAi). Hence, we designed eight small interfering RNAs (siRNAs) targeting the highly conserved 5'-untranslated region (5'-UTR) of SARS-CoV-2. The most promising candidate identified in initial reporter assays, termed siCoV6, targets the leader sequence of the virus, which is present in the genomic as well as in all subgenomic RNAs. In assays with infectious SARS-CoV-2, it reduced replication by two orders of magnitude and prevented the development of a cytopathic effect. Moreover, it retained its activity against the SARS-CoV-2 alpha variant and has perfect homology against all sequences of the delta variant that were analyzed by bioinformatic means. Interestingly, the siRNA was even highly active in virus replication assays with the SARS-CoV-1 family member. This work thus identified a very potent siRNA with a broad activity against various SARS-CoV viruses that represents a promising candidate for the development of new treatment options.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/therapy , RNA Interference , RNA, Small Interfering/pharmacology , SARS-CoV-2/growth & development , Virus Replication/drug effects , 5' Untranslated Regions/genetics , Animals , Cell Line, Tumor , Chlorocebus aethiops , Drug Evaluation, Preclinical , HeLa Cells , Humans , RNA, Small Interfering/genetics , SARS-CoV-2/genetics , Vero Cells , Virus Replication/genetics
4.
Nat Commun ; 12(1): 5553, 2021 09 21.
Article in English | MEDLINE | ID: covidwho-1434104

ABSTRACT

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/metabolism , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Animals , COVID-19/drug therapy , Cell Line , Dipeptides/pharmacology , Humans , Mutation , Myosin-Light-Chain Kinase/antagonists & inhibitors , Myosin-Light-Chain Kinase/genetics , Myosin-Light-Chain Kinase/metabolism , Proteolysis , Proteomics , RNA, Small Interfering/pharmacology , SARS-CoV-2/genetics , Viral Proteases/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Internalization/drug effects , Virus Replication/drug effects , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/genetics , src-Family Kinases/metabolism
5.
Cells ; 10(6)2021 06 08.
Article in English | MEDLINE | ID: covidwho-1264419

ABSTRACT

In late 2019, the betacoronavirus SARS-CoV-2 was identified as the viral agent responsible for the coronavirus disease 2019 (COVID-19) pandemic. Coronaviruses Spike proteins are responsible for their ability to interact with host membrane receptors and different proteins have been identified as SARS-CoV-2 interactors, among which Angiotensin-converting enzyme 2 (ACE2), and Basigin2/EMMPRIN/CD147 (CD147). CD147 plays an important role in human immunodeficiency virus type 1, hepatitis C virus, hepatitis B virus, Kaposi's sarcoma-associated herpesvirus, and severe acute respiratory syndrome coronavirus infections. In particular, SARS-CoV recognizes the CD147 receptor expressed on the surface of host cells by its nucleocapsid protein binding to cyclophilin A (CyPA), a ligand for CD147. However, the involvement of CD147 in SARS-CoV-2 infection is still debated. Interference with both the function (blocking antibody) and the expression (knock down) of CD147 showed that this receptor partakes in SARS-CoV-2 infection and provided additional clues on the underlying mechanism: CD147 binding to CyPA does not play a role; CD147 regulates ACE2 levels and both receptors are affected by virus infection. Altogether, these findings suggest that CD147 is involved in SARS-CoV-2 tropism and represents a possible therapeutic target to challenge COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/physiology , Basigin/physiology , SARS-CoV-2/physiology , Virus Internalization , A549 Cells , Angiotensin-Converting Enzyme 2/metabolism , Animals , Basigin/antagonists & inhibitors , Basigin/genetics , COVID-19/pathology , COVID-19/prevention & control , COVID-19/virology , Caco-2 Cells , Cell Line , Chlorocebus aethiops , Hep G2 Cells , Host-Pathogen Interactions , Humans , Molecular Targeted Therapy , RNA Interference/physiology , RNA, Small Interfering/pharmacology , RNA, Small Interfering/therapeutic use , Receptors, Virus/metabolism , Receptors, Virus/physiology , SARS-CoV-2/metabolism , Vero Cells , Viral Tropism/physiology
6.
Comput Biol Chem ; 92: 107486, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1226281

