SARS-CoV-2 omicron variants harbor spike protein mutations responsible for their attenuated fusogenic phenotype.

Since the emergence of the Omicron variants at the end of 2021, they quickly became the dominant variants globally. The Omicron variants may be more easily transmitted compared to the earlier Wuhan and the other variants. In this study, we aimed to elucidate mechanisms of the altered infectivity associated with the Omicron variants. We systemically evaluated mutations located in the S2 sequence of spike and identified mutations that are responsible for altered viral fusion. We demonstrated that mutations near the S1/S2 cleavage site decrease S1/S2 cleavage, resulting in reduced fusogenicity. Mutations in the HR1 and other S2 sequences also affect cell-cell fusion. Based on nuclear magnetic resonance (NMR) studies and in silico modeling, these mutations affect fusogenicity possibly at multiple steps of the viral fusion. Our findings reveal that the Omicron variants have accumulated mutations that contribute to reduced syncytial formation and hence an attenuated pathogenicity.

Three-Dimensional Reconstruction of the Hepatitis C Virus Envelope Glycoprotein E1E2 Heterodimer by Electron Microscopic Analysis.

Despite the development of highly effective hepatitis C virus (HCV) treatments, an effective prophylactic vaccine is still lacking. HCV infection is mediated by its envelope glycoproteins, E1 and E2, during the entry process, with E2 binding to cell receptors and E1 mediating endosomal fusion. The structure of E1E2 has only been partially resolved by X-ray crystallography of the core domain of E2 protein (E2c) and its complex with various neutralizing antibodies. Structural understanding of the E1E2 heterodimer in its native form can advance the design of candidates for HCV vaccine development. Here, we analyze the structure of the recombinant HCV E1E2 heterodimer with the aid of well-defined monoclonal anti-E1 and E2 antibodies, as well as a small-molecule chlorcyclizine-diazirine-biotin that can target and cross-link the putative E1 fusion domain. Three-dimensional (3D) models were generated after extensive 2D classification analysis with negative-stain single-particle data sets. We modeled the available crystal structures of the E2c and Fabs into 3D volumes of E1E2-Fab complexes based on the shape and dimension of the domain density. The E1E2 heterodimer exists in monomeric form and consists of a main globular body, presumably depicting the E1 and E2 stem/transmembrane domain, and a protruding structure representing the E2c region, based on anti-E2 Fab binding. At low resolution, a model generated from negative-stain analysis revealed the unique binding and orientation of individual or double Fabs onto the E1 and E2 components of the complex. Cryo-electron microscopy (cryo-EM) of the double Fab complexes resulted in a refined structural model of the E1E2 heterodimer, presented here. IMPORTANCE Recombinant HCV E1E2 heterodimer is being developed as a vaccine candidate. Using electron microscopy, we demonstrated unique features of E1E2 in complex with various neutralizing antibodies and small molecule inhibitors that are important to understanding its antigenicity and induction of immune response.

A dual conditional CRISPR-Cas9 system to activate gene editing and reduce off-target effects in human stem cells.

The CRISPR-Cas9 system has emerged as a powerful and efficient tool for genome editing. An important drawback of the CRISPR-Cas9 system is the constitutive endonuclease activity when Cas9 endonuclease and its sgRNA are co-expressed. This constitutive activity results in undesirable off-target effects that hinder studies using the system, such as probing gene functions or its therapeutic use in humans. Here, we describe a convenient method that allows temporal and tight control of CRISPR-Cas9 activity by combining transcriptional regulation of Cas9 expression and protein stability control of Cas9 in human stem cells. To achieve this dual control, we combined the doxycycline-inducible system for transcriptional regulation and FKBP12-derived destabilizing domain fused to Cas9 for protein stability regulation. We showed that approximately 5%-10% of Cas9 expression was observed when only one of the two controls was applied. By combining two systems, we markedly lowered the baseline Cas9 expression and limited the exposure time of Cas9 endonuclease in the cell, resulting in little or no undesirable on- or off-target effects. We anticipate that this dual conditional CRISPR-Cas9 system can serve as a valuable tool for systematic characterization and identification of genes for various pathological processes.

