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Lassa virus (LASV) is the causative agent of human Lassa fever which in severe cases manifests as hemorrhagic fever leading to thousands of deaths annually. However, no approved vaccines or antiviral drugs are currently available. Recently, we screened approximately 2,500 compounds using a recombinant vesicular stomatitis virus (VSV) expressing LASV glycoprotein GP (VSV-LASVGP) and identified a P-glycoprotein inhibitor as a potential LASV entry inhibitor. Here, we show that another identified candidate, hexestrol (HES), an estrogen receptor agonist, is also a LASV entry inhibitor. HES inhibited VSV-LASVGP replication with a 50% inhibitory concentration (IC50) of 0.63 µM. Importantly, HES also inhibited authentic LASV replication with IC50 values of 0.31 µM–0.61 µM. Time-of-addition and cell-based membrane fusion assays suggested that HES inhibits the membrane fusion step during virus entry. Alternative estrogen receptor agonists did not inhibit VSV-LASVGP replication, suggesting that the estrogen receptor itself is unlikely to be involved in the antiviral activity of HES. Generation of a HES-resistant mutant revealed that the phenylalanine at amino acid position 446 (F446) of LASVGP, which is located in the transmembrane region, conferred resistance to HES. Although mutation of F446 enhanced the membrane fusion activity of LASVGP, it exhibited reduced VSV-LASVGP replication, most likely due to the instability of the pre-fusion state of LASVGP. Collectively, our results demonstrated that HES is a promising anti-LASV drug that acts by inhibiting the membrane fusion step of LASV entry. This study also highlights the importance of the LASVGP transmembrane region as a target for anti-LASV drugs. IMPORTANCE Lassa virus (LASV), the causative agent of Lassa fever, is the most devastating mammarenavirus with respect to its impact on public health in West Africa. However, no approved antiviral drugs or vaccines are currently available. Here, we identified hexestrol (HES), an estrogen receptor agonist, as the potential antiviral candidate drug. We showed that the estrogen receptor itself is not involved in the antiviral activity. HES directly bound to LASVGP and blocked membrane fusion, thereby inhibiting LASV infection. Through the generation of a HES-resistant virus, we found that phenylalanine at position 446 (F446) within the LASVGP transmembrane region plays a crucial role in the antiviral activity of HES. The mutation at F446 caused reduced virus replication, likely due to the instability of the pre-fusion state of LASVGP. These findings highlight the potential of HES as a promising candidate for the development of antiviral compounds targeting LASV. [ABSTRACT FROM AUTHOR]

Prenatal administration of monoclonal antibodies (mAbs) is a strategy that could be exploited to prevent viral infections during pregnancy and early life. To reach protective levels in fetuses, mAbs must be transported across the placenta, a selective barrier that actively and specifically promotes the transfer of antibodies (Abs) into the fetus through the neonatal Fc receptor (FcRn). Because FcRn also regulates Ab half-life, Fc mutations like the M428L/N434S, commonly known as LS mutations, and others have been developed to enhance binding affinity to FcRn and improve drug pharmacokinetics. We hypothesized that these FcRn-enhancing mutations could similarly affect the delivery of therapeutic Abs to the fetus. To test this hypothesis, we measured the transplacental transfer of leronlimab, an anti-CCR5 mAb, in clinical development for preventing HIV infections, using pregnant rhesus macaques to model in utero mAb transfer. We also generated a stabilized and FcRn-enhanced form of leronlimab, termed leronlimab-PLS. Leronlimab-PLS maintained higher levels within the maternal compartment while also reaching higher mAb levels in the fetus and newborn circulation. Further, a single dose of leronlimab-PLS led to complete CCR5 receptor occupancy in mothers and newborns for almost a month after birth. These findings support the optimization of FcRn interactions in mAb therapies designed for administration during pregnancy.

