Flavonoids and Nucleotide Analogs Show High Affinity for Viral Proteins of SARS-CoV-2 by in silico Analysis: New Candidates for the Treatment of COVID-19.

The recent epidemic of COVID-19 caused by SARS-CoV-2 was declared by the World Health Organization as a public health emergency of international concern. The absence of an approved vaccine or a specific antiviral drug has made bioinformatic tools crucial for the identification of potential therapeutic targets and drugs for its control. As in other RNA viruses, the protease 3C-like and the RNA-polymerase are two of the SARS-CoV-2 targets to test drugs that can be analyzed in silico. In the present study, compounds derived from plants, fungi, and nucleoside 5'-triphosphate or uridine nucleotide analogs, with anti-DENV activity in vitro or in vivo, were analyzed by molecular docking as potential anti-SARS-CoV-2 drugs. Anthraquinone, with a DENV NS3 protease inhibitory activity; Balapiravir, Fisetin, Hyperoside, and Sofosbuvir, with a DENV NS5 RNA-polymerase inhibitory activity; and Quercetin, with both anti-NS3-NS5 activities, were tested against 3C-like protease and RNA-polymerase of SARS-CoV-2. All these drugs demonstrated a high affinity for the corresponding SARS-CoV-2 proteins, representing excellent candidates for the treatment of COVID-19. Therefore, in vitro or in vivo studies should be carried out using these compounds on models for SARS-CoV-2 infection.

Designing and Validating a New Method the TUNEL Microwave (TUNEL-MW) for Rapid Quantification of Apoptosis in Islets Following Isolation and Post-Thaw

Background: Among the current quality control assays used in islet transplantation, there is an urgent need for more appropriate assays that measure cell damage via apoptosis that are accurate and rapid. Although the Terminal Uridine Nucleotide End Labeling (TUNEL) is a popular marker for apoptosis, the protocol takes 4 hours to complete. In this regard, microwave assisted histoprocessing, which shortens the time taken for processing, holds promise. Keeping this in mind, a new TUNEL Microwave (TUNEL-MW) method, for rapid quantification of apoptosis, was designed, developed and validated. Method: Two lots of post-thaw isolated human islets cultured for 24 hours, 3 days, 5 days and 7 days i.e. 8 samples, were used for the study. Dewaxed and rehydrated tissues were processed for routine histology, stained with haematoxylin and eosin (H&E) and the conventional TUNEL was carried out as per manufacturer’s instructions. For the TUNEL-MW, kit instructions were modified and microwave-assisted histoprocessing was done. The assessment of apoptotic index (AI%) by light microscopy (LM) was carried out by a pathologist who was completely blinded to the study. Results: The new TUNEL-Microwave (TUNEL-MW) developed by us reduced processing time from 4 hours to 30 minutes (saving 3½ hours). Results were validated by univariate linear regression (r2>0.990), coefficient of variation (<5% between all three methods) and the Bland Altman plot comparing AI% determined by the new TUNEL-MW with the conventional TUNEL and with LM (gold standard). Conclusion: TUNEL Microwave appears to be an ideal method. It is simple and takes just 30 minutes to perform and can therefore be used along with existing quality control measures to rule out or measure apoptosis prior to islet release for islet transplantation.

Pseudouridinylation of mRNA coding sequences alters translation

Chemical modifications of RNAs have long been established as key modulators of nonprotein-coding RNA structure and function in cells. There is a growing appreciation that messenger RNA (mRNA) sequences responsible for directing protein synthesis can also be posttranscriptionally modified. The enzymatic incorporation of mRNA modifications has many potential outcomes, including changing mRNA stability, protein recruitment, and translation. We tested how one of the most common modifications present in mRNA coding regions, pseudouridine (Ψ), impacts protein synthesis using a fully reconstituted bacterial translation system and human cells. Our work reveals that replacing a single uridine nucleotide with Ψ in an mRNA codon impedes amino acid addition and EF-Tu GTPase activation. A crystal structure of the Thermus thermophilus 70S ribosome with a tRNAPhe bound to a ΨUU codon in the A site supports these findings. We also find that the presence of Ψ can promote the low-level synthesis of multiple peptide products from a single mRNA sequence in the reconstituted translation system as well as human cells, and increases the rate of near-cognate Val-tRNAVal reacting on a ΨUU codon. The vast majority of Ψ moieties in mRNAs are found in coding regions, and our study suggests that one consequence of the ribosome encountering Ψ can be to modestly alter both translation speed and mRNA decoding.

