Computational Analysis Reveals Monomethylated Triazolopyrimidine as a Novel Inhibitor of SARS-CoV-2 RNA-Dependent RNA Polymerase (RdRp)

The human population is still facing appalling conditions due to several outbreaks of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) virus. The absence of specific drugs, appropriate vaccines for mutants, and knowledge of potential therapeutic agents makes this situation more difficult. Several 1, 2, 4-triazolo [1, 5-a] pyrimidine (TP)-derivative compounds were comprehensively studied for antiviral activities against RNA polymerase of HIV, HCV, and influenza viruses, and showed immense pharmacological interest. Therefore, TP-derivative compounds can be repurposed against the RNA-dependent RNA polymerase (RdRp) protein of SARS-CoV-2. In this study, a meta-analysis was performed to ensure the genomic variability and stability of the SARS-CoV-2 RdRp protein. The molecular docking of natural and synthetic TP compounds to RdRp and molecular dynamic (MD) simulations were performed to analyse the dynamic behaviour of TP compounds at the active site of the RdRp protein. TP compounds were also docked against other non-structural proteins (NSP1, NSP2, NSP3, NSP5, NSP8, NSP13, and NSP15) of SARS-CoV-2. Furthermore, the inhibition potential of TP compounds was compared with Remdesivir and Favipiravir drugs as a positive control. Additionally, TP compounds were analysed for inhibitory activity against SARS-CoV RdRp protein. This study demonstrates that TP analogues (monomethylated triazolopyrimidine and essramycin) represent potential lead molecules for designing an effective inhibitor to control viral replication. Furthermore, in vitro and in vivo studies will strengthen the use of these inhibitors as suitable drug candidates against SARS-CoV-2.     

Protein factors mediating selenoprotein synthesis

The amino acid selenocysteine represents the major biological form of selenium. Both the synthesis of selenocysteine and its co-translational incorporation into selenoproteins in response to an in-frame UGA codon, require a complex molecular machinery. To decode the UGA Sec codon in eubacteria, this machinery comprises the tRNASec, the specialized elongation factor SelB and the SECIS hairpin in the selenoprotein mRNAs. SelB conveys the Sec-tRNASec to the A site of the ribosome through binding to the SECIS mRNA hairpin adjacent to the UGA Sec codon. SelB is thus a bifunctional factor, carrying functional homology to elongation factor EF-Tu in its N-terminal domain and SECIS RNA binding activity via its C-terminal extension. In archaea and eukaryotes, selenocysteine incorporation exhibits a higher degree of complexity because the SECIS hairpin is localized in the 3' untranslated region of the mRNA. In the last couple of years, remarkable progress has been made toward understanding the underlying mechanism in mammals. Indeed, the discovery of the SECIS RNA binding protein SBP2, which is not a translation factor, paved the way for the subsequent isolation of mSelB/EFSec, the mammalian homolog of SelB. In contrast to the eubacterial SelB, the specialized elongation factor mSelB/EFSec the SECIS RNA binding function. The role is carried out by SBP2 that also forms a protein-protein complex with mSelB/EFSec. As a consequence, an important difference between the eubacterial and eukaryal selenoprotein synthesis machineries is that the functions of SelB are divided into two proteins in eukaryotes. Obviously, selenoprotein synthesis represents a higher degree of complexity than anticipated, and more needs to be discovered in eukaryotes. In this review, we will focus on the structural and functional aspects of the SelB and SBP2 factors in selenoprotein synthesis.     

Structure and Biosynthesis of Crocagins: Polycyclic Posttranslationally Modified Ribosomal Peptides from Chondromyces crocatus

Secondary metabolome mining efforts in the myxobacterial multiproducer of natural products, Chondromyces crocatus Cm c5, resulted in the isolation and structure elucidation of crocagins, which are novel polycyclic peptides containing a tetrahydropyrrolo[2,3-b]indole core. The gene cluster was identified through an approach combining genome analysis, targeted gene inactivation in the producer, and in vitro experiments. Based on our findings, we developed a biosynthetic scheme for crocagin biosynthesis. These natural products are formed from the three C-terminal amino acids of a precursor peptide and thus belong to a novel class of ribosomally synthesized and post-translationally modified peptides (RiPPs). We demonstrate that crocagin A binds to the carbon storage regulator protein CsrA, thereby inhibiting the ability of CsrA to bind to its cognate RNA target.     