Targeting the Fusion Process of SARS-CoV-2 Infection by Small Molecule Inhibitors.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a serious threat to global public health, underscoring the urgency of developing effective therapies. Therapeutics and, more specifically, direct-acting antiviral development are still very much in their infancy. Here, we report that two hepatitis C virus (HCV) fusion inhibitors identified in our previous study, dichlorcyclizine and fluoxazolevir, broadly block human coronavirus entry into various cell types. Both compounds were effective against various human-pathogenic CoVs in multiple assays based on vesicular stomatitis virus (VSV) pseudotyped with the spike protein and spike-mediated syncytium formation. The antiviral effects were confirmed in SARS-CoV-2 infection systems. These compounds were equally effective against recently emerged variants, including the delta variant. Cross-linking experiments and structural modeling suggest that the compounds bind to a hydrophobic pocket near the fusion peptide of S protein, consistent with their potential mechanism of action as fusion inhibitors. In summary, these fusion inhibitors have broad-spectrum antiviral activities and may be promising leads for treatment of SARS-CoV-2, its variants, and other pathogenic CoVs. IMPORTANCE SARS-CoV-2 is an enveloped virus that requires membrane fusion for entry into host cells. Since the fusion process is relatively conserved among enveloped viruses, we tested our HCV fusion inhibitors, dichlorcyclizine and fluoxazolevir, against SARS-CoV-2. We performed in vitro assays and demonstrated their effective antiviral activity against SARS-CoV-2 and its variants. Cross-linking experiments and structural modeling suggest that the compounds bind to a hydrophobic pocket in spike protein to exert their inhibitory effect on the fusion step. These data suggest that both dichlorcyclizine and fluoxazolevir are promising candidates for further development as treatment for SARS-CoV-2.

Discovery of Small Molecule Entry Inhibitors Targeting the Fusion Peptide of SARS-CoV-2 Spike Protein.

SARS-CoV-2 entry into host cells relies on the spike (S) protein binding to the human ACE2 receptor. In this study, we investigated the structural dynamics of the viral S protein at the fusion peptide (FP) domain and small molecule binding for therapeutics development. Following comparative modeling analysis and docking studies of our previously identified fusion inhibitor chlorcyclizine, we performed a pharmacophore-based virtual screen and identified two novel chemotypes of entry inhibitors targeting the FP. The compounds were evaluated in the pseudoparticle viral entry assay and SARS-CoV-2 cytopathic effect assay and showed single-digital micromole inhibition against SARS-CoV-2 as well as SARS-CoV-1 and MERS. The characterization of the FP binding site of SARS-CoV-2 S protein provides a promising target for the structure-based development of small molecule entry inhibitors as drug candidates for the treatment of COVID-19.

Modeling PNPLA3-Associated NAFLD Using Human-Induced Pluripotent Stem Cells.

BACKGROUND AND AIMS: NAFLD is a growing public health burden. However, the pathogenesis of NAFLD has not yet been fully elucidated, and the importance of genetic factors has only recently been appreciated. Genomic studies have revealed a strong association between NAFLD progression and the I148M variant in patatin-like phospholipase domain-containing protein 3 (PNPLA3). Nonetheless, very little is known about the mechanisms by which this gene and its variants can influence disease development. To investigate these mechanisms, we have developed an in vitro model that takes advantage of the unique properties of human-induced pluripotent stem cells (hiPSCs) and the CRISPR/CAS9 gene editing technology. APPROACH AND RESULTS: We used isogenic hiPSC lines with either a knockout (PNPLA3KO ) of the PNPLA3 gene or with the I148M variant (PNPLA3I148M ) to model PNPLA3-associated NAFLD. The resulting hiPSCs were differentiated into hepatocytes, treated with either unsaturated or saturated free fatty acids to induce NAFLD-like phenotypes, and characterized by various functional, transcriptomic, and lipidomic assays. PNPLA3KO hepatocytes showed higher lipid accumulation as well as an altered pattern of response to lipid-induced stress. Interestingly, loss of PNPLA3 also caused a reduction in xenobiotic metabolism and predisposed PNPLA3KO cells to be more susceptible to ethanol-induced and methotrexate-induced toxicity. The PNPLA3I148M cells exhibited an intermediate phenotype between the wild-type and PNPLA3KO cells. CONCLUSIONS: Together, these results indicate that the I148M variant induces a loss of function predisposing to steatosis and increased susceptibility to hepatotoxins.

