Enhancer deregulation inTET2-mutant clonal hematopoiesis is associated with increased COVID-19 severity and mortality

Background: COVID-19 causes significant morbidity and mortality, albeit with considerable heterogeneity among affected individuals. It remains unclear which host factors determine disease severity and survival. Given the propensity of clonal hematopoiesis (CH) to promote inflammation in healthy individuals, we investigated its effect on COVID-19 outcomes. Method(s): We performed a multi-omics interrogation of the genome, epigenome, transcriptome, and proteome of peripheral blood mononuclear cells from COVID-19 patients (n=227). We obtained clinical data, laboratory studies, and survival outcomes. We determined CH status and TET2-related DNA methylation. We performed single-cell proteogenomics to understand clonal composition in relation to cell phenotype. We interrogated single-cell gene expression in isolation and in conjunction with DNA accessibility. We integrated these multi-omics data to understand the effect of CH on clonal composition, gene expression, methylation of cis-regulatory elements, and lineage commitment in COVID-19 patients. We performed shRNA knockdowns to validate the effect of one candidate transcription factor in myeloid cell lines. Result(s): The presence of CH was strongly associated with COVID-19 severity and all-cause mortality, independent of age (HR 3.48, 95% CI 1.45-8.36, p=0.005). Differential methylation of promoters and enhancers was prevalent in TET2-mutant, but not DNMT3A-mutant CH. TET2- mutant CH was associated with enhanced classical/intermediate monocytosis and single-cell proteogenomics confirmed an enrichment of TET2 mutations in these cell types. We identified celltype specific gene expression changes associated with TET2 mutations in 102,072 single cells (n=34). Single-cell RNA-seq confirmed the skewing of hematopoiesis towards classical and intermediate monocytes and demonstrated the downregulation of EGR1 (a transcription factor important for monocyte differentiation) along with up-regulation of the lncRNA MALAT1 in monocytes. Combined scRNA-/scATAC-seq in 43,160 single cells (n=18) confirmed the skewing of hematopoiesis and up-regulation of MALAT1 in monocytes along with decreased accessibility of EGR1 motifs in known cis-regulatory elements. Using myeloid cell lines for functional validation, shRNA knockdowns of EGR1 confirmed the up-regulation of MALAT1 (in comparison to wildtype controls). Conclusion(s): CH is an independent prognostic factor in COVID-19 and skews hematopoiesis towards monocytosis. TET2-mutant CH is characterized by differential methylation and accessibility of enhancers binding myeloid transcriptions factors including EGR1. The ensuing loss of EGR1 expression in monocytes causes MALAT1 overexpression, a factor known to promote monocyte differentiation and inflammation. These data provide a mechanistic insight to the adverse prognostic impact of CH in COVID-19.

Antimalarial phytochemicals as potential inhibitors of SARS-CoV-2 guanine N7-methyltransferase (nsp 14): an integrated computational approach.

The inhibition of capping enzymes such as guanine-N7-methyltransferase (GMT) is an attractive target for regulating viral replication, transcription, virulence, and pathogenesis. Thus, compounds that target the Severe Acute Respiratory Syndrome Corona Virus 2 GMT (S2GMT) will enhance drug development against COVID-19. In this study, an in-house library of 249 phytochemicals from African medicinal plants was screened using computational approaches including homology modeling, molecular docking, molecular dynamic simulations, binding free energy calculations based on molecular mechanics/Poisson-Boltzmann surface area (MMPBSA) and Absorption-Distribution-Metabolism-Excretion-Toxicity (ADMET) analysis for inhibitors of S2GMT. The top-ten ranked phytochemicals (TTRP) obtained from the docking analysis to S2GMT were further docked to SARS-COV N7-MTase. Among the TTRP, the top-four ranked phytocompounds (TFRP) viz: 3 alkaloids (Isocryptolepine, 10'-Hydroxyusambarensine and Isostrychnopentamine) and a flavonoid (Mulberrofuran F) interacted strongly with critical catalytic residues whose interference either reduce or completely abolish N7-MTase activity, indicating their potential as capping machinery disruptors. The interactions of TFRP with the catalytic residues of S2GMT were preserved in a 100 ns simulated dynamic environment, thereby, demonstrating high degree of structural stability. The MMPBSA binding free energy calculations corroborated the docking scores with biscryptolepine having the highest binding free energy to S2GMT. The TFRP showed favourable drug-likeness and ADMET properties over a wide range of molecular descriptors. Therefore, the TFRP can be further explored as potential S2GMT inhibitors in in vitro and in vivo experiments.Communicated by Ramaswamy H. Sarma.