Since the beginning of the COVID-19 pandemic, extensive drug repurposing efforts have sought to identify small-molecule antivirals with various mechanisms of action. Here, we aim to review research progress on small-molecule viral entry and fusion inhibitors that directly bind to the SARS-CoV-2 Spike protein. Early in the pandemic, numerous small molecules were identified in drug repurposing screens and reported to be effective in in vitro SARS-CoV-2 viral entry or fusion inhibitors. However, given minimal experimental information regarding the exact location of small-molecule binding sites on Spike, it was unclear what the specific mechanism of action was or where the exact binding sites were on Spike for some inhibitor candidates. The work of countless researchers has yielded great progress, with the identification of many viral entry inhibitors that target elements on the S1 receptor-binding domain (RBD) or N-terminal domain (NTD) and disrupt the S1 receptor-binding function. In this review, we will also focus on highlighting fusion inhibitors that target inhibition of the S2 fusion function, either by disrupting the formation of the postfusion S2 conformation or alternatively by stabilizing structural elements of the prefusion S2 conformation to prevent conformational changes associated with S2 function. We highlight experimentally validated binding sites on the S1/S2 interface and on the S2 subunit. While most substitutions to the Spike protein to date in variants of concern (VOCs) have been localized to the S1 subunit, the S2 subunit sequence is more conserved, with only a few observed substitutions in proximity to S2 binding sites. Several recent small molecules targeting S2 have been shown to have robust activity over recent VOC mutant strains and/or greater broad-spectrum antiviral activity for other more distantly related coronaviruses. [ABSTRACT FROM AUTHOR]

The complement system is an innate immune mechanism against microbial infections. It involves a cascade of effector molecules that is activated via classical, lectin and alternative pathways. Consequently, many pathogens bind to or incorporate in their structures host negative regulators of the complement pathways as an evasion mechanism. Factor H (FH) is a negative regulator of the complement alternative pathway that protects "self" cells of the host from nonspecific complement attack. FH has been shown to bind viruses including human influenza A viruses (IAVs). In addition to its involvement in the regulation of complement activation, FH has also been shown to perform a range of functions on its own including its direct interaction with pathogens. Here, we show that human FH can bind directly to IAVs of both human and avian origin, and the interaction is mediated via the IAV surface glycoprotein haemagglutinin (HA). HA bound to common pathogen binding footprints on the FH structure, complement control protein modules, CCP 5-7 and CCP 15-20. The FH binding to H1 and H3 showed that the interaction overlapped with the receptor binding site of both HAs, but the footprint was more extensive for the H3 HA than the H1 HA. The HA - FH interaction impeded the initial entry of H1N1 and H3N2 IAV strains but its impact on viral multicycle replication in human lung cells was strain-specific. The H3N2 virus binding to cells was significantly inhibited by preincubation with FH, whereas there was no alteration in replicative rate and progeny virus release for human H1N1, or avian H9N2 and H5N3 IAV strains. We have mapped the interaction between FH and IAV, the in vivo significance of which for the virus or host is yet to be elucidated. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has had a significant and lasting impact on the world. Four years on, despite the existence of effective vaccines, the continuous emergence of new SARS-CoV-2 variants remains a challenge for long-term immunity. Additionally, there remain few purpose-built antivirals to protect individuals at risk of severe disease in the event of future coronavirus outbreaks. A promising mechanism of action for novel coronavirus antivirals is the inhibition of viral entry. To facilitate entry, the coronavirus spike glycoprotein interacts with angiotensin converting enzyme 2 (ACE2) on respiratory epithelial cells. Blocking this interaction and consequently viral replication may be an effective strategy for treating infection, however further research is needed to better characterize candidate molecules with antiviral activity before progressing to animal studies and clinical trials. In general, antiviral drugs are developed from purely synthetic compounds or synthetic derivatives of natural products such as plant secondary metabolites. While the former is often favored due to the higher specificity afforded by rational drug design, natural products offer several unique advantages that make them worthy of further study including diverse bioactivity and the ability to work synergistically with other drugs. Accordingly, there has recently been a renewed interest in natural product-derived antivirals in the wake of the COVID-19 pandemic. This review provides a summary of recent research into coronavirus entry inhibitors, with a focus on natural compounds derived from plants, honey, and marine sponges. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has had a significant and lasting impact on the world. Four years on, despite the existence of effective vaccines, the continuous emergence of new SARS-CoV-2 variants remains a challenge for long-term immunity. Additionally, there remain few purpose-built antivirals to protect individuals at risk of severe disease in the event of future coronavirus outbreaks. A promising mechanism of action for novel coronavirus antivirals is the inhibition of viral entry. To facilitate entry, the coronavirus spike glycoprotein interacts with angiotensin converting enzyme 2 (ACE2) on respiratory epithelial cells. Blocking this interaction and consequently viral replication may be an effective strategy for treating infection, however further research is needed to better characterize candidate molecules with antiviral activity before progressing to animal studies and clinical trials. In general, antiviral drugs are developed from purely synthetic compounds or synthetic derivatives of natural products such as plant secondary metabolites. While the former is often favored due to the higher specificity afforded by rational drug design, natural products offer several unique advantages that make them worthy of further study including diverse bioactivity and the ability to work synergistically with other drugs. Accordingly, there has recently been a renewed interest in natural product-derived antivirals in the wake of the COVID-19 pandemic. This review provides a summary of recent research into coronavirus entry inhibitors, with a focus on natural compounds derived from plants, honey, and marine sponges.

