Inhibition potential of natural flavonoids against selected omicron (B.1.19) mutations in the spike receptor binding domain of SARS-CoV-2: a molecular modeling approach.

The omicron (B.1.19) variant of contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is considered a variant of concern (VOC) due to its increased transmissibility and highly infectious nature. The spike receptor-binding domain (RBD) is a hotspot of mutations and is regarded as a prominent target for screening drug candidates owing to its crucial role in viral entry and immune evasion. To date, no effective therapy or antivirals have been reported; therefore, there is an urgent need for rapid screening of antivirals. An extensive molecular modelling study has been performed with the primary goal to assess the inhibition potential of natural flavonoids as inhibitors against RBD from a manually curated library. Out of 40 natural flavonoids, five natural flavonoids, namely tomentin A (-8.7 kcal/mol), tomentin C (-8.6 kcal/mol), hyperoside (-8.4 kcal/mol), catechin gallate (-8.3 kcal/mol), and corylifol A (-8.2 kcal/mol), have been considered as the top-ranked compounds based on their binding affinity and molecular interaction profiling. The state-of-the-art molecular dynamics (MD) simulations of these top-ranked compounds in complex with RBD exhibited stable dynamics and structural compactness patterns on 200 nanoseconds. Additionally, complexes of these molecules demonstrated favorable free binding energies and affirmed the docking and simulation results. Moreover, the post-simulation validation of these interacted flavonoids using principal component analysis (PCA) revealed stable interaction patterns with RBD. The integrated results suggest that tomentin A, tomentin C, hyperoside, catechin gallate, and corylifol A might be effective against the emerging variants of SARS-CoV-2 and should be further evaluated using in-vitro and in-vivo experiments.Communicated by Ramaswamy H. Sarma.

Potential of Natural Alkaloids From Jadwar (Delphinium denudatum) as Inhibitors Against Main Protease of COVID-19: A Molecular Modeling Approach.

The ongoing pandemic coronavirus disease (COVID-19) caused by a novel corona virus, namely, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has had a major impact on global public health. COVID-19 cases continue to increase across the globe with high mortality rates in immunocompromised patients. There is still a pressing demand for drug discovery and vaccine development against this highly contagious disease. To design and develop antiviral drugs against COVID-19, the main protease (Mpro) has emerged as one of the important drug targets. In this context, the present work explored Jadwar (Delphinium denudatum)-derived natural alkaloids as potential inhibitors against Mpro of SARS-CoV-2 by employing a combination of molecular docking and molecular dynamic simulation-based methods. Molecular docking and interaction profile analysis revealed strong binding on the Mpro functional domain with four natural alkaloids viz. panicutine (-7.4 kcal/mol), vilmorrianone (-7.0 kcal/mol), denudatine (-6.0 kcal/mol), and condelphine (-5.9 kcal/mol). The molecular docking results evaluated by using the MD simulations on 200 nanoseconds confirmed highly stable interactions of these compounds with the Mpro. Additionally, mechanics/generalized Born/Poisson-Boltzmann surface area (MM/G/P/BSA) free energy calculations also affirmed the docking results. Natural alkaloids explored in the present study possess the essential drug-likeness properties, namely, absorption, distribution, metabolism, and excretion (ADME), and are in accordance with Lipinski's rule of five. The results of this study suggest that these four bioactive molecules, namely, condelphine, denudatine, panicutine, and vilmorrianone, might be effective candidates against COVID-19 and can be further investigated using a number of experimental methods.

Alzheimer's disease as an autoimmune disorder of innate immunity endogenously modulated by tryptophan metabolites.

