Viral Entry Inhibitors Research Articles

Novel Antivirals in Clinical Development for Chronic Hepatitis B Infection.

Globally, chronic hepatitis B (CHB) infection is one of the leading causes of liver failure, decompensated cirrhosis, and hepatocellular carcinoma. Existing antiviral therapy can suppress viral replication but not fully eradicate the virus nor the risk of liver-related complications. Novel treatments targeting alternative steps of the viral cycle or to intensify/restore the host’s immunity are being developed. We discuss novel drugs that have already entered clinical phases of development. Agents that interfere with specific steps of HBV replication include RNA interference, core protein allosteric modulation, and inhibition of viral entry or viral protein excretion (NAPs and STOPS). Agents that target the host’s immunity include toll-like receptor agonists, therapeutic vaccines, immune checkpoint modulators, soluble T-cell receptors, and monoclonal antibodies. Most have demonstrated favorable results in suppression of viral proteins and genomic materials (i.e., HBV DNA and/or pre-genomic RNA), and/or evidence on host-immunity restoration including cytokine responses and T-cell activation. Given the abundant clinical experience and real-world safety data with the currently existing therapy, any novel agent for CHB should be accompanied by convincing safety data. Combination therapy of nucleos(t)ide analogue, a novel virus-directing agent, and/or an immunomodulatory agent will be the likely approach to optimize the chance of a functional cure in CHB.

Toward a new era of hepatitis B virus therapeutics: The pursuit of a functional cure.

Hepatitis B virus (HBV) infection, although preventable by vaccination, remains a global health problem and a major cause of chronic liver disease. Although current treatment strategies suppress viral replication very efficiently, the optimal endpoint of hepatitis B surface antigen (HBsAg) clearance is rarely achieved. Moreover, the thorny problems of persistent chromatin-like covalently closed circular DNA and the presence of integrated HBV DNA in the host genome are ignored. Therefore, the scientific community has focused on developing innovative therapeutic approaches to achieve a functional cure of HBV, defined as undetectable HBV DNA and HBsAg loss over a limited treatment period. A deeper understanding of the HBV life cycle has led to the introduction of novel direct-acting antivirals that exert their function through multiple mechanisms, including inhibition of viral entry, transcriptional silencing, epigenetic manipulation, interference with capsid assembly, and disruption of HBsAg release. In parallel, another category of new drugs aims to restore dysregulated immune function in chronic hepatitis B accompanied by lethargic cellular and humoral responses. Stimulation of innate immunity by pattern-recognition receptor agonists leads to upregulation of antiviral cytokine expression and appears to contribute to HBV containment. Immune checkpoint inhibitors and adoptive transfer of genetically engineered T cells are breakthrough technologies currently being explored that may elicit potent HBV-specific T-cell responses. In addition, several clinical trials are attempting to clarify the role of therapeutic vaccination in this setting. Ultimately, it is increasingly recognized that elimination of HBV requires a treatment regimen based on a combination of multiple drugs. This review describes the rationale for progressive therapeutic interventions and discusses the latest findings in the field of HBV therapeutics.

An extract of Stephania hernandifolia, an ethnomedicinal plant, inhibits herpes simplex virus 1 entry

Stephania hernandifolia (Nimukho), an ethnomedicinal herb from rural Bengal, has been used traditionally for the management of nerve, skin, urinary, and digestive ailments. Here, we attempted to confirm the antiviral potential of aqueous, methanol, and chloroform extracts of S. hernandifolia against herpes simplex virus type 1 (HSV-1), the causative agent of orolabial herpes in humans, and decipher its underlying mechanism of action. The bioactive extract was standardized and characterized by gas chromatography-mass spectroscopy, while cytotoxicity and antiviral activity were evaluated by MTT and plaque reduction assay, respectively. Two HSV strains, HSV-1F and the clinical isolate VU-09, were inhibited by the chloroform extract (CE) with a median effective concentration (EC50) of 4.32 and 4.50 µg/ml respectively, with a selectivity index (SI) of 11. Time-of-addition assays showed that pre-treatment of virus-infected cells with the CE and its removal before infection reduced the number of plaques without lasting toxicity to the cell, indicating that the CE affected the early stage in the viral life cycle. The number of plaques was also reduced by direct inactivation of virions and by the addition of CE for a short time following attachment of virions. These results together suggest that modification of either the virion surface or the cell surface by the CE inhibits virus entry into the host cell.