CD4-mimetic compounds (CD4mcs) bind the human immunodeficiency virus (HIV-1) gp120 exterior envelope glycoprotein (Env) and compete for binding to CD4, the host receptor. CD4mcs prematurely trigger conformational changes in Env similar to those induced by CD4, leading to transient activation of infectivity followed by irreversible virus inactivation. Natural HIV-1 variants exhibit a wide range of susceptibilities to CD4mc inhibition, only a small fraction of which can be explained by variation in the gp120 Phe-43 cavity/vestibule where CD4mcs bind. Here, we study Envs from the resistant HIV-1BG505 and the more sensitive HIV-1191955_A4 clade A strains. The major determinant of the relative sensitivity of the HIV-1191955_A4 Env to CD4mcs mapped to a single residue change (F317Y) in the tip of the gp120 V3 variable loop. In the Envs of several HIV-1 strains, replacement of the more prevalent Phe 317 with a tyrosine residue increased virus sensitivity to multiple CD4mcs. Tryptophan substitutions at residues 317 and 316 resulted in increases and decreases, respectively, in sensitivity to CD4mcs. Some of the gp120 V3 changes increased virus sensitivity to inactivation by both CD4mc and cold exposure, phenotypes indicative of increased Env triggerability. Infection of CD4-negative cells expressing the CCR5 coreceptor by these Env variants was triggered more efficiently by CD4mcs. For the panel of studied HIV-1 Envs, resistance to the CD4mcs was associated with decreased ability to support virus entry. These studies illustrate how variation in gp120 outside the CD4mc binding site can influence the sensitivity of natural HIV-1 strains to inhibition by these compounds. [ABSTRACT FROM AUTHOR]

The utility of human neuroblastoma cell lines as in vitro model to study neuro-invasiveness and neuro-virulence of SARS-CoV-2 has been demonstrated by our laboratory and others. The aim of this report is to further characterize the associated cellular responses caused by a pre-alpha SARS-CoV-2 strain on differentiated SH-SY5Y and to prevent its cytopathic effect by using a set of entry inhibitors. The susceptibility of SH-SY5Y to SARS-CoV-2 was confirmed at high multiplicity-of-infection, without viral replication or release. Infection caused a reduction in the length of neuritic processes, occurrence of plasma membrane blebs, cell clustering, and changes in lipid droplets electron density. No changes in the expression of cytoskeletal proteins, such as tubulins or tau, could explain neurite shortening. To counteract the toxic effect on neurites, entry inhibitors targeting TMPRSS2, ACE2, NRP1 receptors, and Spike RBD were co-incubated with the viral inoculum. The neurite shortening could be prevented by the highest concentration of camostat mesylate, anti-RBD antibody, and NRP1 inhibitor, but not by soluble ACE2. According to the degree of entry inhibition, the average amount of intracellular viral RNA was negatively correlated to neurite length. This study demonstrated that targeting specific SARS-CoV-2 host receptors could reverse its neurocytopathic effect on SH-SY5Y. [ABSTRACT FROM AUTHOR]