Introduction: Alzheimer's disease (AD) is characterized by neurotoxic immuno-inflammation concomitant with cytotoxic oligomerization of amyloid beta (Aß) and tau, culminating in concurrent, interdependent immunopathic and proteopathic pathogeneses. Methods: We performed a comprehensive series of in silico, in vitro, and in vivo studies explicitly evaluating the atomistic-molecular mechanisms of cytokine-mediated and Aß-mediated neurotoxicities in AD.  Next, 471 new chemical entities were designed and synthesized to probe the pathways identified by these molecular mechanism studies and to provide prototypic starting points in the development of small-molecule therapeutics for AD. Results: In response to various stimuli (e.g., infection, trauma, ischemia, air pollution, depression), Aß is released as an early responder immunopeptide triggering an innate immunity cascade in which Aß exhibits both immunomodulatory and antimicrobial properties (whether bacteria are present, or not), resulting in a misdirected attack upon "self" neurons, arising from analogous electronegative surface topologies between neurons and bacteria, and rendering them similarly susceptible to membrane-penetrating attack by antimicrobial peptides (AMPs) such as Aß. After this self-attack, the resulting necrotic (but not apoptotic) neuronal breakdown products diffuse to adjacent neurons eliciting further release of Aß, leading to a chronic self-perpetuating autoimmune cycle.  AD thus emerges as a brain-centric autoimmune disorder of innate immunity. Based upon the hypothesis that autoimmune processes are susceptible to endogenous regulatory processes, a subsequent comprehensive screening program of 1137 small molecules normally present in human brain identified tryptophan metabolism as a regulator of brain innate immunity and a source of potential endogenous anti-AD molecules capable of chemical modification into multi-site therapeutic modulators targeting AD's complex immunopathic-proteopathic pathogenesis. Discussion:  Conceptualizing AD as an autoimmune disease, identifying endogenous regulators of this autoimmunity, and designing small molecule drug-like analogues of these endogenous regulators represents a novel therapeutic approach for AD.

Heterologous immunization with Covishield and Pfizer vaccines against SARS-CoV-2 elicits a robust humoral immune response.

Understanding the efficacy and durability of heterologous immunization schedules against SARS-CoV-2 is critical, as supply demands and vaccine choices become significant issues in the global vaccination strategy. Here we characterize the neutralizing antibodies produced in two subjects who received combination immunizations against SARS-CoV-2, first with Covishield (Oxford-AstraZeneca) vaccine, followed 33 days later with a second dose (booster) shot of the Pfizer-BioNTech vaccine. Serum samples were collected 25 days following the primary vaccination and 13 days after the secondary Pfizer vaccination. Both subjects exhibited increased levels of isotype IgG and IgM antibodies directed against the entire spike protein following immunizations. These antibodies also exhibited increased reactivity with the receptor binding domain (RBD) in the spike protein and neutralized the infectivity of replicating vesicular stomatitis virus (VSV) that contains the COVID-19 coronavirus S protein gene in place of its normal G glycoprotein. This VSV pseudovirus also contains the reporter gene for enhanced green fluorescent protein (eGFP). Antibody titers against the spike protein and serum neutralization titers against the reporter virus are reported for the 2 heterologous vaccinated individuals and compared to a positive control derived from a convalescent patient and a negative control from an unexposed individual. The Pfizer-BioNTech vaccine increased antibody binding to the spike protein and RBD, and approached levels found in the convalescent positive control. Neutralizing antibodies against the VSV-S pseudovirus in the 2 subjects also approached levels in the convalescent sera. These results firmly validate the value of the Pfizer-BioNTech vaccine in boosting immunity following initial Covishield inoculation.

Ursolic Acid and Its Nanoparticles Are Potentiators of Oncolytic Measles Virotherapy against Breast Cancer Cells.