Structural analysis of inhibition mechanisms of aurintricarboxylic acid on SARS-CoV polymerase and other proteins

We recently published experimental results that indicated Aurintricarboxylic Acid (ATA) could selectively inhibit SARS-CoV replication inside host cells by greater than 1000 times. This inhibition suggested that ATA could be developed as potent anti-viral drug. Here, to extend our experimental observation, we have incorporated protein structural studies (with positive/negative controls) to investigate the potential binding modes/sites of ATA onto RNA-dependent RNA polymerase (RdRp) from SARS-CoV and other pathogenic positive-strand RNA-viruses, as well as other proteins in SARS-CoV based on the fact that ATA binds to Ca2+-activated neutral protease (m-calpain), the protein tyrosine phosphatase (PTP) and HIV integrase which have existing crystal structures. Eight regions with homologous 3D-conformation were derived for 10 proteins of interest. One of the region, Rbinding (754-766 in SARS-CoV's RdRp), located in the palm sub-domain mainly constituted of anti-parallel beta-strand-turn-beta-strand hairpin structures that covers two of the three RdRp catalytic sites (Asp 760, Asp761), was also predicted by molecular docking method (based on free energy of binding DeltaG) to be important binding motif recognized by ATA. The existence of this strictly conserved region that incorporated catalytic residues, coupled with the homologous ATA binding pockets and their consistent DeltaG values, suggested strongly ATA may be involved in an analogous inhibition mechanism of SARS-COV's RdRp in concomitant to the case in m-calpain, PTP and HIV integrase.     

Effect of steric exclusion on the separation of proteins by hydrophilic size-exclusion chromatography

On the basis of studying the retention and band broadening of proteins on the TSK SW column, diffusion coefficients (Ds) of solute in stationary phase were obtained which elucidate the hydrodynamic process of chromatographic resolution of proteins by hydrophilic size-exclusion chromatography (SEC). After calculating the correlation between Ds and the molecular weight of the solute, the molecular dimensions of proteins in the process of chromatographic separation can be predicted. Deviations in diffusion coefficient of a protein from the calculated value reflect differences of measured molecular dimensions from molecular volumes predicted from the calibration curve of the SEC column. This study illustrates a convenient method for estimating the purity of proteins by SEC. Deviations from 2 lambda dp (where dp is the particle diameter) in the intercept of the theoretical plate height (H) versus flow-rate (U) curve from the band broadening equation H = CsU + 2 lambda dp + f(alpha M)T (where CsU represents mass transfer resistance caused by solute diffusion in the stationary phase and f(alpha M)T an added term for polydisperse solutes as proposed by Knox and McLennan [Chromatographia, 10 (1977) 75]) reflect impurities in the proteins.     

Computational Study of SARS-CoV-2 RNA Dependent RNA Polymerase Allosteric Site Inhibition

The COVID-19 pandemic has caused millions of fatalities since 2019. Despite the availability of vaccines for this disease, new strains are causing rapid ailment and are a continuous threat to vaccine efficacy. Here, molecular docking and simulations identify strong inhibitors of the allosteric site of the SARS-CoV-2 virus RNA dependent RNA polymerase (RdRp). More than one hundred different flavonoids were docked with the SARS-CoV-2 RdRp allosteric site through computational screening. The three top hits were Naringoside, Myricetin and Aureusidin 4,6-diglucoside. Simulation analyses confirmed that they are in constant contact during the simulation time course and have strong association with the enzyme’s allosteric site. Absorption, distribution, metabolism, excretion and toxicity (ADMET) data provided medicinal information of these top three hits. They had good human intestinal absorption (HIA) concentrations and were non-toxic. Due to high mutation rates in the active sites of the viral enzyme, these new allosteric site inhibitors offer opportunities to drug SARS-CoV-2 RdRp. These results provide new information for the design of novel allosteric inhibitors against SARS-CoV-2 RdRp.     

Molecular weight determination of recombinant interleukin 2 and interferon gamma by electrospray ionization mass spectroscopy.