In silico identification of the potential natural inhibitors of SARS-CoV-2 Guanine-N7 methyltransferase

Background: The outbreak of the COVID-19 pandemic caused by the SARS-CoV-2 has triggered intense scientific research into the possible therapeutic strategies that can combat the ravaging disease. One of such strategies is the inhibition of an important enzyme that affects an important physiological process of the virus. The enzyme, Guanine-N7 Methyltransferase is responsible for the capping of the SARS-CoV-2 mRNA to conceal it from the host's cellular defense. The aim of the study: This study aims at computationally identifying the potential natural inhibitors of the SARS-CoV-2 Guanine-N7 methyltransferase binding at the active site (Pocket 41). Material(s) and Method(s): A library of small molecules was obtained from edible African plants and was molecularly docked against the SARS-CoV-2 Guanine-N7 methyltransferase (QHD43415_13. pdb) using the Pyrx software. Sinefungin, an approved antiviral drug had a binding score of -7.6 kcal/ mol with the target was chosen as a standard. Using the molecular descriptors of the compounds, virtual screening for oral availability was performed using the Pubchem and SWISSADME web tools. The online servers pkCSM and Molinspiration were used for further screening for the pharmacokinetic properties and bioactivity respectively. The molecular dynamic simulation and analyses of the Apo and Holo proteins were performed using the GROMACS software on the Galaxy webserver. Result(s): With a total RMSD of 77.78, average RMSD of 3.704, total regional (active site) RMSF of 30.61, average regional RMSF of 1.91, gyration of 6.9986, and B factor of 696.14, Crinamidine showed the greatest distortion of the target. Conclusion(s): All the lead compounds performed better than the standard while Crinamidine is predicted to show the greatest inhibitory activity. Further tests are required to further investigate the inhibitory activities of the lead compounds.Copyright © 2022 Belgorod State National Research University. All right reserved.

Assembling a structural model of the SARS-CoV-2 replication and transcription complex from 60+ subunits

The SARS-CoV-2 replication and transcription complex (RTC) is made up of nine distinct non-structural viral proteins encoded by the ORF1ab gene. These proteins house seven enzymatic sites that synthesize new viral genomic and subgenomic RNA, proofread and correct errors in the synthesis, add a 5'-cap to the nascent RNA, and truncate the intermediate negative sense 5'-poly-U tail. While x-ray crystallography and cryo-EM have provided high resolution structures of each of the individual proteins of the RTC and have shed light on how subsets of the proteins associate, a full picture of the RTC has remained elusive. Using molecular modeling tools, including protein-protein docking, we have generated a model of the RTC centered around hexameric nsp15, which is capped on two faces by trimers of nsp14/nsp16/(nsp10)2. A conformational change of nsp14, necessary to facilitate binding to nsp15, then recruits six nsp12/nsp7/(nsp8)2 polymerase subunits. To this, six nsp13 subunits are distributed around the complex. The resulting superstructure is composed of 60 subunits total and positions the nsp14 exonuclease and nsp15 endonuclease sites in line with the dsRNA exiting the nsp12 polymerase site. Nsp10 acts to separate the RNA strands, directing the nascent strand to the nsp12 NiRAN site, where a transiently associated nsp9 facilitates the first step in mRNA capping. The RNA is then directed to the nsp14 N7-methyltransferase site and the nsp16 2'O-methyltransferase site to complete the capping. Additionally, template switching during transcription is proposed to be facilitated by positioning of the TRS-L RNA-bound N-protein above the polymerase active site, between two subunits of nsp13. The model, while constructed based on structural considerations, offers a unifying set of hypotheses to explain the diverse set of processes involved in coronavirus genome replication and transcription. All work presented was funded by Gilead Sciences.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Structure-guided optimization of adenosine mimetics as selective and potent inhibitors of coronavirus nsp14 N7-methyltransferases.