Oncolytic viruses (OVs) and phytochemical ursolic acid (UA) are two efficacious therapeutic candidates in development against breast cancer, the deadliest women's cancer worldwide. However, as single agents, OVs and UA have limited clinical efficacies. As a common strategy of enhancing monotherapeutic anticancer efficacy, we explored the combinatorial chemovirotherapeutic approach of combining oncolytic measles virus (MV), which targets the breast tumor marker Nectin-4, and the anticancer UA against breast adenocarcinoma. Our findings revealed that in vitro co-treatment with UA synergistically potentiated the killing of human breast cancer cells by oncolytic MV, without UA interfering the various steps of the viral infection. Mechanistic studies revealed that the synergistic outcome from the combined treatment was mediated through UA's potentiation of apoptotic killing by MV. To circumvent UA's poor solubility and bioavailability and strengthen its clinical applicability, we further developed UA nanoparticles (UA-NP) by nanoemulsification. Compared to the non-formulated UA, UA-NP exhibited improved drug dissolution property and similarly synergized with oncolytic MV in inducing apoptotic breast cancer cell death. This oncolytic potentiation was partly attributed to the enhanced autophagic flux induced by the UA-NP and MV combined treatment. Finally, the synergistic effect from the UA-NP and MV combination was also observed in BT-474 and MDA-MB-468 breast cancer cells. Our study thus highlights the potential value of oncolytic MV and UA-based chemovirotherapy for further development as a treatment strategy against breast cancer, and the feasibility of employing nanoformulation to enhance UA's applicability.

Measles Virus Infects and Programs MAIT Cells for Apoptosis.

Measles virus (MeV) binds, infects, and kills CD150+ memory T cells, leading to immune amnesia. Whether MeV targets innate, memory-like T cells is unknown. We demonstrate that human peripheral blood and hepatic mucosa-associated invariant T (MAIT) cells and invariant natural killer T cells express surprisingly high levels of CD150, more than other lymphocyte subsets. Furthermore, exposing MAIT cells to MeV results in their efficient infection and rapid apoptosis. This constitutes the first report of direct MAIT cell infection by a viral pathogen. Given MAIT cells' antimicrobial properties, their elimination by MeV may contribute to measles-induced immunosuppression and heightened vulnerability to unrelated infections.

The Histone Chaperone FACT Induces Cas9 Multi-turnover Behavior and Modifies Genome Manipulation in Human Cells.

Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-free Xenopus laevis egg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interactor of substrate-bound Cas9. FACT is both necessary and sufficient to displace dCas9, and FACT immunodepletion converts Cas9's activity from multi-turnover to single turnover. In human cells, FACT depletion extends dCas9 residence times, delays genome editing, and alters the balance between indel formation and homology-directed repair. FACT knockdown also increases epigenetic marking by dCas9-based transcriptional effectors with a concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.

Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs.

CRISPR-Cas9 nucleases are powerful genome engineering tools, but unwanted cleavage at off-target and previously edited sites remains a major concern. Numerous strategies to reduce unwanted cleavage have been devised, but all are imperfect. Here, we report that off-target sites can be shielded from the active Cas9•single guide RNA (sgRNA) complex through the co-administration of dead-RNAs (dRNAs), truncated guide RNAs that direct Cas9 binding but not cleavage. dRNAs can effectively suppress a wide-range of off-targets with minimal optimization while preserving on-target editing, and they can be multiplexed to suppress several off-targets simultaneously. dRNAs can be combined with high-specificity Cas9 variants, which often do not eliminate all unwanted editing. Moreover, dRNAs can prevent cleavage of homology-directed repair (HDR)-corrected sites, facilitating scarless editing by eliminating the need for blocking mutations. Thus, we enable precise genome editing by establishing a flexible approach for suppressing unwanted editing of both off-targets and HDR-corrected sites.

Targeting Autophagy Augments BBR-Mediated Cell Death in Human Hepatoma Cells Harboring Hepatitis C Virus RNA.