The molecular weights of the recombinant proteins interleukin 2 and interferon gamma were determined by electrospray ionization (ESI) mass spectroscopy on the whole molecules. For interleukin 2 an average experimental mass of 15,549.4 u, was observed in excellent agreement with the theoretical average mass of 15,547.2 u. The mass observed for interferon gamma (16,908.4 u) agreed with the theoretical molecular weight of the monomeric species (16,907.3 u). By ESI mass spectrometry it was possible to determine the presence of proteins lacking one or two amino acids.     

Unveiling of Pyrimidindinones as Potential Anti-Norovirus Agents—A Pharmacoinformatic-Based Approach

The RNA-dependent RNA polymerase (RdRp) receptor is an attractive target for treating human norovirus (HNV). A computer-aided approach like e-pharmacophore, molecular docking, and single point energy calculations were performed on the compounds retrieved from the Development Therapeutics Program (DTP) AIDS Antiviral Screen Database to identify the antiviral agent that could target the HNV RdRp receptor. Induced-fit docking (IFD) results showed that compounds ZINC1617939, ZINC1642549, ZINC6425208, ZINC5887658 and ZINC32068149 bind with the residues in the active site-B of HNV RdRp receptor via hydrogen bonds, salt bridge, and electrostatic interactions. During the molecular dynamic simulations, compounds ZINC6425208, ZINC5887658 and ZINC32068149 displayed an unbalanced backbone conformation with HNV RdRp protein, while ZINC1617939 and ZINC1642549 maintained stability with the protein backbone when interacting with the residues. Hence, the two new concluding compounds discovered by the computational approach can be used as a chemotype to design promising antiviral agents aimed at HNV RdRp.     

Traveling Time of a translating ribosome along messenger RNA monitored directly on a quartz crystal microbalance

During translation, the biosynthesis of polypeptides is dynamically regulated. The translation rate along messenger RNA (mRNA), which is dependent on the codon, structure, and sequence, is not always constant. However, methods for measuring the duration required for polypeptide elongation on an mRNA of interest have not been developed. In this work, we used a quartz crystal microbalance (QCM) technique to monitor mRNA translation in an Escherichia coli cell-free translation system in real time. This method permitted us to evaluate the translation of proteins of interest fused upstream of a streptavidin-binding peptide (SBP) fusion protein. The translation of mRNA encoding the SBP fusion protein alone was observed as a mass increase on a streptavidin-modified QCM plate. Addition of the protein of interest resulted in a delay in the mass change corresponding to the traveling time of the ribosome along the coding region of the protein of interest. With this technique, the lengths of coding sequences, codon usages, influences of unique sequences, and various protein-coding sequences were evaluated. The results showed that the traveling time of the translating ribosome depends on the length of the coding region translated but is also affected by the sequence itself. Differences in the time lags for various proteins imply that mRNA coding sequences may regulate gene expression.     

Targeting the RdRp of Emerging RNA Viruses: The Structure-Based Drug Design Challenge

The RNA-dependent RNA polymerase (RdRp) is an essential enzyme for the viral replication process, catalyzing the viral RNA synthesis using a metal ion-dependent mechanism. In recent years, RdRp has emerged as an optimal target for the development of antiviral drugs, as demonstrated by recent approvals of sofosbuvir and remdesivir against Hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respectively. In this work, we overview the main sequence and structural features of the RdRp of emerging RNA viruses such as Coronaviruses, Flaviviruses, and HCV, as well as inhibition strategies implemented so far. While analyzing the structural information available on the RdRp of emerging RNA viruses, we provide examples of success stories such as for HCV and SARS-CoV-2. In contrast, Flaviviruses’ story has raised attention about how the lack of structural details on catalytically-competent or ligand-bound RdRp strongly hampers the application of structure-based drug design, either in repurposing and conventional approaches.     

Error compensation of tRNA misacylation by codon-anticodon mismatch prevents translational amino acid misinsertion