The COVID-19 pandemic reveals the urgent need to develop new therapeutics targeting the SARS-CoV-2 replication machinery. The first antiviral drugs were nucleoside analogues targeting RdRp and protease inhibitors active on nsp5 Mpro. In addition to these common antiviral targets, SARS-CoV-2 codes for the highly conserved protein nsp14 harbouring N7-methyltransferase (MTase) activity. Nsp14 is involved in cap N7-methylation of viral RNA and its inhibition impairs viral RNA translation and immune evasion, making it an attractive new antiviral target. In this work, we followed a structure-guided drug design approach to design bisubstrates mimicking the S-adenosylmethionine methyl donor and RNA cap. We developed adenosine mimetics with an N-arylsulfonamide moiety in the 5'-position, recently described as a guanine mimicking the cap structure in a potent adenosine-derived nsp14 inhibitor. Here, the adenine moiety was replaced by hypoxanthine, N6-methyladenine, or C7-substituted 7-deaza-adenine. 26 novel adenosine mimetics were synthesized, one of which selectively inhibits nsp14 N7-MTase activity with a subnanomolar IC50 (and seven with a single-digit nanomolar IC50). In the most potent inhibitors, adenine was replaced by two different 7-deaza-adenines bearing either a phenyl or a 3-quinoline group at the C7-position via an ethynyl linker. These more complex compounds are barely active on the cognate human N7-MTase and docking experiments reveal that their selectivity of inhibition might result from the positioning of their C7 substitution in a SAM entry tunnel present in the nsp14 structure and absent in the hN7-MTase. These compounds show moderate antiviral activity against SARS-CoV-2 replication in cell culture, suggesting delivery or stability issue.

A universal fluorescence polarization high throughput screening assay to target the SAM-binding sites of SARS-CoV-2 and other viral methyltransferases.

SARS-CoV-2 has caused a global pandemic with significant humanity and economic loss since 2020. Currently, only limited options are available to treat SARS-CoV-2 infections for vulnerable populations. In this study, we report a universal fluorescence polarization (FP)-based high throughput screening (HTS) assay for SAM-dependent viral methyltransferases (MTases), using a fluorescent SAM-analogue, FL-NAH. We performed the assay against a reference MTase, NSP14, an essential enzyme for SARS-CoV-2 to methylate the N7 position of viral 5'-RNA guanine cap. The assay is universal and suitable for any SAM-dependent viral MTases such as the SARS-CoV-2 NSP16/NSP10 MTase complex and the NS5 MTase of Zika virus (ZIKV). Pilot screening demonstrated that the HTS assay was very robust and identified two candidate inhibitors, NSC 111552 and 288387. The two compounds inhibited the FL-NAH binding to the NSP14 MTase with low micromolar IC50. We used three functional MTase assays to unambiguously verified the inhibitory potency of these molecules for the NSP14 N7-MTase function. Binding studies indicated that these molecules are bound directly to the NSP14 MTase with similar low micromolar affinity. Moreover, we further demonstrated that these molecules significantly inhibited the SARS-CoV-2 replication in cell-based assays at concentrations not causing cytotoxicity. Furthermore, NSC111552 significantly synergized with known SARS-CoV-2 drugs including nirmatrelvir and remdesivir. Finally, docking suggested that these molecules bind specifically to the SAM-binding site on the NSP14 MTase. Overall, these molecules represent novel and promising candidates to further develop broad-spectrum inhibitors for the management of viral infections.

Identification and structural studies of natural inhibitors against SARS-CoV-2 viral RNA methyltransferase (NSP16).

Pathogenic RNA viruses are emerging as one of the major threats and posing challenges to human community. RNA viruses have an exceptionally shorter generation time and easy to adapt in host cells. The recent emergence of SARS-CoV-2, a long RNA virus, has shown us how difficult it is to overcome this kind of pandemic without understanding the viral infection and replication mechanisms. It is essential to comprehend replications of the viral genome, including RNA polymerization and the final capping process. The mRNAs of SARS-CoV-2 coronaviruses are protected at their 5'-ends by cap structure. The cap-like system plays a significant role in viral translational process, viral RNA stability, and scatting in detecting innate immune recognition in host cells. Two coronavirus enzymes, Nsp14 and Nsp16, critically help in the formation of capping and are considered as potential drug targets for antiviral therapy. Natural and herbal medicines have a past record of treating various acute respiratory diseases. In this work, we have exploited 56000 natural compounds to screen potential inhibitors against NSP16. In silico virtual screening, docking and Molecular Dynamics (MD) simulation studies were performed to understand how these potential inhibitors are bound to NSP16. We observed that the most highly screened compound binds to protein molecules with a high dock score, primarily through hydrophobic interactions and hydrogen bonding, as previously reported for NSP16. Compound-13 (2-hydroxy-N-({1-[2-hydroxy-1-(hydroxymethyl)ethyl]piperidin-3-yl}methyl)-5-methylbenzamide) and compound-51 (N-(2-isobutoxybenzyl)-N,2-dimethyl-2,8-diazaspiro[4.5]decane-3-carboxamide) occupied in active site along with good pharmokinetices properties. In conclusion, the selected compounds could be used as a novel therapeutic against SARS-CoV-2.Communicated by Ramaswamy H. Sarma.