Disruption of disulfides within RBD of SARS-CoV-2 spike protein prevents fusion and represents a target for viral entry inhibition by registered drugs.

The SARS‐CoV‐2 pandemic imposed a large burden on health and society. Therapeutics targeting different components and processes of the viral infection replication cycle are being investigated, particularly to repurpose already approved drugs. Spike protein is an important target for both vaccines and therapeutics. Insights into the mechanisms of spike‐ACE2 binding and cell fusion could support the identification of compounds with inhibitory effects. Here, we demonstrate that the integrity of disulfide bonds within the receptor‐binding domain (RBD) plays an important role in the membrane fusion process although their disruption does not prevent binding of spike protein to ACE2. Several reducing agents and thiol‐reactive compounds are able to inhibit viral entry. N‐acetyl cysteine amide, L‐ascorbic acid, JTT‐705, and auranofin prevented syncytia formation, viral entry into cells, and infection in a mouse model, supporting disulfides of the RBD as a therapeutically relevant target.

Differential Inhibition of HIV Replication by the 12 Interferon Alpha Subtypes.

Type I interferons (IFNs) are a family of cytokines that represent a first line of defense against virus infections. The 12 different IFN-α subtypes share a receptor on target cells and trigger similar signaling cascades. Several studies have collectively shown that this apparent redundancy conceals qualitatively different responses induced by individual subtypes, which display different efficacies of inhibition of HIV replication. Some studies, however, provided evidence that the disparities are quantitative rather than qualitative. Since RNA expression analyses show a large but incomplete overlap of the genes induced, they may support both models. To explore if the IFN-α subtypes induce functionally relevant different anti-HIV activities, we have compared the efficacies of inhibition of all 12 subtypes on HIV spread and on specific steps of the viral replication cycle, including viral entry, reverse transcription, protein synthesis, and virus release. Finding different hierarchies of inhibition would validate the induction of qualitatively different responses. We found that while most subtypes similarly inhibit virus entry, they display distinctive potencies on other early steps of HIV replication. In addition, only some subtypes were able to target effectively the late steps. The extent of induction of known anti-HIV factors helps to explain some, but not all differences observed, confirming the participation of additional IFN-induced anti-HIV effectors. Our findings support the notion that different IFN-α subtypes can induce the expression of qualitatively different antiviral activities. IMPORTANCE The initial response against viruses relies in large part on type I interferons, which include 12 subtypes of IFN-α. These cytokines bind to a common receptor on the cell surface and trigger the expression of incompletely overlapping sets of genes. Whether the anti-HIV responses induced by IFN-α subtypes differ in the extent of expression or in the nature of the genes involved remains debated. Also, RNA expression profiles led to opposite conclusions, depending on the importance attributed to the induction of common or distinctive genes. To explore if relevant anti-HIV activities can be differently induced by the IFN-α subtypes, we compared their relative efficacies on specific steps of the replication cycle. We show that the hierarchy of IFN potencies depends on the step analyzed, supporting qualitatively different responses. This work will also prompt the search for novel IFN-induced anti-HIV factors acting on specific steps of the replication cycle.

Viral Entry Inhibitors Targeting Six-Helical Bundle Core against Highly Pathogenic Enveloped Viruses with Class I Fusion Proteins.