Discovery and Optimization of a 4-Aminopiperidine Scaffold for Inhibition of Hepatitis C Virus Assembly.

The majority of FDA-approved HCV therapeutics target the viral replicative machinery. An automated high-throughput phenotypic screen identified several small molecules as potent inhibitors of hepatitis C virus replication. Here, we disclose the discovery and optimization of a 4-aminopiperidine (4AP) scaffold targeting the assembly stages of the HCV life cycle. The original screening hit (1) demonstrates efficacy in the HCVcc assay but does not show potency prior to or during viral replication. Colocalization and infectivity studies indicate that the 4AP chemotype inhibits the assembly and release of infectious HCV. Compound 1 acts synergistically with FDA-approved direct-acting antiviral compounds Telaprevir and Daclatasvir, as well as broad spectrum antivirals Ribavirin and cyclosporin A. Following an SAR campaign, several derivatives of the 4AP series have been identified with increased potency against HCV, reduced in vitro toxicity, as well as improved in vitro and in vivo ADME properties.

Fluoxazolevir inhibits hepatitis C virus infection in humanized chimeric mice by blocking viral membrane fusion.

Fluoxazolevir is an aryloxazole-based entry inhibitor of hepatitis C virus (HCV). We show that fluoxazolevir inhibits fusion of HCV with hepatic cells by binding HCV envelope protein 1 to prevent fusion. Nine of ten fluoxazolevir resistance-associated substitutions are in envelope protein 1, and four are in a putative fusion peptide. Pharmacokinetic studies in mice, rats and dogs revealed that fluoxazolevir localizes to the liver. A 4-week intraperitoneal regimen of fluoxazolevir in humanized chimeric mice infected with HCV genotypes 1b, 2a or 3 resulted in a 2-log reduction in viraemia, without evidence of drug resistance. In comparison, daclatasvir, an approved HCV drug, suppressed more than 3 log of viraemia but is associated with the emergence of resistance-associated substitutions in mice. Combination therapy using fluoxazolevir and daclatasvir cleared HCV genotypes 1b and 3 in mice. Fluoxazolevir combined with glecaprevir and pibrentasvir was also effective in clearing multidrug-resistant HCV replication in mice. Fluoxazolevir may be promising as the next generation of combination drug cocktails for HCV treatment.

Chlorcyclizine Inhibits Viral Fusion of Hepatitis C Virus Entry by Directly Targeting HCV Envelope Glycoprotein 1.

Chlorcyclizine (CCZ) is a potent hepatitis C virus (HCV) entry inhibitor, but its molecular mechanism is unknown. Here, we show that CCZ directly targets the fusion peptide of HCV E1 and interferes with the fusion process. Generation of CCZ resistance-associated substitutions of HCV in vitro revealed six missense mutations in the HCV E1 protein, five being in the putative fusion peptide. A viral fusion assay demonstrated that CCZ blocked HCV entry at the membrane fusion step and that the mutant viruses acquired resistance to CCZ's action in blocking membrane fusion. UV cross-linking of photoactivatable CCZ-diazirine-biotin in both HCV-infected cells and recombinant HCV E1/E2 protein demonstrated direct binding to HCV E1 glycoprotein. Mass spectrometry analysis revealed that CCZ cross-linked to an E1 sequence adjacent to the putative fusion peptide. Docking simulations demonstrate a putative binding model, wherein CCZ binds to a hydrophobic pocket of HCV E1 and forms extensive interactions with the fusion peptide.