Methyltransferases of Riboviria.

Many viruses from the realm Riboviria infecting eukaryotic hosts encode protein domains with sequence similarity to S-adenosylmethionine-dependent methyltransferases. These protein domains are thought to be involved in methylation of the 5'-terminal cap structures in virus mRNAs. Some methyltransferase-like domains of Riboviria are homologous to the widespread cellular FtsJ/RrmJ-like methyltransferases involved in modification of cellular RNAs; other methyltransferases, found in a subset of positive-strand RNA viruses, have been assigned to a separate "Sindbis-like" family; and coronavirus-specific Nsp13/14-like methyltransferases appeared to be different from both those classes. The representative structures of proteins from all three groups belong to a specific variety of the Rossmann fold with a seven-stranded ß-sheet, but it was unclear whether this structural similarity extends to the level of conserved sequence signatures. Here I survey methyltransferases in Riboviria and derive a joint sequence alignment model that covers all groups of virus methyltransferases and subsumes the previously defined conserved sequence motifs. Analysis of the spatial structures indicates that two highly conserved residues, a lysine and an aspartate, frequently contact a water molecule, which is located in the enzyme active center next to the methyl group of S-adenosylmethionine cofactor and could play a key role in the catalytic mechanism of the enzyme. Phylogenetic evidence indicates a likely origin of all methyltransferases of Riboviria from cellular RrmJ-like enzymes and their rapid divergence with infrequent horizontal transfer between distantly related viruses.

SS148 and WZ16 inhibit the activities of nsp10-nsp16 complexes from all seven human pathogenic coronaviruses.

Seven coronaviruses have infected humans (HCoVs) to-date. SARS-CoV-2 caused the current COVID-19 pandemic with the well-known high mortality and severe socioeconomic consequences. MERS-CoV and SARS-CoV caused epidemic of MERS and SARS, respectively, with severe respiratory symptoms and significant fatality. However, HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43 cause respiratory illnesses with less severe symptoms in most cases. All coronaviruses use RNA capping to evade the immune systems of humans. Two viral methyltransferases, nsp14 and nsp16, play key roles in RNA capping and are considered valuable targets for development of anti-coronavirus therapeutics. But little is known about the kinetics of nsp10-nsp16 methyltransferase activities of most HCoVs, and reliable assays for screening are not available. Here, we report the expression, purification, and kinetic characterization of nsp10-nsp16 complexes from six HCoVs in parallel with previously characterized SARS-CoV-2. Probing the active sites of all seven by SS148 and WZ16, the two recently reported dual nsp14 / nsp10-nsp16 inhibitors, revealed pan-inhibition. Overall, our study show feasibility of developing broad-spectrum dual nsp14 / nsp10-nsp16-inhibitor therapeutics.

NSUN2-mediated M5c methylation of IRF3 mRNA negatively regulates type I interferon responses during various viral infections.