Type Ⅰ enveloped viruses bind to cell receptors through surface glycoproteins to initiate infection or undergo receptor-mediated endocytosis and initiate membrane fusion in the acidic environment of endocytic compartments, releasing genetic material into the cell. In the process of membrane fusion, envelope protein exposes fusion peptide, followed by an insertion into the cell membrane or endosomal membrane. Further conformational changes ensue in which the type 1 envelope protein forms a typical six-helix bundle structure, shortening the distance between viral and cell membranes so that fusion can occur. Entry inhibitors targeting viral envelope proteins, or host factors, are effective antiviral agents and have been widely studied. Some have been used clinically, such as T20 and Maraviroc for human immunodeficiency virus 1 (HIV-1) or Myrcludex B for hepatitis D virus (HDV). This review focuses on entry inhibitors that target the six-helical bundle core against highly pathogenic enveloped viruses with class I fusion proteins, including retroviruses, coronaviruses, influenza A viruses, paramyxoviruses, and filoviruses.

SARS‐CoV‐2 Viral Budding and Entry can be Modeled Using BSL‐2 Level Virus‐Like Particles

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) was first discovered in December 2019 in Wuhan, China and expeditiously spread across the globe causing a global pandemic. Research on SARS‐CoV‐2, as well as the closely related SARS‐CoV‐1 and MERS coronaviruses is restricted to BSL‐3 facilities. Such BSL‐3 classification makes SARS‐CoV‐2 research inaccessible to the majority of functioning research laboratories in the US; this becomes problematic when the collective scientific effort needs to be focused on such in the face of a pandemic. However, a minimal system capable of recapitulating different steps of the viral life cycle without using the virus' genetic material could increase accessibility. In this work, we assessed the four structural proteins from SARS‐CoV‐2 for their ability to form virus‐like particles (VLPs) from human cells to form a competent system for BSL‐2 studies of SARS‐CoV‐2. After establishing the minimal system requirements for VLP production, we examined their morphological relevance with transmission electron microscopy (TEM). VLPs produced with all four viral structural proteins were approximately 100 nm in diameter and bared the characteristic coronavirus crown or ‘corona’. We next sought to evaluate the entry competency of our VLPs. GFP‐tagged VLPs were generated by fluorescently tagging one of the four structural proteins used to produce VLPs. Once incorporation of the GFP‐tagged protein into VLPs was confirmed, we used these GFP‐VLPs to infect HEK293 cells. GFP‐VLPs indeed did enter HEK293 cells and properly colocalized with endocytic markers Rab5 and LAMP1 in accordance with live virus data. To further evaluate viral entry, we made the VLP entry assay accessible to TEM analysis by replacing the GFP tag with an ascorbate peroxidase (APEX2) tag, which when oxidized produces a dark brown precipitate visible on micrographs. APEX2‐VLPs were found to be entry‐competent as well. In addition to entry, APEX2‐VLPs yield the ability to visualize VLP assembly at the ER‐Golgi intermediary complex (ERGIC) and for the first time we show localization of the structural proteins during SARS‐CoV‐2 VLP assembly, budding, and egress. In total, this research provides ample resources for other BSL‐2 laboratories interested in joining the growing field to try and understand SARS‐CoV‐2 assembly, budding, and entry dynamics, biochemical and biophysical questions on the four structural proteins, and drug screening of viral assembly, budding, and/or entry inhibitors.