ABSTRACT

SARS-CoV-2 is a single-stranded RNA (+) virus first identified in China and then became an ongoing global outbreak. In most cases, it is fatal in humans due to respiratory malfunction. Extensive researches are going to find an effective therapeutic technique for the treatment of SARS-CoV-2 infected individuals. In this study, we attempted to design a siRNA molecule to silence the most suitable nucleocapsid(N) gene of SARS-CoV-2, which play a major role during viral pathogenesis, replication, encapsidation and RNA packaging. At first, 270 complete N gene sequences of different strains in Bangladesh of these viruses were retrieved from the NCBI database. Different computational methods were used to design siRNA molecules. A siRNA molecule was built against these strains using the SiDirect 2.0 server. Using Mfold and the OligoCalc server, the siRNA molecule was tested for its secondary structure and GC material. The Clustal Omega tool was employed to evaluate any off-target harmony of the planned siRNA molecule. Herein, we proposed a duplex siRNA molecule that does not fit any off-target sequences for the gene silencing of SARS-CoV-2. To treat SARS-CoV-2 infections, currently, any effective therapy is not available. Our engineered siRNA molecule could give an alternative therapeutic approach against various sequenced SARS-CoV-2 strains in Bangladesh.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/metabolism , RNA Interference , RNA, Small Interfering/chemistry , RNA, Small Interfering/pharmacology , SARS-CoV-2/genetics , Bangladesh/epidemiology , COVID-19/epidemiology , Computer Simulation , Coronavirus Nucleocapsid Proteins/genetics , Gene Expression Regulation, Viral , Humans , Models, Chemical
7.
Genomics ; 113(1 Pt 1): 331-343, 2021 01.
Article in English | MEDLINE | ID: covidwho-972544

ABSTRACT

An outbreak, caused by an RNA virus, SARS-CoV-2 named COVID-19 has become pandemic with a magnitude which is daunting to all public health institutions in the absence of specific antiviral treatment. Surface glycoprotein and nucleocapsid phosphoprotein are two important proteins of this virus facilitating its entry into host cell and genome replication. Small interfering RNA (siRNA) is a prospective tool of the RNA interference (RNAi) pathway for the control of human viral infections by suppressing viral gene expression through hybridization and neutralization of target complementary mRNA. So, in this study, the power of RNA interference technology was harnessed to develop siRNA molecules against specific target genes namely, nucleocapsid phosphoprotein gene and surface glycoprotein gene. Conserved sequence from 139 SARS-CoV-2 strains from around the globe was collected to construct 78 siRNA that can inactivate nucleocapsid phosphoprotein and surface glycoprotein genes. Finally, based on GC content, free energy of folding, free energy of binding, melting temperature, efficacy prediction and molecular docking analysis, 8 siRNA molecules were selected which are proposed to exert the best action. These predicted siRNAs should effectively silence the genes of SARS-CoV-2 during siRNA mediated treatment assisting in the response against SARS-CoV-2.


Subject(s)
COVID-19/therapy , Computational Chemistry , Coronavirus Nucleocapsid Proteins/genetics , Drug Design , Genetic Therapy/methods , Molecular Docking Simulation , RNA Interference , RNA, Messenger/antagonists & inhibitors , RNA, Small Interfering/chemistry , RNA, Viral/antagonists & inhibitors , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Argonaute Proteins/chemistry , Argonaute Proteins/genetics , Base Composition , COVID-19/drug therapy , COVID-19/virology , Evolution, Molecular , Gene Expression Regulation, Viral/drug effects , Humans , Pandemics , Phosphoproteins/genetics , Phylogeny , RNA Folding , RNA, Guide/chemistry , RNA, Guide/genetics , RNA, Messenger/genetics , RNA, Small Interfering/pharmacology , RNA, Small Interfering/therapeutic use , RNA, Viral/genetics , SARS-CoV-2/drug effects , Sequence Alignment , Thermodynamics
8.
Nanoscale ; 12(47): 23959-23966, 2020 Dec 21.
Article in English | MEDLINE | ID: covidwho-947558