5-Methylcytosine (m5C) is a widespread post-transcriptional RNA modification and is reported to be involved in manifold cellular responses and biological processes through regulating RNA metabolism. However, its regulatory role in antiviral innate immunity has not yet been elucidated. Here, we report that NSUN2, a typical m5C methyltransferase, negatively regulates type I interferon responses during various viral infections, including SARS-CoV-2. NSUN2 specifically mediates m5C methylation of IRF3 mRNA and accelerates its degradation, resulting in low levels of IRF3 and downstream IFN-ß production. Knockout or knockdown of NSUN2 enhanced type I interferon and downstream ISGs during various viral infection in vitro. And in vivo, the antiviral innate response is more dramatically enhanced in Nsun2+/- mice than in Nsun2+/+ mice. The highly m5C methylated cytosines in IRF3 mRNA were identified, and their mutation enhanced cellular IRF3 mRNA levels. Moreover, infection with Sendai virus (SeV), vesicular stomatitis virus (VSV), herpes simplex virus 1 (HSV-1), or Zika virus (ZIKV) resulted in a reduction of endogenous NSUN2 levels. Especially, SARS-CoV-2 infection (WT strain and BA.1 omicron variant) also decreased endogenous levels of NSUN2 in COVID-19 patients and K18-hACE2 KI mice, further increasing type I interferon and downstream ISGs. Together, our findings reveal that NSUN2 serves as a negative regulator of interferon response by accelerating the fast turnover of IRF3 mRNA, while endogenous NSUN2 levels decrease during SARS-CoV-2 and various viral infections to boost antiviral responses for effective elimination of viruses.

SARS-CoV-2 Uses Nonstructural Protein 16 To Evade Restriction by IFIT1 and IFIT3.

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'-O-methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'-O-MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo, using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive than wild-type SARS-CoV-2 to type I interferon (IFN-I) in vitro. Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'-O-methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, an MTase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment and attenuates viral replication. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a target for future antiviral therapies. IMPORTANCE Similar to other coronaviruses, disruption of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo, our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1 but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'-O-methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

3-(Adenosylthio)benzoic Acid Derivatives as SARS-CoV-2 Nsp14 Methyltransferase Inhibitors.

SARS-CoV-2 nsp14 guanine-N7-methyltransferase plays an important role in the viral RNA translation process by catalyzing the transfer of a methyl group from S-adenosyl-methionine (SAM) to viral mRNA cap. We report a structure-guided design and synthesis of 3-(adenosylthio)benzoic acid derivatives as nsp14 methyltransferase inhibitors resulting in compound 5p with subnanomolar inhibitory activity and improved cell membrane permeability in comparison with the parent inhibitor. Compound 5p acts as a bisubstrate inhibitor targeting both SAM and mRNA-binding pockets of nsp14. While the selectivity of 3-(adenosylthio)benzoic acid derivatives against human glycine N-methyltransferase was not improved, the discovery of phenyl-substituted analogs 5p,t may contribute to further development of SARS-CoV-2 nsp14 bisubstrate inhibitors.

Identification and characterization of alternative sites and molecular probes for SARS-CoV-2 target proteins.

Three protein targets from SARS-CoV-2, the viral pathogen that causes COVID-19, are studied: the main protease, the 2'-O-RNA methyltransferase, and the nucleocapsid (N) protein. For the main protease, the nucleophilicity of the catalytic cysteine C145 is enabled by coupling to three histidine residues, H163 and H164 and catalytic dyad partner H41. These electrostatic couplings enable significant population of the deprotonated state of C145. For the RNA methyltransferase, the catalytic lysine K6968 that serves as a Brønsted base has significant population of its deprotonated state via strong coupling with K6844 and Y6845. For the main protease, Partial Order Optimum Likelihood (POOL) predicts two clusters of biochemically active residues; one includes the catalytic H41 and C145 and neighboring residues. The other surrounds a second pocket adjacent to the catalytic site and includes S1 residues F140, L141, H163, E166, and H172 and also S2 residue D187. This secondary recognition site could serve as an alternative target for the design of molecular probes. From in silico screening of library compounds, ligands with predicted affinity for the secondary site are reported. For the NSP16-NSP10 complex that comprises the RNA methyltransferase, three different sites are predicted. One is the catalytic core at the conserved K-D-K-E motif that includes catalytic residues D6928, K6968, and E7001 plus K6844. The second site surrounds the catalytic core and consists of Y6845, C6849, I6866, H6867, F6868, V6894, D6895, D6897, I6926, S6927, Y6930, and K6935. The third is located at the heterodimer interface. Ligands predicted to have high affinity for the first or second sites are reported. Three sites are also predicted for the nucleocapsid protein. This work uncovers key interactions that contribute to the function of the three viral proteins and also suggests alternative sites for ligand design.

A Comprehensive Study to Unleash the Putative Inhibitors of Serotype2 of Dengue Virus: Insights from an In Silico Structure-Based Drug Discovery.

