ABSTRACT
Inorganic polyphosphate (polyP) is a widely distributed polymer found from bacteria to animals, including marine species. This polymer exhibits morphogenetic as well as antiviral activity and releases metabolic energy after enzymatic hydrolysis also in human cells. In the pathogenesis of the coronavirus disease 2019 (COVID-19), the platelets are at the frontline of this syndrome. Platelets release a set of molecules, among them polyP. In addition, the production of airway mucus, the first line of body defense, is impaired in those patients. Therefore, in this study, amorphous nanoparticles of the magnesium salt of polyP (Mg-polyP-NP), matching the size of the coronavirus SARS-CoV-2, were prepared and loaded with the secondary plant metabolite quercetin or with dexamethasone to study their effects on the respiratory epithelium using human alveolar basal epithelial A549 cells as a model. The results revealed that both compounds embedded into the polyP nanoparticles significantly increased the steady-state-expression of the MUC5AC gene. This mucin species is the major mucus glycoprotein present in the secreted gel-forming mucus. The level of gene expression caused by quercetin or with dexamethasone, if caged into polyP NP, is significantly higher compared to the individual drugs alone. Both quercetin and dexamethasone did not impair the growth-supporting effect of polyP on A549 cells even at concentrations of quercetin which are cytotoxic for the cells. A possible mechanism of the effects of the two drugs together with polyP on mucin expression is proposed based on the scavenging of free oxygen species and the generation of ADP/ATP from the polyP, which is needed for the organization of the protective mucin-based mucus layer.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Antioxidants/pharmacology , Dexamethasone/pharmacology , Mucin 5AC/biosynthesis , Mucin 5AC/drug effects , Quercetin/pharmacology , A549 Cells , Anti-Inflammatory Agents/chemistry , Antioxidants/chemistry , COVID-19 , Dexamethasone/chemistry , Free Radical Scavengers/pharmacology , Gene Expression Regulation/drug effects , Humans , Magnesium/chemistry , Mucin 5AC/genetics , Mucins/biosynthesis , Mucins/chemistry , Nanoparticles , Particle Size , Plants/chemistry , Polyphosphates/chemistry , Quercetin/chemistry , Reactive Oxygen SpeciesABSTRACT
SARS-CoV-2 currently lacks effective first-line drug treatment. We present promising data from in silico docking studies of new Methisazone compounds (modified with calcium, Ca; iron, Fe; magnesium, Mg; manganese, Mn; or zinc, Zn) designed to bind more strongly to key proteins involved in replication of SARS-CoV-2. In this in silico molecular docking study, we investigated the inhibiting role of Methisazone and the modified drugs against SARS-CoV-2 proteins: ribonucleic acid (RNA)-dependent RNA polymerase (RdRp), spike protein, papain-like protease (PlPr), and main protease (MPro). We found that the highest binding interactions were found with the spike protein (6VYB), with the highest overall binding being observed with Mn-bound Methisazone at -8.3 kcal/mol, followed by Zn and Ca at -8.0 kcal/mol, and Fe and Mg at -7.9 kcal/mol. We also found that the metal-modified Methisazone had higher affinity for PlPr and MPro. In addition, we identified multiple binding pockets that could be singly or multiply occupied on all proteins tested. The best binding energy was with Mn-Methisazone versus spike protein, and the largest cumulative increases in binding energies were found with PlPr. We suggest that further studies are warranted to identify whether these compounds may be effective for treatment and/or prophylaxis.
Subject(s)
Antiviral Agents/chemistry , Metals/chemistry , Methisazone/chemistry , Molecular Docking Simulation , SARS-CoV-2/chemistry , Antiviral Agents/metabolism , Calcium/chemistry , Calcium/metabolism , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Drug Design , Humans , Iron/chemistry , Iron/metabolism , Magnesium/chemistry , Magnesium/metabolism , Manganese/chemistry , Manganese/metabolism , Metals/metabolism , Methisazone/metabolism , Models, Molecular , Molecular Dynamics Simulation , Protein Binding , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Zinc/chemistry , Zinc/metabolism , COVID-19 Drug TreatmentABSTRACT
The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.
Subject(s)
Magnesium/chemistry , RNA Caps/metabolism , RNA, Viral/metabolism , SARS-CoV-2/genetics , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Amino Acid Sequence , Binding Sites , Biocatalysis , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Viral , Humans , Magnesium/metabolism , Methylation , Methyltransferases , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , RNA Caps/chemistry , RNA Caps/genetics , RNA, Viral/chemistry , RNA, Viral/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/ultrastructure , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/geneticsABSTRACT
Nucleotide analogues are used for treating viral infections such as HIV, hepatitis B, hepatitis C, influenza, and SARS-CoV-2. To become polymerase substrates, a nucleotide analogue must be phosphorylated by cellular kinases which is rate-limiting. The goal of this study is to develop dNTP/NTP analogues directly from nucleotides. Tenofovir (TFV) analogues were synthesized by conjugating with amino acids. We demonstrate that some conjugates act as dNTP analogues and HIV-1 reverse transcriptase (RT) catalytically incorporates the TFV part as the chain terminator. X-ray structures in complex with HIV-1 RT/dsDNA showed binding of the conjugates at the polymerase active site, however, in different modes in the presence of Mg2+ versus Mn2+ ions. The adaptability of the compounds is seemingly essential for catalytic incorporation of TFV by RT. 4d with a carboxyl sidechain demonstrated the highest incorporation. 4e showed weak incorporation and rather behaved as a dNTP-competitive inhibitor. This result advocates the feasibility of designing NTP/dNTP analogues by chemical substitutions to nucleotide analogues.