Current Status of Direct Acting Antiviral Agents against Hepatitis C Virus Infection in Pakistan

In Pakistan, the burden of the hepatitis C virus (HCV) infection is the second highest in the world with the development of chronic hepatitis. Interferon-based combination therapy with ribavirin was the only available treatment until a few years back, with severe side-effects and high failure rates against different genotypes of HCV. Interferon-free all-oral direct-acting antiviral agents (DAAs) approved by the FDA have revolutionized the HCV therapeutic landscape due to their efficiency in targeting different genotypes in different categories of patients, including treatment naïve, treatment failure and relapsing patients, as well as patients with compensated and decompensated cirrhosis. The availability and use of these DAAs is limited in the developing world. Sofosbuvir (SOF), a uridine nucleotide analogue and inhibitor of HCV encoded NS5B polymerase, is now a widely available and in-use DAA in Pakistan; whereas daclatasvir was recently added in the list. According to the documented results, there is hope that this disease can be effectively cured in Pakistan, although a few concerns still remain. The aim of this article is to review the effectiveness of DAAs and the current status of this treatment against HCV genotype 3 infection in Pakistan; various factors associated with SVR; its limitations as an effective treatment regime; and future implications.

De Novo Pyrimidine Biosynthesis Connects Cell Integrity to Amphotericin B Susceptibility in Cryptococcus neoformans

ABSTRACT Synergy between AmB and nucleotide biosynthetic pathways has been documented, but the mechanism of this interaction has not been delineated. Results from this study suggest a correlation between uridine nucleotide biosynthesis and cell integrity likely mediated through the pool of nucleotide-sugar conjugates, which are precursor molecules for both capsule and cell wall of C. neoformans. Thus, we propose a mechanism by which structural defects in the cell wall resulting from perturbation of pyrimidine biosynthesis allow faster and increased penetration of AmB molecules into the cell membrane. Overall, our work demonstrates that impairment of pyrimidine biosynthesis in C. neoformans could be a potential target for antifungal therapy, either alone or in combination with AmB. The use of amphotericin B (AmB) in conjunction with 5-fluorocytosine (5-FC) is known to be the optimal therapy for treating cryptococcosis, but the mechanism by which 5-FC synergizes with AmB is unknown. In this study, we generated a Cryptococcus neoformans ura1Δ mutant lacking dihydroorotate dehydrogenase (DHODH), which demonstrated temperature-sensitive growth due to a defect in cell integrity and sensitivity to cell wall-damaging agents. In addition, sensitivity to AmB was greatly increased. Inclusion of uracil or uridine in the medium did not suppress the cell wall or AmB phenotype, whereas complementation with the wild-type URA1 gene complemented the mutant phenotype. As a measure of membrane accessibility, we assayed the rate of association of the lipid-binding dye 3,3′-dihexyloxacarbocyanine iodide (DiOC6) and saw more rapid association in the ura1Δ mutant. We likewise saw an increased rate of DiOC6 association in other AmB-sensitive mutants, including a ura − spontaneous URA5 mutant made by 5-fluoroorotic acid (5-FOA) selection and a bck1Δ mutant defective in cell integrity signaling. Similar results were also obtained by using a specific plasma membrane-binding CellMask live stain, with cell integrity mutants that exhibited increased and faster association of the dye with the membrane. Chitin synthase mutants (chs5Δ and chs6Δ) that lack any reported cell wall defects, in turn, demonstrate neither any increased susceptibility to AmB nor a greater accessibility to either of the dyes. Finally, perturbation of the cell wall of the wild type by treatment with the β-1,6-glucan synthase inhibitor caspofungin was synergistic with AmB in vitro. IMPORTANCE Synergy between AmB and nucleotide biosynthetic pathways has been documented, but the mechanism of this interaction has not been delineated. Results from this study suggest a correlation between uridine nucleotide biosynthesis and cell integrity likely mediated through the pool of nucleotide-sugar conjugates, which are precursor molecules for both capsule and cell wall of C. neoformans. Thus, we propose a mechanism by which structural defects in the cell wall resulting from perturbation of pyrimidine biosynthesis allow faster and increased penetration of AmB molecules into the cell membrane. Overall, our work demonstrates that impairment of pyrimidine biosynthesis in C. neoformans could be a potential target for antifungal therapy, either alone or in combination with AmB.