Dengue fever is a mosquito-borne disease that claims the lives of millions of people around the world. A number of factors like disease's non-specific symptoms, increased viral mutation, growing antiviral drug resistance due to reduced susceptibility, unavailability of an effective vaccine for dengue, weak immunity against the virus, and many more are involved. Dengue belongs to the Flaviviridae family of viruses. The two species of the vector transmitting dengue are Aedes aegypti and Aedes albopictus, with the former one being dominant. Serotypes 2 of dengue fever are spread to the human body and cause severe illness. Recently, dengue has imposed an aggressive effect synergistically with the COVID-19 pandemic. As a result, we concentrated our efforts on finding a potential therapeutic. For this, we chose natural compounds to fight dengue fever, which is currently regarded as successful among many drug therapies. Following this, we started the in silico experiment with 922 plant extracts as lead compounds to fight serotype 2. In this study, we used SwissADME for analyzing ligand drug-likeness, pkCSM for designing an ADMET profile, Autodock vina 4.2 and Swissdock tools for molecular docking, and finally Desmond for molecular dynamics simulation. Ultimately 45 were found effective against the 2'O methyltransferase protein of serotype 2. CHEMBL376820 was found as possible therapeutic candidates for inhibiting methyltransferase protein in this thorough analysis. Nevertheless, more in vitro and in vivo research are required to substantiate their potential therapeutic efficacy.

Nsp16 shields SARS-CoV-2 from efficient MDA5 sensing and IFIT1-mediated restriction.

Methylation of the mRNA 5' cap by cellular methyltransferases enables efficient translation and avoids recognition by innate immune factors. Coronaviruses encode viral 2'-O-methyltransferases to shield their RNA from host factors. Here, we generate recombinant SARS-CoV-2 harboring a catalytically inactive 2'-O-methyltransferase Nsp16, Nsp16mut, and analyze viral replication in human lung epithelial cells. Although replication is only slightly attenuated, we find SARS-CoV-2 Nsp16mut to be highly immunogenic, resulting in a strongly enhanced release of type I interferon upon infection. The elevated immunogenicity of Nsp16mut is absent in cells lacking the RNA sensor MDA5. In addition, we report that Nsp16mut is highly sensitive to type I IFN treatment and demonstrate that this strong antiviral effect of type I IFN is mediated by the restriction factor IFIT1. Together, we describe a dual role for the 2'-O-methyltransferase Nsp16 during SARS-CoV-2 replication in avoiding efficient recognition by MDA5 and in shielding its RNA from interferon-induced antiviral responses, thereby identifying Nsp16 as a promising target for generating attenuated and highly immunogenic SARS-CoV-2 strains and as a potential candidate for therapeutic intervention.

A perspective influence of the Epsilon variant of COVID-19 on Catechol-O-Methyltransferase Val158Met leads to the emergence of human papilloma virus-Gueye, which is wrongly described as monkey pox

A perspective study discussed how the epsilon variant of COVID-19 induced mutation to the existed human papilloma virus via mutagenic cadmium content of the COVID-19, and arsenic content of epsilon variant lead to appearance of new strain of human papilloma virus, we named it Human Papilloma Virus-Gueye. As this virus is most common among gay men, and due to the absence of infection with it among HIV-controlled patients. We suggest that this virus disturbs Catechol-O-Methyltransferase Val158Met which is the region which is responsible for male sexual orientation and has been linked to executive dysfunction, which might increase sexual risk behaviours favouring HIV transmission, which is damaged by mycotoxin of myecetoma fungal species and by cadmium contents of epsilon variant of COVID-19. We conclude that COVID-19 is not hazardous just because of its severe symptoms, but also because of its action as a mutagen on other microbes that may be present in a subclinical state. Also, we suggest that immunization against COVID-19 may lead to unexpected complications, especially mutations in other microbes. Finally, we claim that the scientific community named it monkey pox. Even among individuals who do not travel to the endemic, it is just mutant form of Human Papilloma virus.

Synthesis and characterization of two known and one new impurities of dolutegravir: In silico evaluation of certain intermediates against SARS CoV-2 O-ribose methyltransferase (OMTase).