Since the beginning of the COVID-19 pandemic, extensive drug repurposing efforts have sought to identify small-molecule antivirals with various mechanisms of action. Here, we aim to review research progress on small-molecule viral entry and fusion inhibitors that directly bind to the SARS-CoV-2 Spike protein. Early in the pandemic, numerous small molecules were identified in drug repurposing screens and reported to be effective in in vitro SARS-CoV-2 viral entry or fusion inhibitors. However, given minimal experimental information regarding the exact location of small-molecule binding sites on Spike, it was unclear what the specific mechanism of action was or where the exact binding sites were on Spike for some inhibitor candidates. The work of countless researchers has yielded great progress, with the identification of many viral entry inhibitors that target elements on the S1 receptor-binding domain (RBD) or N-terminal domain (NTD) and disrupt the S1 receptor-binding function. In this review, we will also focus on highlighting fusion inhibitors that target inhibition of the S2 fusion function, either by disrupting the formation of the postfusion S2 conformation or alternatively by stabilizing structural elements of the prefusion S2 conformation to prevent conformational changes associated with S2 function. We highlight experimentally validated binding sites on the S1/S2 interface and on the S2 subunit. While most substitutions to the Spike protein to date in variants of concern (VOCs) have been localized to the S1 subunit, the S2 subunit sequence is more conserved, with only a few observed substitutions in proximity to S2 binding sites. Several recent small molecules targeting S2 have been shown to have robust activity over recent VOC mutant strains and/or greater broad-spectrum antiviral activity for other more distantly related coronaviruses.

The threat to global health caused by three highly pathogenic human coronaviruses (HCoV), SARS-CoV-2, MERS-CoV and SARS-CoV, calls for the development of pan-HCoV therapeutics and vaccines. This study reports the design and engineering of a recombinant protein designated HR1LS. It contains three linked molecules, each consisting of three structural domains, including a heptad repeat 1 (HR1), a central helix (CH), and a stem helix (SH) region, in the S2 subunit of SARS-CoV-2 spike (S) protein. It was found that HR1LS protein automatically formed a trimer able to bind with heptad repeat 2 (HR2) region in the SARS-CoV-2 S2 subunit, thus potently inhibiting HCoV fusion and entry into host cells. Furthermore, immunization of mice with HR1LS, when combined with CF501 adjuvant, resulted in the production of neutralizing antibodies against infection of SARS-CoV-2 and its variants, as well as SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63 and MjHKU4r-CoV-1. These results suggest that HR1LS is a promising candidate for further development as a novel HR1-trimer-based pan-HCoV entry inhibitor or vaccine for the treatment and prevention of infection by SARS-CoV-2 and its variants, but also other HCoVs with the potential to cause future emerging and re-emerging infectious coronavirus diseases.

The COVID-19 pandemic evolves constantly, requiring adaptable solutions to combat emerging SARS-CoV-2 variants. To address this, we created a pentameric scaffold based on a mammalian protein, which can be customized with up to 10 protein binding modules. This molecular scaffold spans roughly 20 nm and can simultaneously neutralize SARS-CoV-2 Spike proteins from one or multiple viral particles. Using only two different modules targeting the Spike's RBD domain, this construct outcompetes human antibodies from vaccinated individuals' serum and blocks in vitro cell attachment and pseudotyped virus entry. Additionally, the multibodies inhibit viral replication at low picomolar concentrations, regardless of the variant. This customizable multibody can be easily produced in procaryote systems, providing a new avenue for therapeutic development and detection devices, and contributing to preparedness against rapidly evolving pathogens. [ABSTRACT FROM AUTHOR]

Chronic hepatitis delta (CHD) affects approximately 10–20 million people worldwide and represents the most severe form of chronic viral hepatitis, as it is characterized by high rates of progression to cirrhosis and its complications (end‐stage liver disease, hepatocellular carcinoma). In the last 30 years, the only treatment option for CHD has been represented by the off‐label administration of Interferon (or Pegylated Interferon)‐alpha: antiviral treatment, however, resulted in suboptimal (20–30%) virological response and was burdened by several side effects, de facto contraindicating Interferon (IFN) administration in patients with more advanced liver disease. Recently, Bulevirtide (BLV), a first‐in‐class HBV‐HDV entry inhibitor blocking Na+‐taurocholate co‐transporting polypeptide (NTCP), has provided very promising efficacy data in Phase II and Phase III (interim analysis) trials as well as in preliminary real‐life reports. In July 2020, BLV has granted conditional approval by EMA for treatment of compensated CHD, at the dose of 2 mg/day by self‐administered subcutaneous injections. In Phase II and Phase III trials, BLV was evaluated at different doses (2 vs. 10 mg/day) for 24 or 48 weeks, either in monotherapy or in combination with PegIFN. Administration of BLV monotherapy for 24 or 48 weeks resulted in 50%–83% virological response (HDV RNA ≥ 2 Log decline) rates and 45%–78% ALT normalization. Combination therapy with PegIFN provided synergistic effects. These results were replicated in real‐life studies and confirmed also in patients with advanced cirrhosis and clinically significant portal hypertension. BLV treatment was optimally tolerated, resulting only in an asymptomatic increase of bile acids. [ABSTRACT FROM AUTHOR]