731 FAILURE TO DEVELOP NEUTRALIZING ANTIBODIES FOLLOWING SARS-COV-2 INFECTION IN YOUNG PATIENTS WITH INFLAMMATORY BOWEL DISEASE RECEIVING BIOLOGICS

Objectives and Study: SARS-CoV-2, a novel coronavirus causing the pandemic clinical disease COVID-19, is generally associated with mild disease in most young patients with inflammatory bowel disease (IBD) despite their receiving immunosuppressive therapy. The aim of this study was to longitudinally evaluate characteristics of clinical and serologic response to SARS-CoV-2 infection in a cohort of young patients with IBD receiving biologics. Methods: Starting May 2020 we obtained serum on patients with IBD at the time of infusion with either infliximab or vedolizumab, along with baseline demographic and clinical data, as well possible SARSCoV2 exposure history (patient, family member). To measure antibodies to SARS-CoV-2, we used a fluorescent bead-based immunoassay that takes advantage of the high dynamic range of fluorescent molecules using flow cytometry. We immobilized biotinylated SARS-CoV-2 Spike protein receptor binding domain (S-RBD) or Nucleocapsid protein of SARS-CoV-2 (N) to detect specific IgG antibodies to the virus in patient serum. Spike protein-RBD-specific antibodies were detectable in serial dilutions up to 100,000-fold of serum samples. Titration curves from COVID-19 convalescent and healthy controls were used to normalize the area under the curve (AUC) values to quantitate the antibody levels. Antibody isotypes were measured using anti-Ig (IgG, IgA, IgM, IgG1-4) specific secondary antibodies conjugated to a fluorescent tag. We used a sensitive and high throughput SARS-CoV-2 neutralization assay using a lentivirus that expresses Spike protein to assess specific inhibition of viral entry. The results shown reflect the first serum sample obtained from unique patients. Results: 410 subjects were studied (mean age 17 years, 59% male, 305 (74%) Crohn's disease, 105 (25%) ulcerative colitis/IBD-U, 341 (83%) on infliximab, 69 (17%) vedolizumab, 13% concomitant methotrexate. 27/410 (6.6%) were positive for S-RBD and Nucleocapsid specific IgG. AUC values varied from 3150 to 285724 (Fig 1). S-RBD specific IgA+ 4/27 (15%) and IgG1+ 20/27 (74%) were found. Other isotypes undetectable. Patients' serum efficiently neutralized the virus at up to 10,000-fold serial dilution in only 10/27 (37%). (Fig 2). No differences in age, gender, diagnosis, or specific therapies were noted for (+) vs (-) anti-SARS-CoV-2 antibody status. 13/27 (48%) patients were asymptomatic. Non-exclusive symptoms were rhinorrhea 9 (33%), headache 8 (30%), sore throat 4 (15%). cough 4 (15%), diarrhea 4 (15%), chills 3 (11%), loss of smell/taste 2 (7%), fever 1 (4%). No patient was hospitalized. 4 (15%) had a family member with PCR+ COVID-19. Conclusions: We found a significant prevalence of anti SARS-CoV-2 antibody in our IBD population with the majority of + patients having non-neutralizing antibody. The role of biologics in mitigating clinical and serologic response to SARS-CoV-2 requires exploration.

Recent Advances in Hepatitis B Treatment.

Hepatitis B virus infection affects over 250 million chronic carriers, causing more than 800,000 deaths annually, although a safe and effective vaccine is available. Currently used antiviral agents, pegylated interferon and nucleos(t)ide analogues, have major drawbacks and fail to completely eradicate the virus from infected cells. Thus, achieving a “functional cure” of the infection remains a real challenge. Recent findings concerning the viral replication cycle have led to development of novel therapeutic approaches including viral entry inhibitors, epigenetic control of cccDNA, immune modulators, RNA interference techniques, ribonuclease H inhibitors, and capsid assembly modulators. Promising preclinical results have been obtained, and the leading molecules under development have entered clinical evaluation. This review summarizes the key steps of the HBV life cycle, examines the currently approved anti-HBV drugs, and analyzes novel HBV treatment regimens.