The clinically approved antiviral drug sofosbuvir impairs brazilian zika virus replication

SummaryZika virus (ZIKV) is a member of Flaviviridae family, as other agents of clinical significance, such as dengue (DENV) and hepatitis C (HCV) viruses. ZIKV spread from Africa to Pacific and South American territories, emerging as an etiological pathogen of neurological disorders, during fetal development and in adulthood. Therefore, antiviral drugs able to inhibit ZIKV replication are necessary. Broad spectrum antivirals, such as interferon, ribavirin and favipiravir, are harmful for pregnant animal models and women. The clinically approved uridine nucleotide analog anti-HCV drug, sofosbuvir, has not been affiliated to teratogenicity. Sofosbuvir target the most conserved protein over the members of the Flaviviridae family, the viral RNA polymerase. We thus studied ZIKV susceptibility to sofosbovir. We initially characterized a Brazilian ZIKV strain for use in experimental assays. Sofosbuvir inhibits the Brazilian ZIKV replication in a dose-dependent manner, both in BHK-21 cells and SH-Sy5y, by targeting ZIKV RNA polymerase activity, with the involvement of conserved amino acid residues over the members of Flaviviridae family. The identification of clinically approved antiviral drugs endowed with anti-ZIKV could reduce the time frame in pre-clinical development. Altogether, our data indicates that sofosbuvir chemical structure is endowed with anti-ZIKV activity.

Validation and Application of a Liquid Chromatography-Tandem Mass Spectrometry Method To Determine the Concentrations of Sofosbuvir Anabolites in Cells

ABSTRACTSofosbuvir (SOF) is a highly efficacious and well-tolerated uridine nucleotide analog that inhibits the hepatitis C virus (HCV) NS5B polymerase enzyme. SOF is administered as a prodrug, which undergoes intracellular phosphorylation by host enzymes to a monophosphate, diphosphate, and finally a pharmacologically active triphosphate. In order to fully understand the clinical pharmacology of SOF, there is a great need to determine the intracellular phosphate concentrations of the drug. We describe the validation and utilization of a method to characterize SOF's disposition into variousin vivocell types, including hepatocytes, peripheral blood mononuclear cells (PBMC), and red blood cells (RBC). Standard bioanalytical validation criteria were applied to lysed cellular matrices, with a validated linear range of 50 to 50,000 fmol/sample for each phosphate moiety. The assay was utilized to collect the first data demonstrating concentrations of phosphorylated anabolites formed in PBMC, hepatocytes, and RBCin vivoduring SOF therapy. Median concentrations in PBMC were 220 (range, 51.5 to 846), 70.2 (range, 25.8 to 275), and 859 (range, 54.5 to 6,756) fmol/106cells in the monophosphate, diphosphate, and triphosphate fractions, respectively. In contrast, RBC triphosphate concentrations were much lower than those of PBMC, as the median concentration was 2.91 (range, 1.14 to 10.4) fmol/106cells. The PBMC triphosphate half-life was estimated at 26 h using noncompartmental approaches, while nonlinear mixed-effect modeling was used to estimate a 69 h half-life for this moiety in RBC. The validated method and the data it generates provide novel insight into the cellular disposition of SOF and its phosphorylated anabolitesin vivo.