ABSTRACT

Lipid nanoparticle (LNP) formulations of nucleic acid are leading vaccine candidates for COVID-19, and enabled the first approved RNAi therapeutic, Onpattro. LNPs are composed of ionizable cationic lipids, phosphatidylcholine, cholesterol, and polyethylene glycol (PEG)-lipids, and are produced using rapid-mixing techniques. These procedures involve dissolution of the lipid components in an organic phase and the nucleic acid in an acidic aqueous buffer (pH 4). These solutions are then combined using a continuous mixing device such as a T-mixer or microfluidic device. In this mixing step, particle formation and nucleic acid entrapment occur. Previous work from our group has shown that, in the absence of nucleic acid, the particles formed at pH 4 are vesicular in structure, a portion of these particles are converted to electron-dense structures in the presence of nucleic acid, and the proportion of electron-dense structures increases with nucleic acid content. What remained unclear from previous work was the mechanism by which vesicles form electron-dense structures. In this study, we use cryogenic transmission electron microscopy and dynamic light scattering to show that efficient siRNA entrapment occurs in the absence of ethanol (contrary to the established paradigm), and suggest that nucleic acid entrapment occurs through inversion of preformed vesicles. We also leverage this phenomenon to show that specialized mixers are not required for siRNA entrapment, and that preformed particles at pH 4 can be used for in vitro transfection.


Subject(s)
COVID-19 , Lab-On-A-Chip Devices , Lipids , Nanoparticles , RNA, Small Interfering , SARS-CoV-2 , Animals , Cell Line , Hydrogen-Ion Concentration , Lipids/chemistry , Lipids/pharmacology , Mice , Nanoparticles/chemistry , Nanoparticles/therapeutic use , RNA, Small Interfering/chemistry , RNA, Small Interfering/pharmacology
9.
Pharmacol Rev ; 72(4): 862-898, 2020 10.
Article in English | MEDLINE | ID: covidwho-767668

ABSTRACT

RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed. SIGNIFICANCE STATEMENT: With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.


Subject(s)
RNA/drug effects , RNA/pharmacology , Aptamers, Nucleotide/pharmacology , Aptamers, Nucleotide/therapeutic use , Betacoronavirus , COVID-19 , Chemistry Techniques, Analytical/methods , Chemistry Techniques, Analytical/standards , Clustered Regularly Interspaced Short Palindromic Repeats , Coronavirus Infections/drug therapy , Drug Delivery Systems/methods , Drug Development/organization & administration , Drug Discovery , Humans , MicroRNAs/pharmacology , MicroRNAs/therapeutic use , Oligonucleotides, Antisense/pharmacology , Oligonucleotides, Antisense/therapeutic use , Pandemics , Pneumonia, Viral/drug therapy , RNA/adverse effects , RNA, Antisense/pharmacology , RNA, Antisense/therapeutic use , RNA, Guide/pharmacology , RNA, Guide/therapeutic use , RNA, Messenger/drug effects , RNA, Messenger/pharmacology , RNA, Ribosomal/drug effects , RNA, Ribosomal/pharmacology , RNA, Small Interfering/pharmacology , RNA, Small Interfering/therapeutic use , RNA, Viral/drug effects , Ribonucleases/metabolism , Riboswitch/drug effects , SARS-CoV-2
11.
Virol J ; 17(1): 71, 2020 06 03.
Article in English | MEDLINE | ID: covidwho-505653

ABSTRACT

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) of the family Coronaviridae has caused substantial economic losses in the swine husbandry industry. There's currently no specific drug available for treatment of coronaviruses or PEDV. METHOD: In the current study, we use coronavirus PEDV as a model to study antiviral agents. Briefly, a fusion inhibitor tHR2, recombinant lentivirus-delivered shRNAs targeted to conserved M and N sequences, homoharringtonine (HHT), and hydroxychloroquine (HCQ) were surveyed for their antiviral effects. RESULTS: Treatment with HCQ at 50 µM and HHT at 150 nM reduced virus titer in TCID50 by 30 and 3.5 fold respectively, and the combination reduced virus titer in TCID50 by 200 fold. CONCLUSION: Our report demonstrates that the combination of HHT and HCQ exhibited higher antiviral activity than either HHT or HCQ exhibited. The information may contribute to the development of antiviral strategies effective in controlling PEDV infection.


Subject(s)
Antiviral Agents/pharmacology , Homoharringtonine/pharmacology , Hydroxychloroquine/pharmacology , Porcine epidemic diarrhea virus/drug effects , RNA, Small Interfering/pharmacology , Animals , Chlorocebus aethiops , Coronavirus Infections/drug therapy , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Coronavirus M Proteins , Coronavirus Nucleocapsid Proteins , Drug Synergism , Nucleocapsid Proteins/genetics , Peptides/pharmacology , RNA, Small Interfering/genetics , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells , Viral Load/drug effects , Viral Matrix Proteins/genetics
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