Computational techniques in designing a series of 1,3,4-trisubstituted pyrazoles as unique hepatitis C virus entry inhibitors

In this study, we developed a QSAR model for studying the antiviral activity of 1,3,4-trisubstituted pyrazoles derivatives on hepatitis C virus infected in human HuH-7 cell lines. We employed random analysis to split the data sets. Statistically robust model was generated with R2, Q2 and R2pred values of 0.777, 0.731 and 0.774 respectively. The reliability of this model was confirmed by acceptable validation parameters, and this model also fulfilled the Golbraikh and Tropsha standard model conditions. Through the evaluation of selected molecular descriptors we observed that, topological charge index of order 4 (GGI4), mean topological charge index of order 1 (JGI1), octanol water partition coefficient (XlogP), 3D topological distance based autocorrelation lag5/weighted by polarizabilities (TDB5p) and total molecular surface area (FPSA-2) are the molecular properties determining biological activities of the study compounds, which shed light on the vital features that aid in the design of unique potent hepatitis C virus entry inhibitors using computer-aided drug design tools.

Computational drug repurposing study elucidating simultaneous inhibition of entry and replication of novel corona virus by Grazoprevir

Outcomes of various clinical studies for the coronavirus disease 2019 (COVID-19) treatment indicated that the drug acts via inhibition of multiple pathways (targets) is likely to be more successful and promising. Keeping this hypothesis intact, the present study describes for the first-time, Grazoprevir, an FDA approved anti-viral drug primarily approved for Hepatitis C Virus (HCV), mediated multiple pathway control via synergistic inhibition of viral entry targeting host cell Angiotensin-Converting Enzyme 2 (ACE-2)/transmembrane serine protease 2 (TMPRSS2) and viral replication targeting RNA-dependent RNA polymerase (RdRP). Molecular modeling followed by in-depth structural analysis clearly demonstrated that Grazoprevir interacts with the key residues of these targets. Futher, Molecular Dynamics (MD) simulations showed stability and burial of key residues after the complex formation. Finally, Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) analysis identified the governing force of drug-receptor interactions and stability. Thus, we believe that Grazoprevir could be an effective therapeutics for the treatment of the COVID-19 pandemic with a promise of unlikely drug resistance owing to multiple inhibitions of eukaryotic and viral proteins, thus warrants further clinical studies.

Identification of SARS-CoV-2 viral entry inhibitors using machine learning and cell-based pseudotyped particle assay

In response to the pandemic caused by SARS-CoV-2, we constructed a hybrid support vector machine (SVM) classification model using a set of publicly posted SARS-CoV-2 pseudotyped particle (PP) entry assay repurposing screen data to identify novel potent compounds as a starting point for drug development to treat COVID-19 patients. Two different molecular descriptor systems, atom typing descriptors and 3D fingerprints (FPs), were employed to construct the SVM classification models. Both models achieved reasonable performance, with the area under the curve of receiver operating characteristic (AUC-ROC) of 0.84 and 0.82, respectively. The consensus prediction outperformed the two individual models with significantly improved AUC-ROC of 0.91, where the compounds with inconsistent classifications were excluded. The consensus model was then used to screen the 173,898 compounds in the NCATS annotated and diverse chemical libraries. Of the 255 compounds selected for experimental confirmation, 116 compounds exhibited inhibitory activities in the SARS-CoV-2 PP entry assay with IC50 values ranged between 0.17 µM and 62.2 µM, representing an enrichment factor of 3.2. These 116 active compounds with diverse and novel structures could potentially serve as starting points for chemistry optimization for COVID-19 drug discovery.

Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein Based Novel Epitopes Induce Potent Immune Responses in vivo and Inhibit Viral Replication in vitro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) initiates infection by attachment of the surface-exposed spike glycoprotein to the host cell receptors. The spike glycoprotein (S) is a promising target for inducing immune responses and providing protection; thus the ongoing efforts for the SARS-CoV-2 vaccine and therapeutic developments are mostly spiraling around S glycoprotein. The matured functional spike glycoprotein is presented on the virion surface as trimers, which contain two subunits, such as S1 (virus attachment) and S2 (virus fusion). The S1 subunit harbors the N-terminal domain (NTD) and the receptor-binding domain (RBD). The RBD is responsible for binding to host-cellular receptor angiotensin-converting enzyme 2 (ACE2). The NTD and RBD of S1, and the S2 of S glycoprotein are the major structural moieties to design and develop spike-based vaccine candidates and therapeutics. Here, we have identified three novel epitopes (20-amino acid peptides) in the regions NTD, RBD, and S2 domains, respectively, by structural and immunoinformatic analysis. We have shown as a proof of principle in the murine model, the potential role of these novel epitopes in-inducing humoral and cellular immune responses. Further analysis has shown that RBD and S2 directed epitopes were able to efficiently inhibit the replication of SARS-CoV-2 wild-type virus in vitro suggesting their role as virus entry inhibitors. Structural analysis revealed that S2-epitope is a part of the heptad repeat 2 (HR2) domain which might have plausible inhibitory effects on virus fusion. Taken together, this study discovered novel epitopes that might have important implications in the development of potential SARS-CoV-2 spike-based vaccine and therapeutics.

Comparison of the immunologic response to herpes simplex virus type 1 entry glycoproteins induced in humans after primary infection of oral or genital sites

<h3>Objectives</h3> We sought to determine potential differences in the humoral immune response to herpes simplex virus type 1 (HSV-1) in patients after primary infection in either oral or genital sites. <h3>Methods</h3> Serum samples from 20 patients with primary HSV-1 infection (10 oral, 10 genital) were compared. A neutralization assay categorized samples by their ability to inhibit virus entry into cells. An enzyme-linked immunosorbent assay determined the quantity of antibodies against each of the major HSV-1 glycoproteins (gD, gB, gC, gH/gL). Surface plasmon resonance imaging (SPRi) was used to estimate the quality of the response by determining the epitope specificity of antibodies against the receptor-binding glycoprotein gD. Information from these assays was combined to develop a comprehensive profile of immunologic response to HSV-1 infection. <h3>Results</h3> All 10 oral site serum samples contained significant levels of virus-specific antibodies to block virus entry and demonstrated excellent immune response to the major virion glycoproteins. In contrast, only 4 of the 10 genital site serum samples were able to block virus entry. The 6 genital site sera that failed to block entry had markedly reduced antiglycoprotein antibody levels, indicating a poor overall HSV-1 immunologic response. The quantity of antiglycoprotein antibodies in the 4 genital (protective) sera mirrored that induced by oral infection. Subsequently, SPRi was used to determine whether the qualitative response was equivalent between the oral and genital site sera. All 10 oral site sera targeted gD epitopes at levels significantly higher and more varied than those generated for the 4 protective genital site samples. Interestingly, the gD epitope profiles of the 6 nonprotective genital site samples were directed at a single non-neutralizing antigenic site on gD that is involved in cell-to-cell spread. <h3>Conclusions</h3> Based on a comprehensive profile of immunologic response to HSV-1 infection for individuals infected at either oral or genital sites, results of this study suggest that the site of primary infection is important in driving the overall humoral immune-protective response. Findings of this study will have direct implications for the future development of a glycoprotein-epitope–based HSV-1 vaccine.

Potential Antiviral Target for SARS-CoV-2: A Key Early Responsive Kinase during Viral Entry

Currently, there is no effective antiviral medication for coronavirus disease 2019 (COVID-19) and the knowledge on the potential therapeutic target is in great need. Guided by a time-course transmi...

Withanone from Withania somnifera Attenuates SARS-CoV-2 RBD and Host ACE2 Interactions to Rescue Spike Protein Induced Pathologies in Humanized Zebrafish Model.