Hepatocellular carcinoma (HCC), including hepatitis C virus (HCV)-induced HCC, is a deadly disease highly refractory to chemotherapy, thus requiring the continuous identification of novel treatment strategies. Berberine (BBR) has been previously reported to inhibit hepatoma cell growth, but the main type of cell death elicited by BBR, and whether the alkaloid can inhibit hepatoma cells carrying HCV genomes, is unclear. Herein, we show that BBR treatment induced a biphasic cell death irrespective of the presence of HCV subgenomic replicon RNA, first triggering apoptosis that then progressed to necrosis between 24 and 48 h post-treatment. Furthermore, BBR treatment potentiated the HCV replicon-induced reactive oxygen species (ROS) production, inhibition of which with an antioxidant attenuated the cell death that was elicited by BBR in these cells. Moreover, BBR dampened the autophagic response in HCV RNA-positive or negative hepatoma cells, and pharmacological inhibition of autophagy conversely augmented the BBR-induced cell death. Finally, BBR inhibited the growth of Huh-7 cells that were persistently infected with the full-length genome HCV particles, and concomitant pharmacological inhibition of autophagy potentiated the killing of these cells by BBR. Our findings suggest that combining BBR with the inhibition of autophagy could be an attractive treatment strategy against HCC, irrespective of the presence of the HCV genome.

2019-nCoV (Wuhan virus), a novel Coronavirus: human-to-human transmission, travel-related cases, and vaccine readiness.

On 31 December 2019 the Wuhan Health Commission reported a cluster of atypical pneumonia cases that was linked to a wet market in the city of Wuhan, China. The first patients began experiencing symptoms of illness in mid-December 2019. Clinical isolates were found to contain a novel coronavirus with similarity to bat coronaviruses. As of 28 January 2020, there are in excess of 4,500 laboratory-confirmed cases, with > 100 known deaths. As with the SARS-CoV, infections in children appear to be rare. Travel-related cases have been confirmed in multiple countries and regions outside mainland China including Germany, France, Thailand, Japan, South Korea, Vietnam, Canada, and the United States, as well as Hong Kong and Taiwan. Domestically in China, the virus has also been noted in several cities and provinces with cases in all but one provinence. While zoonotic transmission appears to be the original source of infections, the most alarming development is that human-to-human transmission is now prevelant. Of particular concern is that many healthcare workers have been infected in the current epidemic. There are several critical clinical questions that need to be resolved, including how efficient is human-to-human transmission? What is the animal reservoir? Is there an intermediate animal reservoir? Do the vaccines generated to the SARS-CoV or MERS-CoV or their proteins offer protection against 2019-nCoV? We offer a research perspective on the next steps for the generation of vaccines. We also present data on the use of in silico docking in gaining insight into 2019-nCoV Spike-receptor binding to aid in therapeutic development. Diagnostic PCR protocols can be found at https://www.who.int/health-topics/coronavirus/laboratory-diagnostics-for-novel-coronavirus.

The Ubiquitin-Specific Protease 18 Promotes Hepatitis C Virus Production by Increasing Viral Infectivity.

BACKGROUND AND AIMS: Ubiquitin-specific protease 18 (USP18) is involved in immunoregulation and response to interferon- (IFN-) based treatment in patients chronically infected with hepatitis C virus (HCV). We investigated whether and how its upregulation alters HCV infection. METHODS: Overexpression of wild-type (USP18 WT) or catalytically inactive mutant (USP18 C64S) USP18 was examined for effects on HCV replication in the absence and presence of IFNα or IFNλ using both the HCV-infective model and replicon cells. The IFN signaling pathway was assessed via STAT1 phosphorylation (western blot) and downstream ISG expression (real-time PCR). Mechanistic roles were sought by quantifying microRNA-122 levels and J6/JFH1 infectivity of Huh7.5 cells. RESULTS: We found that overexpression of either USP18 WT or USP18 C64S stimulated HCV production and blunted the anti-HCV effect of IFNα and IFNλ in the infective model but not in the replicon system. Overexpressed USP18 showed no effect on Jak/STAT signaling nor on microRNA-122 expression. However, USP18 upregulation markedly increased J6/JFH1 infectivity and promoted the expression of the key HCV entry factor CD81 on Huh7.5 cells. CONCLUSIONS: USP18 stimulates HCV production and blunts the effect of both type I and III IFNs by fostering a cellular environment characterized by upregulation of CD81, promoting virus entry and infectivity.

Advances in genome editing through control of DNA repair pathways.