Changes in Allergenicity of Ovalbumin in Vitro and in Vivo on Conjugation with Quercetin.

A previous study demonstrated decreased allergenicity in vitro of some food allergens after conjugation with polyphenols. However, little is known about how polyphenol conjugation with food allergens affects in vivo allergenicity. We conjugated a well-known food allergen, ovalbumin (OVA), with quercetin (QUE) to assess the potential allergenicity of OVA in vitro and in vivo in a BALB/c mouse model. QUE could covalently conjugate with OVA and changed the protein structure, which might destroy and/or mask OVA epitopes. Conjugation with QUE decreased IgE binding properties and the release capacity of the conjugated OVA. In vivo, as compared with native protein, conjugation with QUE decreased the levels of IgE, IgG1, IgG, plasma histamine, and mast cell protease-1 (mMCP-1) on the surface of sensitized mast cells, along with decreased FcεRI+ and c-kit+ expression. The levels of Th2-related cytokines (IL-4, IL-5, IL-13) decreased and that of a Th1-related cytokine (IFN-γ) increased slightly, which suggests that conjugation with QUE modulated the imbalance of the Th1/Th2 immune response. Conjugation of OVA with QUE could reduce OVA allergenicity in vitro and in vivo, which could provide information for reducing food allergenicity by conjugation with polyphenols.

Discovery and characterization of a novel HCV inhibitor targeting the late stage of HCV life cycle.

BACKGROUND: Currently approved anti-HCV drugs, the direct-acting antivirals (DAAs), are highly effective and target the viral RNA replication stage of the HCV life cycle. Due to high mutation rate of HCV, drug resistant variants can arise during DAA monotherapy. Thus, a combination of DAAs is necessary to achieve a high response rate. Novel HCV inhibitors targeting the HCV late stage such as assembly and release may further improve combination therapy with the DAAs. Here we characterize one late stage-targeting candidate compound, 6-(4-chloro-3-methylphenoxy)-pyridin-3-amine (MLS000833705). METHODS: We treated HCV-infected cells with MLS000833705 and other HCV inhibitors and examined HCV RNA and infectious titres. We evaluated the colocalization of HCV core and lipid droplets by confocal microscopy. We performed HCV core-proteinase K digestion assay and several lipid assays to study the mechanism of MLS000833705. RESULTS: We showed that MLS000833705 decreased extracellular HCV RNA levels more than intracellular HCV RNA levels in HCV infectious cell culture. Similarly, MLS000833705 reduced infectious HCV titres substantially more in the culture supernatant than intracellularly. Confocal microscopy showed that MLS000833705 did not affect the colocalization of HCV core protein with cellular lipid droplets where HCV assembles. HCV core-proteinase K digestion assay showed that MLS000833705 inhibited the envelopment of HCV capsid. CONCLUSIONS: Our study demonstrates that MLS000833705 is a late-stage HCV inhibitor targeting HCV morphogenesis and maturation. Therefore, MLS000833705 can be used as a molecular probe to study HCV maturation and secretion and possibly guide development of a new class of HCV antivirals.

Preclinical Pharmacological Development of Chlorcyclizine Derivatives for the Treatment of Hepatitis C Virus Infection.

Hepatitis C virus (HCV) is a small, single-stranded, positive-sense RNA virus that infects more than an estimated 70 million people worldwide. Untreated, persistent HCV infection often results in chronic hepatitis, cirrhosis, or liver failure, with progression to hepatocellular carcinoma. Current anti-HCV regimens comprising direct acting antivirals (DAAs) can provide curative treatment; however, due to high costs there remains a need for effective, shorter-duration, and affordable treatments. Recently, we disclosed anti-HCV activity of the cheap antihistamine chlorcyclizine, targeting viral entry. Following our hit-to-lead optimization campaign, we report evaluation of preclinical in vitro absorption, distribution, metabolism, and excretion properties, and in vivo pharmacokinetic profiles of lead compounds. This led to selection of a new lead compound and evaluation of efficacy in chimeric mice engrafted with primary human hepatocytes infected with HCV. Further development and incorporation of this compound into DAA regimens has the potential to improve treatment efficacy, affordability, and accessibility.