Besides its use against HIV infection the marketed anti-retroviral drug dolutegravir attracted attention as a potential agent against COVID-19 in multiple AI (artificial intelligence) based studies. Due to our interest in accessing the impurities of this drug we report the synthesis and characterization of three impurities of dolutegravir one of which is new. The synthesis of O-methyl ent-dolutegravir was accomplished in three-steps the first one involved the construction of fused 1,3-oxazinane ring. The cleavage of -OEt ether moiety followed by methylation afforded the target compound. The second impurity i.e. N-(2,4-difluorobenzyl)-4-methoxy-3-oxobutanamide was synthesized via a multi-step method involving sequentially the keto group protection, ester hydrolysis, acid chloride formation followed by the reaction with amine and finally keto group deprotection. The synthesis of new or dimer impurity was carried out via another multi-step method similar to the previous one starting from ethyl 4-chloro acetoacetate. The methodology involved preparation of ether derivative, keto group protection, ester hydrolysis, preparation of amide derivative via acid chloride formation in situ and then keto group deprotection for a longer duration. The last step afforded the target compound for which a plausible reaction mechanism has been proposed. All three impurities were prepared in gram scale (minimum 2 g and maximum 8 g). The in silico evaluation of three selected synthesized intermediates e.g. 7, 8 and 9 (structurally similar to dolutegravir) against SARS CoV-2 O-ribose methyltransferase (OMTase) (PDB: 3R24) indicated that compound 7 could be of interest as a possible inhibitor of this protein.

VIRTUAL SCREENING AND MOLECULAR DOCKING ANALYSIS ON THREE SARS-COV-2 DRUG TARGETS BY MULTIPLE COMPUTATIONAL APPROACH

Objective: SARS-CoV-2 is a pandemic virus characterized by upper respiratory tract infection and can range from mild symptoms to severe complications. In this case, drug repurposing and computer-aided studies have become very important to find emergency solutions. In this study, drug-target interactions on three nonstructural protein structures of SARS-CoV-2 of 8820 drug candidates or drug molecules obtained from the DrugBank database were analyzed. Material and Method: Comprehensive virtual screening and molecular docking studies from 8820 drug molecules or candidates obtained from the DrugBank database were performed on the RNA binding protein, 2'-O-methyltransferase, and endoribonuclease of SARS-CoV-2;and potential drug candidates were determined for each target. Virtual screening studies have been done with High-Throughput Virtual Screening (HTVS), Standard Precision (SP), Extra Precision (XP), and Molecular Mechanics Generalized Born Surface Area (MM-GBSA). Also, information about the clinical findings, transmission, pathogenesis, and treatment of SARS-CoV-2 has been given. Result and Discussion: Drug-target interactions on three nonstructural protein structures of SARS-CoV-2 of 8820 drug candidates or drug molecules obtained from the DrugBank database were analyzed. Potential compound recommendations for each drug target were presented. Information was given about key amino acids where active sites of drug target proteins interact with ligands. This study is expected to be useful in target-based drug development studies on the proteins of SARS-CoV-2.

Evaluation of a pharmacist-led virtual thiopurine clinic

Introduction: Monitoring of patients on immunomodulator therapy for inflammatory bowel disease (IBD) and auto-immune hepatitis (AIH) significantly increases clinical workload in gastroenterology outpatient clinics owing to blood test monitoring requirements. Aim(s): This study conducted at Barnsley Hospital NHS Foundation Trust aimed to determine the clinical impact of a pharmacist-led virtual thiopurine clinic on drug monitoring, safety and quality of service in a cohort of IBD and AIH patients. Method(s): Patients attended an initial face-to-face outpatient consultation and were then followed-up via remote telephone consultations, as part of a virtual clinic, at two-week intervals. Patient biochemical data were analysed alongside anonymous postal questionnaires to provide an assessment of the quality of the service over a three-month period between 1 May and 31 July 2014. A second survey was performed in December 2019 to re-assess patient satisfaction. Result(s): 81 patients were assessed;clinical indications for thiopurines were IBD (n=75) and AIH (n=6). Overall, 39.5% (n=32) patients failed to reach the thiopurine therapeutic target (2mg/kg with normal thiopurine methyltransferase) owing to drug intolerance, lack of clinical response or hepatotoxicity. Thioguanine nucleotide analysis was performed in 29.6% (n=24) of patients who failed initial treatment, to allow dose optimisation. Hepatotoxicity developed in 9.9% (n=8) of patients, which required shunting of thiopurine metabolism with allopurinol. Myelosuppression developed in 6.9% (n=21) of the total number of blood tests performed (n=304), leading to discontinuation of thiopurines in two patients. The questionnaire response rate was 51.9% (n=42);92.9% of respondents (n=39) found the virtual clinic to be convenient;and 95.2% (n=40) were satisfied with the service. The follow-up survey included 373 patients with a 35.9% response rate (n=134);88% (n=118) found the service easily or very easily accessible, and 82% (n=109) found it very helpful or extremely helpful. Overall satisfaction was highly rated (average 4.46/5). Conclusion(s): Pharmacist-led virtual thiopurine clinics were shown to be safe and regarded favourably by patients. The clinics identified patients exposed to the risks of myelosuppression, as well as allowing thiopurine dose optimisation during the three-month period from May to July 2014. The followup survey in 2019 showed patients valued telemedicine as an alternative to face-to-face visits. Virtual clinics offer a viable alternative to traditional outpatient-driven thiopurine monitoring, especially during the ongoing COVID-19 pandemic. Copyright © 2020 Pharmaceutical Press. All rights reserved.