Codon-anticodon mismatches and tRNA misloadings cause translational amino acid misinsertions, producing dysfunctional proteins. Here I explore the original hypothesis whether mismatches tend to compensate misacylation, so as to insert the amino acid coded by the codon. This error compensation is promoted by the fact that codon-anticodon mismatch stabilities increase with tRNA misacylation potentials (predicted by 'tfam') by non-cognate amino acids coded by the mismatched codons for most tRNAs examined. Error compensation is independent of preferential misacylation by non-cognate amino acids physico-chemically similar to cognate amino acids, a phenomenon that decreases misinsertion impacts. Error compensation correlates negatively with (a) codon/anticodon abundance (in human mitochondria and Escherichia coli); (b) developmental instability (estimated by fluctuating asymmetry in bilateral counts of subdigital lamellae, in each of two lizard genera, Anolis and Sceloporus); and (c) pathogenicity of human mitochondrial tRNA polymorphisms. Patterns described here suggest that tRNA misacylation is sometimes compensated by codon-anticodon mismatches. Hence translation inserts the amino acid coded by the mismatched codon, despite mismatch and misloading. Results suggest that this phenomenon is sufficiently important to affect whole organism phenotypes, as shown by correlations with pathologies and morphological estimates of developmental stability.     

Trichobitacin-a new ribosome-inactivating protein Ⅰ.The isolation,physicochemical and biological properties of trichobitacin

From the press-residue of the fresh root tuber of Trichosanthes kirilowii Maxim (Cu-curbitaceae),a new ribosome-inactivating protein (RIP),trichobitacin,was isolated.It has the activity of RNA N-glycosidase and can inhibit the growth of human placental trophoblastic cells.Its molecular weight is 27,228 Da (ES-MS) and pI 9.6.It is a single chain basic RIP.Its amino acid composition was determined.It is a new RIP.It consists of 0.7~0.9% galactose and may be a glycoprotein.Its A'-and C-terminal amino acid is Asp and Ala,respectively.Its N-terminal preliminary amino acid sequence has been determined.     

Molecular docking revealed the binding of nucleotide/side inhibitors to Zika viral polymerase solved structures

A new Zika virus (ZIKV) outbreak started in 2015. According to the World Health Organization, 84 countries confirmed ZIKV infection. RNA-dependent RNA polymerase (RdRp) was an appealing target for drug designers during the last two decades. Through molecular docking, we screened 16 nucleotide/side inhibitors against ZIKV RdRp. While the mode of interaction with ZIKV is different from that in the hepatitis C virus (HCV), nucleotide/side inhibitors in this study (mostly anti-HCV) showed promising binding affinities (?6.2 to ?9.7 kcal/mol calculated by AutoDock Vina) to ZIKV RdRp. Setrobuvir, YAK and, to a lesser extent, IDX-184 reveal promising results compared to other inhibitors in terms of binding ZIKV RdRp. These candidates would be powerful anti-ZIKV drugs.     

Modular and Versatile Trans-Encoded Genetic Switches

Current bacterial RNA switches suffer from lack of versatile inputs and are difficult to engineer. We present versatile and modular RNA switches that are trans-encoded and based on tRNA-mimicking structures (TMSs). These switches provide a high degree of freedom for reengineering and can thus be designed to accept a wide range of inputs, including RNA, small molecules, and proteins. This powerful approach enables control of the translation of protein expression from plasmid and genome DNA.     

Searching Anti-Zika Virus Activity in 1H-1,2,3-Triazole Based Compounds

Zika virus (ZIKV) is a mosquito-borne virus belonging to the Flaviviridae family and is responsible for an exanthematous disease and severe neurological manifestations, such as microcephaly and Guillain-Barré syndrome. ZIKV has a single strand positive-sense RNA genome that is translated into structural and non-structural (NS) proteins. Although it has become endemic in most parts of the tropical world, Zika still does not have a specific treatment. Thus, in this work we evaluate the cytotoxicity and antiviral activities of 14 hybrid compounds formed by 1H-1,2,3-triazole, naphthoquinone and phthalimide groups. Most compounds showed low cytotoxicity to epithelial cells, specially the 3b compound. After screening with all compounds, 4b was the most active against ZIKV in the post-infection test, obtaining a 50% inhibition concentration (IC₅₀) of 146.0 µM and SI of 2.3. There were no significant results for the pre-treatment test. According to the molecular docking compound, 4b was suggested with significant binding affinity for the NS5 RdRp protein target, which was further corroborated by molecular dynamic simulation studies.     