Development of an Aryloxazole Class of Hepatitis C Virus Inhibitors Targeting the Entry Stage of the Viral Replication Cycle.

Reliance on hepatitis C virus (HCV) replicon systems and protein-based screening assays has led to treatments that target HCV viral replication proteins. The model does not encompass other viral replication cycle steps such as entry, processing, assembly and secretion, or viral host factors. We previously applied a phenotypic high-throughput screening platform based on an infectious HCV system and discovered an aryloxazole-based anti-HCV hit. Structure-activity relationship studies revealed several compounds exhibiting EC50 values below 100 nM. Lead compounds showed inhibition of the HCV pseudoparticle entry, suggesting a different mode of action from existing HCV drugs. Hit 7a and lead 7ii both showed synergistic effects in combination with existing HCV drugs. In vivo pharmacokinetics studies of 7ii showed high liver distribution and long half-life without obvious hepatotoxicity. The lead compounds are promising as preclinical candidates for the treatment of HCV infection and as molecular probes to study HCV pathogenesis.

High expression of prostate tumor overexpressed 1 (PTOV1) is a potential prognostic biomarker for cervical cancer.

BACKGROUND: To investigate the role of prostate tumor overexpressed 1 (PTOV1) in the development and progression of human cervical cancer. METHODS: Real-time quantitative PCR, Western blot, and immunohistochemistry were used to explore PTOV1 expression in cervical cancer tissues and cell lines. Cell proliferation capability was examined by MTT assay. Statistical analyzes were applied to evaluate the correlation of PTOV1 expression with clinical parameters and prognosis. RESULTS: The expression level of PTOV1 was markedly higher in cervical cancer tissues and cell lines than that in adjacent noncancerous tissues and the normal cervical epithelial cells. PTOV1 overexpression was correlated with higher tumor stage (P = 0.001), larger tumor size (P = 0.004), and lymph node involvement (P = 0.036). Moreover, patients with high PTOV1 expression showed shorter overall and recurrence-free survival time (P = 0.013 and P = 0.010, respectively). PTOV1 knockdown by short hairpin RNAi inhibited cancer cell growth in vitro. CONCLUSION: PTOV1 may be an important factor associated with proliferation of cervical cancer.

Hepatic differentiation of human pluripotent stem cells in miniaturized format suitable for high-throughput screen.

The establishment of protocols to differentiate human pluripotent stem cells (hPSCs) including embryonic (ESC) and induced pluripotent (iPSC) stem cells into functional hepatocyte-like cells (HLCs) creates new opportunities to study liver metabolism, genetic diseases and infection of hepatotropic viruses (hepatitis B and C viruses) in the context of specific genetic background. While supporting efficient differentiation to HLCs, the published protocols are limited in terms of differentiation into fully mature hepatocytes and in a smaller-well format. This limitation handicaps the application of these cells to high-throughput assays. Here we describe a protocol allowing efficient and consistent hepatic differentiation of hPSCs in 384-well plates into functional hepatocyte-like cells, which remain differentiated for more than 3weeks. This protocol affords the unique opportunity to miniaturize the hPSC-based differentiation technology and facilitates screening for molecules in modulating liver differentiation, metabolism, genetic network, and response to infection or other external stimuli.

Evaluation of antiviral drug synergy in an infectious HCV system.