PurposeSARS-CoV-2 engages human ACE2 through its spike (S) protein receptor binding domain (RBD) to enter the host cell. Recent computational studies have reported that withanone and withaferin A, phytochemicals found in Withania somnifera, target viral main protease (MPro) and host transmembrane TMPRSS2, and glucose related protein 78 (GRP78), respectively, implicating their potential as viral entry inhibitors. Absence of specific treatment against SARS-CoV-2 infection has encouraged exploration of phytochemicals as potential antivirals.AimThis study aimed at in silico exploration, along with in vitro and in vivo validation of antiviral efficacy of the phytochemical withanone.MethodsThrough molecular docking, molecular dynamic (MD) simulation and electrostatic energy calculation the plausible biochemical interactions between withanone and the ACE2-RBD complex were investigated. These in silico observations were biochemically validated by ELISA-based assays. Withanone-enriched extract from W. somnifera was tested for its ability to ameliorate clinically relevant pathological features, modelled in humanized zebrafish through SARS-CoV-2 recombinant spike (S) protein induction.ResultsWithanone bound efficiently at the interacting interface of the ACE2-RBD complex and destabilized it energetically. The electrostatic component of binding free energies of the complex was significantly decreased. The two intrachain salt bridge interactions (K31-E35) and the interchain long-range ion-pair (K31-E484), at the ACE2-RBD interface were completely abolished by withanone, in the 50 ns simulation. In vitro binding assay experimentally validated that withanone efficiently inhibited (IC50=0.33 ng/mL) the interaction between ACE2 and RBD, in a dose-dependent manner. A withanone-enriched extract, without any co-extracted withaferin A, was prepared from W. somnifera leaves. This enriched extract was found to be efficient in ameliorating human-like pathological responses induced in humanized zebrafish by SARS-CoV-2 recombinant spike (S) protein.ConclusionIn conclusion, this study provided experimental validation for computational insight into the potential of withanone as a potent inhibitor of SARS-CoV-2 coronavirus entry into the host cells.

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity.

BackgroundAntivirals are needed to combat the COVID-19 pandemic, which is caused by SARS-CoV-2. The clinically-proven protease inhibitor Camostat mesylate inhibits SARS-CoV-2 infection by blocking the virus-activating host cell protease TMPRSS2. However, antiviral activity of Camostat mesylate metabolites and potential viral resistance have not been analyzed. Moreover, antiviral activity of Camostat mesylate in human lung tissue remains to be demonstrated.MethodsWe used recombinant TMPRSS2, reporter particles bearing the spike protein of SARS-CoV-2 or authentic SARS-CoV-2 to assess inhibition of TMPRSS2 and viral entry, respectively, by Camostat mesylate and its metabolite GBPA.FindingsWe show that several TMPRSS2-related proteases activate SARS-CoV-2 and that two, TMPRSS11D and TMPRSS13, are robustly expressed in the upper respiratory tract. However, entry mediated by these proteases was blocked by Camostat mesylate. The Camostat metabolite GBPA inhibited recombinant TMPRSS2 with reduced efficiency as compared to Camostat mesylate. In contrast, both inhibitors exhibited similar antiviral activity and this correlated with the rapid conversion of Camostat mesylate into GBPA in the presence of serum. Finally, Camostat mesylate and GBPA blocked SARS-CoV-2 spread in human lung tissue ex vivo and the related protease inhibitor Nafamostat mesylate exerted augmented antiviral activity.InterpretationOur results suggest that SARS-CoV-2 can use TMPRSS2 and closely related proteases for spread in the upper respiratory tract and that spread in the human lung can be blocked by Camostat mesylate and its metabolite GBPA.FundingNIH, Damon Runyon Foundation, ACS, NYCT, DFG, EU, Berlin Mathematics center MATH+, BMBF, Lower Saxony, Lundbeck Foundation, Novo Nordisk Foundation.