Eukaryotic cells deploy overlapping repair pathways to resolve DNA damage. Advancements in genome editing take advantage of these pathways to produce permanent genetic changes. Despite recent improvements, genome editing can produce diverse outcomes that can introduce risks in clinical applications. Although homology-directed repair is attractive for its ability to encode precise edits, it is particularly difficult in human cells. Here we discuss the DNA repair pathways that underlie genome editing and strategies to favour various outcomes.

Chemovirotherapeutic Treatment Using Camptothecin Enhances Oncolytic Measles Virus-Mediated Killing of Breast Cancer Cells.

Oncolytic virotherapy represents an emerging development in anticancer therapy. Although it has been tested against a variety of cancers, including breast cancer, the efficacy of oncolytic viral vectors delivered as a monotherapy is limited. Enhancing viral oncolytic therapies through combination treatment with anticancer agents is a feasible strategy. In this study, we considered a chemovirotherapeutic approach for treating breast adenocarcinoma using oncolytic measles virus (MV) and the chemotherapeutic agent camptothecin (CPT). Our results demonstrated that co-treatment of MV with CPT yielded enhanced cytotoxicity against breast cancer cells. Low dosage CPT combined with MV was also found to elicit the same therapeutic effect as high doses of CPT. At the lower dosage used, CPT did not inhibit the early stages of MV entry, nor reduce viral replication. Further studies revealed that co-treatment induced significantly enhanced apoptosis of the breast cancer cells compared to either MV or CPT alone. Overall, our findings demonstrate the potential value of MV plus CPT as a novel chemovirotherapeutic treatment against breast cancer and as a strategy to enhance MV oncolytic activity.

Hepatitis C Virus Non-Structural Protein 5A (NS5A) Disrupts Mitochondrial Dynamics and Induces Mitophagy.

Mitophagy is a selective form of autophagy, targeting damaged mitochondria for lysosomal degradation. Although HCV infection has been shown to induce mitophagy, the precise underlying mechanism and the effector protein responsible remain unclear. Herein, we demonstrated that the HCV non-structural protein 5A (NS5A) plays a key role in regulating cellular mitophagy. Specifically, the expression of HCV NS5A in the hepatoma cells triggered hallmarks of mitophagy including mitochondrial fragmentation, loss of mitochondrial membrane potential, and Parkin translocation to the mitochondria. Furthermore, mitophagy induction through the expression of NS5A led to an increase in autophagic flux as demonstrated by an accumulation of LC3II in the presence of bafilomycin and a time-dependent decrease in p62 protein level. Intriguingly, the expression of NS5A concomitantly enhanced reactive oxygen species (ROS) production, and treatment with an antioxidant attenuated the NS5A-induced mitophagy event. These phenomena are similarly recapitulated in the NS5A-expressing HCV subgenomic replicon cells. Finally, we demonstrated that expression of HCV core, which has been documented to inhibit mitophagy, blocked the mitophagy induction both in cells harboring HCV replicating subgenomes or expressing NS5A alone. Our results, therefore, identified a new role for NS5A as an important regulator of HCV-induced mitophagy and have implications to broadening our understanding of the HCV-mitophagy interplay.

Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq.

CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.

Small molecules targeting coxsackievirus A16 capsid inactivate viral particles and prevent viral binding.

Coxsackievirus A16 (CVA16) is an etiologic agent of hand, foot, and mouth disease (HFMD) that affects young children, and although typically self-limited, severe complications, and fatal cases have been reported. Due to the lack of specific medication and vaccines against CVA16, there is currently a need to develop effective antivirals to better control CVA16 infections in epidemic areas. In this study, we identified the tannins chebulagic acid (CHLA) and punicalagin (PUG) as small molecules that can efficiently disrupt the CVA16 infection of human rhabdomyosarcoma cells. Both compounds significantly reduced CVA16 infectivity at micromolar concentrations without apparent cytotoxicity. A mechanistic analysis revealed that the tannins particularly targeted the CVA16 entry phase by inactivating cell-free viral particles and inhibiting viral binding. Further examination by molecular docking analysis pinpointed the targets of the tannins in the fivefold axis canyon region of the CVA16 capsid near the pocket entrance that functions in cell surface receptor binding. We suggest that CHLA and PUG are efficient antagonists of CVA16 entry and could be of value as antiviral candidates or as starting points for developing molecules to treat CVA16 infections.