Novel coronavirus disease 2019 (COVID-19), a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has brought an unprecedented public health crisis and continues to threaten humanity due to the persistent emergence of new variants. Therefore, developing more effective and broad-spectrum therapeutic and prophylactic drugs against infection by SARS-CoV-2 and its variants, as well as future emerging CoVs, is urgently needed. In this study, we screened several US FDA-approved drugs and identified phenothiazine derivatives with the ability to potently inhibit the infection of pseudotyped SARS-CoV-2 and distinct variants of concern (VOCs), including B.1.617.2 (Delta) and currently circulating Omicron sublineages XBB and BQ.1.1, as well as pseudotyped SARS-CoV and MERS-CoV. Mechanistic studies suggested that phenothiazines predominantly inhibited SARS-CoV-2 pseudovirus (PsV) infection at the early stage and potentially bound to the spike (S) protein of SARS-CoV-2, which may prevent the proteolytic cleavage of the S protein, thereby exhibiting inhibitory activity against SARS-CoV-2 infection. In summary, our findings suggest that phenothiazines can serve as a potential broad-spectrum therapeutic drug for the treatment of SARS-CoV-2 infection as well as the infection of future emerging human coronaviruses (HCoVs). [ABSTRACT FROM AUTHOR]

With increasing resistance of SARS-CoV-2 variants to antibodies, there is interest in developing entry inhibitors that target essential receptor-binding regions of the viral Spike protein and thereby present a high bar for viral resistance. Such inhibitors could be derivatives of the viral receptor, ACE2, or peptides engineered to interact specifically with the Spike receptor-binding pocket. We compared the efficacy of a series of both types of entry inhibitors, constructed as fusions to an antibody Fc domain. Such a design can increase protein stability and act to both neutralize free virus and recruit effector functions to clear infected cells. We tested the reagents against prototype variants of SARS-CoV-2, using both Spike pseudotyped vesicular stomatitis virus vectors and replication-competent viruses. These analyses revealed that an optimized ACE2 derivative could neutralize all variants we tested with high efficacy. In contrast, the Spike-binding peptides had varying activities against different variants, with resistance observed in the Spike proteins from Beta, Gamma, and Omicron (BA.1 and BA.5). The resistance mapped to mutations at Spike residues K417 and N501 and could be overcome for one of the peptides by linking two copies in tandem, effectively creating a tetrameric reagent in the Fc fusion. Finally, both the optimized ACE2 and tetrameric peptide inhibitors provided some protection to human ACE2 transgenic mice challenged with the SARS-CoV-2 Delta variant, which typically causes death in this model within 7-9 days. [ABSTRACT FROM AUTHOR]

Ebola virus (EBOV) infection leads to staggeringly high mortality rate. Effective and low-cost treatments are urgently needed to control frequent EBOV outbreaks in Africa. In this study, we report that a natural compound called berbamine hydrochloride strongly inhibits EBOV replication in vitro and in vivo. Our work further showed that berbamine hydrochloride acts by directly binding to the cleaved EBOV glycoprotein (GPcl), disrupting GPcl interaction with viral receptor Niemann-Pick C1, thus blocking the fusion of viral and cellular membranes. Our data support the probability of developing anti-EBOV small molecule drugs by targeting viral GPcl. More importantly, since berbamine hydrochloride has been used in clinic to treat leukopenia, it holds great promise of being quickly repurposed as an anti-EBOV drug.