BACKGROUND: Direct-acting antivirals (DAAs) have greatly improved the treatment of HCV infection. To improve response and prevent resistance, combination regimens have been the focus of clinical development. Regimens are often first assessed in vitro, with most combination studies to date using subgenomic replicon systems, which do not replicate the complete HCV life cycle and preclude study of entry and assembly inhibitors. Infectious full-length HCV systems have been developed and are being used to test drug efficacy. METHODS: Using cell-based HCV Con1b replicon and an infectious full-length HCV (HCVcc-Luc) infection system, we systematically tested the synergy, additivity or antagonism of combinations of protease, NS5A and nucleotide NS5B inhibitor classes as well as the combination of these DAAs with host-targeting agent cyclosporin A or non-antibody entry inhibitor (S)-chlorcyclizine. Two computational software packages, MacSynergyII and CalcuSyn, were used for data analysis. RESULTS: Combinations between different classes showed good consistency across the two viral assay systems and two software platforms. Combinations between NS5A and nucleotide NS5B inhibitors were synergistic, while combinations of protease inhibitors with the other two classes were additive to slightly antagonistic. As expected, combinations of antivirals of the same class were additive. Combination studies between these DAA classes and cyclosporin A or (S)-chlorcyclizine demonstrated additive to synergistic effects and highly synergistic effects, respectively. Combinations of these drugs did not show any added or unexpected cytotoxicity. CONCLUSIONS: Our results show that in vitro combination studies of anti-HCV DAAs in the HCVcc system may provide useful guidance for drug combination designs in clinical studies. We also demonstrate that these DAAs in combination with host-targeting agents or entry inhibitors may improve HCV treatment response.

Discovery, Optimization, and Characterization of Novel Chlorcyclizine Derivatives for the Treatment of Hepatitis C Virus Infection.

Recently, we reported that chlorcyclizine (CCZ, Rac-2), an over-the-counter antihistamine piperazine drug, possesses in vitro and in vivo activity against hepatitis C virus. Here, we describe structure-activity relationship (SAR) efforts that resulted in the optimization of novel chlorcyclizine derivatives as anti-HCV agents. Several compounds exhibited EC50 values below 10 nM against HCV infection, cytotoxicity selectivity indices above 2000, and showed improved in vivo pharmacokinetic properties. The optimized molecules can serve as lead preclinical candidates for the treatment of hepatitis C virus infection and as probes to study hepatitis C virus pathogenesis and host-virus interaction.

Identification of novel anti-hepatitis C virus agents by a quantitative high throughput screen in a cell-based infection assay.

Hepatitis C virus (HCV) poses a major health threat to the world. The recent development of direct-acting antivirals (DAAs) against HCV has markedly improved the response rate of HCV and reduced the side effects in comparison to the interferon-based therapy. Despite this therapeutic advance, there is still a need to develop new inhibitors that target different stages of the HCV life cycle because of various limitations of the current regimens. In this study, we performed a quantitative high throughput screening of the Molecular Libraries Small Molecule Repository (MLSMR) of ∼350,000 chemicals for novel HCV inhibitors using our previously developed cell-based HCV infection assay. Following confirmation and structural clustering analysis, we narrowed down to 158 compounds from the initial ∼3000 molecules that showed inhibitory activity for further structural and functional analyses. We were able to assign the majority of these compounds to specific stage(s) in the HCV life cycle. Three of them are direct inhibitors of NS3/4A protease. Most of the compounds appear to act on novel targets in HCV life cycle. Four compounds with novel structure and excellent drug-like properties, three targeting HCV entry and one targeting HCV assembly/secretion, were advanced for further development as lead hits. These compounds represent diverse chemotypes that are potential lead compounds for further optimization and may offer promising candidates for the development of novel therapeutics against HCV infection. In addition, they represent novel molecular probes to explore the complex interactions between HCV and the cells.

High-Throughput Screening, Discovery, and Optimization To Develop a Benzofuran Class of Hepatitis C Virus Inhibitors.

Using a high-throughput, cell-based HCV luciferase reporter assay to screen a diverse small-molecule compound collection (∼ 300,000 compounds), we identified a benzofuran compound class of HCV inhibitors. The optimization of the benzofuran scaffold led to the identification of several exemplars with potent inhibition (EC50 < 100 nM) of HCV, low cytotoxicity (CC50 > 25 µM), and excellent selectivity (selective index = CC50/EC50, > 371-fold). The structure-activity studies culminated in the design and synthesis of a 45-compound library to comprehensively explore the anti-HCV activity. The identification, design, synthesis, and biological characterization for this benzofuran series is discussed.