Transmission genetics of drug-resistant hepatitis C virus.

Antiviral development is plagued by drug resistance and genetic barriers to resistance are needed. For HIV and hepatitis C virus (HCV), combination therapy has proved life-saving. The targets of direct-acting antivirals for HCV infection are NS3/4A protease, NS5A phosphoprotein and NS5B polymerase. Differential visualization of drug-resistant and -susceptible RNA genomes within cells revealed that resistant variants of NS3/4A protease and NS5A phosphoprotein are cis-dominant, ensuring their direct selection from complex environments. Confocal microscopy revealed that RNA replication complexes are genome-specific, rationalizing the non-interaction of wild-type and variant products. No HCV antivirals yet display the dominance of drug susceptibility shown for capsid proteins of other viruses. However, effective inhibitors of HCV polymerase exact such high fitness costs for drug resistance that stable genome selection is not observed. Barriers to drug resistance vary with target biochemistry and detailed analysis of these barriers should lead to the use of fewer drugs.

Mutations in the Fusion Protein of Measles Virus That Confer Resistance to the Membrane Fusion Inhibitors Carbobenzoxy-d-Phe-l-Phe-Gly and 4-Nitro-2-Phenylacetyl Amino-Benzamide.

The inhibitors carbobenzoxy (Z)-d-Phe-l-Phe-Gly (fusion inhibitor peptide [FIP]) and 4-nitro-2-phenylacetyl amino-benzamide (AS-48) have similar efficacies in blocking membrane fusion and syncytium formation mediated by measles virus (MeV). Other homologues, such as Z-d-Phe, are less effective but may act through the same mechanism. In an attempt to map the site of action of these inhibitors, we generated mutant viruses that were resistant to the inhibitory effects of Z-d-Phe-l-Phe-Gly. These 10 mutations were localized to the heptad repeat B (HRB) region of the fusion protein, and no changes were observed in the viral hemagglutinin, which is the receptor attachment protein. Mutations were validated in a luciferase-based membrane fusion assay, using transfected fusion and hemagglutinin expression plasmids or with syncytium-based assays in Vero, Vero-SLAM, and Vero-Nectin 4 cell lines. The changes I452T, D458N, D458G/V459A, N462K, N462H, G464E, and I483R conferred resistance to both FIP and AS-48 without compromising membrane fusion. The inhibitors did not block hemagglutinin protein-mediated binding to the target cell. Edmonston vaccine/laboratory and IC323 wild-type strains were equally affected by the inhibitors. Escape mutations were mapped upon a three-dimensional (3D) structure modeled from the published crystal structure of parainfluenzavirus 5 fusion protein. The most effective mutations were situated in a region located near the base of the globular head and its junction with the alpha-helical stalk of the prefusion protein. We hypothesize that the fusion inhibitors could interfere with the structural changes that occur between the prefusion and postfusion conformations of the fusion protein.IMPORTANCE Due to lapses in vaccination worldwide that have caused localized outbreaks, measles virus (MeV) has regained importance as a pathogen. Antiviral agents against measles virus are not commercially available but could be useful in conjunction with MeV eradication vaccine programs and as a safeguard in oncolytic viral therapy. Three decades ago, the small hydrophobic peptide Z-d-Phe-l-Phe-Gly (FIP) was shown to block MeV infections and syncytium formation in monkey kidney cell lines. The exact mechanism of its action has yet to be determined, but it does appear to have properties similar to those of another chemical inhibitor, AS-48, which appears to interfere with the conformational change in the viral F protein that is required to elicit membrane fusion. Escape mutations were used to map the site of action for FIP. Knowledge gained from these studies could help in the design of new inhibitors against morbilliviruses and provide additional knowledge concerning the mechanism of virus-mediated membrane fusion.