Evaluation of flavonoids as 2019-nCoV cell entry inhibitor through molecular docking and pharmacological analysis

The outbreak of Coronavirus Disease 2019 (COVID-19) has been declared as a Public Health Emergency of International Concern (PHEIC) by the World Health Organization (WHO), which is being rapidly spread by the extremely spreadable and pathogenic 2019 novel coronavirus (2019-nCoV), also known as SARS-CoV-2. Pandemic incidence of COVID-19 has created a severe threat to global public health, necessitating the development of effective drugs or inhibitors or therapeutics agents against SARS-CoV-2. Spike protein (S) of the SARS-CoV-2 plays a crucial role in entering viruses into the host cell by binding to angiotensin-converting enzyme 2 (ACE-2), and this specific interaction represents a promising drug target for the identification of potential drugs. This study aimed at the receptor-binding domain of S protein (RBD of nCoV-SP) and the ACE-2 receptor as a promising target for developing drugs against SARS-CoV-2. Over 100 different flavonoids with antioxidant, anti-inflammatory, and antiviral properties from different literatures were taken as a ligand or inhibitor for molecular docking against target protein RBD of nCoV-SP and ACE-2 using PyRX and iGEMDOCK. Top flavonoids based on docking scores were selected for the pharmacokinetic study. Selected flavonoids (hesperidin, naringin, ECGC, and quercetin) showed excellent pharmacokinetics with proper absorption, solubility, permeability, distribution, metabolism, minimal toxicity, and excellent bioavailability. Molecular dynamics simulation studies up to 100 ns exhibited strong binding affinity of selected flavonoids to RBD of nCoV-SP and ACE-2, and the protein-ligand complexes were structurally stable. These identified lead flavonoids may act as potential compounds for developing effective drugs against SARS-CoV-2 by potentially inhibiting virus entry into the host cell.

Biological activity-based modeling identifies antiviral leads against SARS-CoV-2.

Computational approaches for drug discovery, such as quantitative structure-activity relationship, rely on structural similarities of small molecules to infer biological activity but are often limited to identifying new drug candidates in the chemical spaces close to known ligands. Here we report a biological activity-based modeling (BABM) approach, in which compound activity profiles established across multiple assays are used as signatures to predict compound activity in other assays or against a new target. This approach was validated by identifying candidate antivirals for Zika and Ebola viruses based on high-throughput screening data. BABM models were then applied to predict 311 compounds with potential activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of the predicted compounds, 32% had antiviral activity in a cell culture live virus assay, the most potent compounds showing a half-maximal inhibitory concentration in the nanomolar range. Most of the confirmed anti-SARS-CoV-2 compounds were found to be viral entry inhibitors and/or autophagy modulators. The confirmed compounds have the potential to be further developed into anti-SARS-CoV-2 therapies.

Mechanisms of COVID-19 Entry into the Cell: Potential Therapeutic Approaches Based on Virus Entry Inhibition in COVID-19 Patients with Underlying Diseases.

The Coronavirus disease 2019 (COVID-19) virus spread from Wuhan, China, in 2019 and is spreading rapidly around the world. COVID-19 victims are almost associated with cardiovascular disease, high blood pressure, diabetes, and other underlying diseases. Concerning the high prevalence of these disorders, widespread mortality threatens global society, and its fatality rate may increase with increasing COVID-19 prevalence in countries with older populations. Therefore, evaluating patients' clinical status with severe COVID-19 infection and their medical history can help manage treatment. Currently, one of the considered treatments is angiotensin-converting enzyme 2 (ACE2) inhibition. This study investigated virus entry mechanisms through membrane receptors, their role in the pathogenesis of COVID-19 and underlying diseases, and treatment methods based on the viral entrance inhibition. According to existing studies, inhibition of ACE2 can increase oxidative stress, inflammation, fibrosis and ultimately exacerbate underlying diseases such as cardiovascular disease, kidney disease, diabetes, and hypertension in individuals with COVID-19. The ACE2 inhibition is not suitable for patients with COVID-19 with underlying diseases, but it seems that the recombinant ACE2 solution is more appropriate for inhibiting the virus in these patients if hypotension would be monitored.