Pharmacological and biological antiviral therapeutics for cardiac coxsackievirus infections

Subtype B coxsackieviruses (CVB) represent the most commonly identified infectious agents associated with acute and chronic myocarditis, with CVB3 being the most common variant. Damage to the heart is induced both directly by virally mediated cell destruction and indirectly due to the immune and autoimmune processes reacting to virus infection. This review addresses antiviral therapeutics for cardiac coxsackievirus infections discovered over the last 25 years. One group represents pharmacologically active low molecular weight substances that inhibit virus uptake by binding to the virus capsid (e.g., pleconaril) or inactivate viral proteins (e.g., NO-metoprolol and ribavirin) or inhibit cellular proteins which are essential for viral replication (e.g., ubiquitination inhibitors). A second important group of substances are interferons. They have antiviral but also immunomodulating activities. The third and most recently discovered group includes biological and cellular therapeutics. Soluble receptor analogues (e.g., sCAR-Fc) bind to the virus capsid and block virus uptake. Small interfering RNAs, short hairpin RNAs and antisense oligonucleotides bind to and led to degradation of the viral RNA genome or cellular RNAs, thereby preventing their translation and viral replication. Most recently mesenchymal stem cell transplantation has been shown to possess antiviral activity in CVB3 infections. Taken together, a number of antiviral therapeutics has been developed for the treatment of myocardial CVB infection in recent years. In addition to low molecular weight inhibitors, biological therapeutics have become promising anti-viral agents.     

Caged phosphoproteins

We present the chemical and biological synthesis of caged phosphoproteins using the in vitro nonsense codon suppression methodology. Specifically, phosphoamino acid analogues of serine, threonine, and tyrosine with a single photocleavable o-nitrophenylethyl caging group were synthesized as the amino acyl tRNA adducts for insertion into full-length proteins. For this purpose, a novel phosphitylating agent was developed. The successful incorporation of these bulky and charged amino acids into the alpha-subunit of the nicotinic acetyl choline receptor (nAChR) and the vasodilator-stimulated phosphoprotein (VASP) using an in vitro translation system is reported.     

Target prediction of candidate miRNAs from Oryza sativa for silencing the RYMV genome

In order to maintain a consistent supply of rice globally, control of pathogens affecting crop production is a matter of due concern. Rice yellow mottle virus(RYMV) is known to cause a variety of symptoms which can result in reduced yield. Four ORFs can be identified in the genome of RYMV encoding for P1 (ORF1), Polyprotein (processed to produce VPg, protease, helicase, RdRp4) (ORF2), putative RdRp (ORF3) and capsid/coat protein (ORF4). This research was aimed at identifying genome encoded miRNAs of O. sativa that are targeted to the genome of Rice Yellow Mottle Virus (RYMV). A consensus of four miRNA target prediction algorithms (RNA22, miRanda, TargetFinder and psRNATarget) was computed, followed by calculation of free energies of miRNA-mRNA duplex formation. A phylogenetic tree was constructed to portray the evolutionary relationships between RYMV strains isolated to date. From the consensus of algorithms used, a total of seven O. sativa miRNAs were predicted and conservation of target site was finally evaluated. Predicted miRNAs can be further evaluated by experiments involving the testing of the success of in vitro gene silencing of RYMV genome; this can pave the way for development of RYMV resistant rice varieties in the future.     

Absorption spectra of viral components of Sendai virus in the wavelength region from 130 to 320 nm.

Using synchrotron radiation as a light source, the absorption spectra of purified viral components of the Sendai virus, i.e. messenger RNA, lipids, spike (envelope) proteins, reconstructed envelopes, core proteins and whole virions, were obtained in the wavelength region 130-320 nm by measuring the transmission of thin films. Viral (messenger) RNA two peaks at 260 and 190 nm, and a large increase below 160 nm. The absorption spectrum of lipids exhibited a broad peak at 190 nm and a very sharp increase below 160 nm. With spike proteins, a slight peak at 280 nm and a shoulder at 230 nm were observed in addition to a sharper peak at 190 nm and a rather slow increasing absorption below 160 nm. Reconstructed envelopes showed the features of a combination of lipids and proteins. The absorption spectra of core proteins and whole virions exhibited similar characteristics to spike proteins. Conventional UV data were also obtained in the wavelength range 210-320 nm with RNA and lipids. The UV and synchrotron radiation data were in good agreement in terms of the mass absorption coefficients. The molecular splitting of spike proteins was also examined. Proteins gave more diffuse reflection than their subunits, causing a reduction in absorption. This was explained by a loss of transparency with increasing molecular weight.