Delayed leukopenia following intentional azathioprine ingestion

Background: Azathioprine is a purine analog metabolized to 6- mercaptopurine (6-MP) utilizing glutathione. Its high oral bioavailability and longer duration of action make it viable as a treatment for ulcerative colitis or as an anti-rejection medication for renal transplant patients. Specific experience in overdose with this agent is limited although toxicity mimics 6-MP including hepatotoxicity, delayed leukopenia, and acute interstitial nephritis. Case report: A 46 year old female (64 kg) with a history of ulcerative colitis, migraines, and anxiety presented with a selfreported intentional ingestion of 1000mg azathioprine and presented to care approximately 8 h post-ingestion. Her compliance with azathioprine preceding the ingestion was unclear. She reported taking her other medications as prescribed (tadalafil, sulfasalazine, fioricet, alprazolam) the day prior to presentation. Other than one episode of emesis without pill fragments, myalgias, headache she had no other symptoms. Her presenting vital signs were HR 84, RR 22, BP 90/63, T 36.2 degreeC. Initial labs included a normal chemistry profile, undetectable serum acetaminophen and salicylates, an ethanol level of 50 mg/dL and venous lactate of 1.6mmol/L. She received a total of 3 L of crystalloid IV fluids with improvement in blood pressure to 125/66 and was transferred for higher level of care. Due to the delay in presentation and well appearance, activated charcoal and hemodialysis were considered but deferred. While inpatient she had laboratory evaluation including CBC and differential every 8 h. In the ED she developed a fever, 38.1 degreeC. PCR testing for COVID-19 was negative. Whole blood thiopurine metabolites (Prometheus Biosciences, Test 3200) were sent approximately 33 h from time of ingestion. 6-thioguanine levels were 108 pmol/8x10degree8 RBC, below the therapeutic reference range (230-400 pmol/8x10degree8 RBC). 6-methylmercaptopurine metabolites were below the lower limit of quantification (761pmol/8x10degree8 RBC). Genetic testing for thiopurine S-methyltransferase was declined by the patient. She was hospitalized for 4 days and did not develop any substantial vital sign abnormalities or creatinine elevation. Her absolute neutrophil count dropped to 500/mm3 approximately 76 h post-ingestion, but started to improve 84 h post-ingestion and granulocyte-macrophage colony-stimulating factor was deferred. Her peak AST was 113 IU/L, approximately 46 h post-ingestion and returned to normal (16 IU/L) upon follow-up 7 days postingestion. White blood cell count 7 days post-ingestion was 4.3 K/mm3. Discussion(s): Azathioprine overdose is rarely reported in the literature. Case reports describe delayed leukopenia and hepatotoxicity from repeat supratherapeutic ingestions, however, based upon limited experience serious toxicity from single acute ingestions appears rare. A report of a single acute ingestion of 7500mg of azathioprine resulted in moderate leukopenia (4.1 K/ mm3) 3 days post-ingestion. Peak immunosuppressive effects can take up to 2 weeks from initiation or change in dose. Symptoms in this case are consistent with effects from azathioprine including vomiting, transient hypotension, and myalgias. Conclusion(s): Intentional ingestions of azathioprine are infrequently reported and can result in serious delayed myelosuppression. We report a case of a single acute ingestion of >15 mg/kg resulting in delayed myelosuppression managed conservatively.