Na+/taurocholate cotransporting polypeptide (NTCP) is a member of the solute carrier (SLC) family 10 transporters (gene symbol SLC10A1) and is responsible for the sodium-dependent uptake of bile salts across the basolateral membrane of hepatocytes. In addition to its primary transporter function, NTCP is the high-affinity hepatic receptor for hepatitis B (HBV) and hepatitis D (HDV) viruses and, therefore, is a prerequisite for HBV/HDV virus entry into hepatocytes. The inhibition of HBV/HDV binding to NTCP and internalization of the virus/NTCP receptor complex has become a major concept in the development of new antiviral drugs called HBV/HDV entry inhibitors. Hence, NTCP has emerged as a promising target for therapeutic interventions against HBV/HDV infections in the last decade. In this review, recent findings on protein–protein interactions (PPIs) between NTCP and cofactors relevant for entry of the virus/NTCP receptor complex are summarized. In addition, strategies aiming to block PPIs with NTCP to dampen virus tropism and HBV/HDV infection rates are discussed. Finally, this article suggests novel directions for future investigations evaluating the functional contribution of NTCP-mediated PPIs in the development and progression of HBV/HDV infection and subsequent chronic liver disorders. [ABSTRACT FROM AUTHOR]

Respiratory viruses including the respiratory syncytial virus (RSV) aggravate the global burden of virus-inflicted morbidity and mortality. Entry inhibitors are a promising class of antiviral drugs for combating these viruses, as they can prevent infection at the site of viral entry, i.e., the respiratory tract. Here we used a broad-spectrum entry inhibitor, composed of a β-cyclodextrin backbone, functionalized with 11-mercapto-1-undecanesulfonate (CD-MUS) that is capable of neutralizing a variety of viruses that employ heparan sulfate proteoglycans (HSPG) to infect host cells. CD-MUS inactivates viral particles irreversibly by binding to viral attachment proteins through a multivalent binding mechanism. In the present study, we show that CD-MUS is well tolerated when administered to the respiratory tract of mice. Based on this, we developed an inhalable spray-dried powder formulation that fits the size requirements for lung deposition and disperses well upon use with the Cyclops dry powder inhaler (DPI). Using an in vitro dose-response assay, we show that the compound retained its activity against RSV after the spray drying process. Our study sets the stage for further in vivo studies, exploring the efficacy of pulmonary administered CD-MUS in animal models of RSV infection. [Display omitted] • The novel antiviral entry inhibitor CD-MUS is well-tolerated in vivo. • CD-MUS shows suitable pharmacokinetic behavior after pulmonary administration. • CD-MUS can successfully be formulated into an inhalable dry powder formulation. • CD-MUS retains its activity against respiratory syncytial virus after spray drying. • CD-MUS is well dispersed from the Cyclops dry powder inhaler. [ABSTRACT FROM AUTHOR]

Chronic hepatitis Delta (CHD) is a rare and severe form of chronic viral hepatitis. Until recently, the only therapeutic approach has been the off‐label use of a 48 weeks course of PegInterferon alpha (PegIFNα), that was characterized by suboptimal efficacy and burdened by significant side effects that limited treatment applicability in patients with advanced liver disease. In July 2020, European Medicines Agency (EMA) conditionally approved the entry inhibitor Bulevirtde (BLV) at the dose of 2 mg/day for the treatment of adult patients with compensated CHD. Efficacy and safety of BLV in CHD have been evaluated in clinical trials either as monotherapy or in combination with PegIFNα. These results were confirmed by real‐life studies, which also evaluated long‐term BLV monotherapy in patients with advanced compensated cirrhosis. Notwithstanding these promising results there are still several issues to be addressed, such as the optimal duration of the treatment, the rates of off‐therapy responses, as well as the long‐term clinical benefits. This review summarizes updated and current literature data about clinical trials and real‐life studies with BLV monotherapy and/or in combination with PegIFNα. [ABSTRACT FROM AUTHOR]

Summary: Chronic infection with hepatitis delta virus (HDV) affects between 12-20 million people worldwide and represents the most severe form of viral hepatitis, leading to accelerated liver disease progression, cirrhosis and its complications, such as end-stage-liver disease and hepatocellular carcinoma. From the discovery of HDV in 1977 by Prof. Mario Rizzetto, knowledge on the HDV life cycle and mechanisms of viral spread has expanded. However, little is still known about the natural history of the disease, host-viral interactions, and the role of the immune system in HDV persistence. Diagnosis of HDV is still challenging due to a lack of standardised assays, while accurate viral load quantification is needed to assess response and endpoints of antiviral treatment. Until recently, interferon has represented the only treatment option in patients with chronic hepatitis delta; however, it is associated with low efficacy and a high burden of side effects. The discovery of the entry inhibitor bulevirtide has represented a breakthrough in HDV treatment, by demonstrating high rates of viral suppression in phase II and III trials, results which have been confirmed in real-world settings and in patients with compensated advanced liver disease. In the meantime, other compounds (i.e. lonafarnib, new anti-hepatitis B virus drugs) are under development to provide alternative or combined strategies for HDV cure. The first international Delta Cure meeting was organised in Milan in October 2022 with the aim of sharing and disseminating the latest data; this review summarises key takeaway messages from state-of-the-art lectures and research data on HDV.

The COVID-19 pandemic evolves constantly, requiring adaptable solutions to combat emerging SARS-CoV-2 variants. To address this, we created a pentameric scaffold based on a mammalian protein, which can be customized with up to 10 protein binding modules. This molecular scaffold spans roughly 20 nm and can simultaneously neutralize SARS-CoV-2 Spike proteins from one or multiple viral particles. Using only two different modules targeting the Spike’s RBD domain, this construct outcompetes human antibodies from vaccinated individuals’ serum and blocks in vitro cell attachment and pseudotyped virus entry. Additionally, the multibodies inhibit viral replication at low picomolar concentrations, regardless of the variant. This customizable multibody can be easily produced in procaryote systems, providing a new avenue for therapeutic development and detection devices, and contributing to preparedness against rapidly evolving pathogens.

Influenza A virus (IAV) leads to significant morbidity and mortality due to the seasonal epidemics and spread. We have demonstrated that oleanolic acid (OA) C28 glucose conjugates and OA trimers are capable of effectively blocking the recognition and interaction between the influenza virus and host cells. In this study, a series of OA-glucose trimers were designed and synthesized through the CuAAC reaction. All trimers underwent screening for anti-IAV activities in vitro. Among these, compounds 13a and 13b showed inhibitory activity against the influenza virus, with IC 50 values of 0.68 μM and 0.47 μM, respectively, demonstrating greater potency than oseltamivir (IC 50 = 1.36 μM). Results from the time-of-addition experiment and hemagglutination inhibition assay suggest that these OA-glucose trimers may disrupt the recognition between the HA protein of IAV and sialic acid receptors on host cells, thus blocking viral entry. Furthermore, it was found that compound 13b effectively inhibits IAV infection in BALB/c mice. This study has elucidated the structure-activity relationships of OA trimers against the influenza virus and highlighted the utility of multivalent OA conjugates for enhancing ligand-target interactions in anti-influenza virus drug design, laying a groundwork for future research into the antiviral applications of these natural products. [Display omitted] • A series of OA-glucose trimers were designed and synthesized via CuAAC reaction. • Compounds 13a and 13b exhibited superior inhibitory activity against the influenza virus compared to oseltamivir in vitro. • The mechanism of action was identified, confirming that these compounds target the viral HA protein. • In vivo studies showed that Compound 13b effectively inhibits influenza A virus (IAV) infection in BALB/c mice. [ABSTRACT FROM AUTHOR]

[Display omitted] Increasing resistance to antiviral drugs approved for the treatment of influenza urges the development of novel compounds. Ideally, this should be complemented by a careful consideration of the administration route. 6′siallyllactosamine-functionalized β-cyclodextrin (CD-6′SLN) is a novel entry inhibitor that acts as a mimic of the primary attachment receptor of influenza, sialic acid. In this study, we aimed to develop a dry powder formulation of CD-6′SLN to assess its in vivo antiviral activity after administration via the pulmonary route. By means of spray drying the compound together with trileucine, a dispersion enhancer, we created a powder that retained the antiviral effect of the drug, remained stable under elevated temperature conditions and performed well in a dry powder inhaler. To test the efficacy of the dry powder drug against influenza infection in vivo, infected mice were treated with CD-6′SLN using an aerosol generator that allowed for the controlled administration of powder formulations to the lungs of mice. CD-6′SLN was effective in mitigating the course of the disease compared to the control groups, reflected by lower disease activity scores and by the prevention of virus-induced IL-6 production. Our data show that CD-6′SLN can be formulated as a stable dry powder that is suitable for use in a dry powder inhaler and is effective when administered via the pulmonary route to influenza-infected mice. [ABSTRACT FROM AUTHOR]

The global spread of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the continuously emerging new variants underscore an urgent need for effective therapeutics for the treatment of coronavirus disease 2019 (COVID-19). Here, we screened several FDA-approved amphiphilic drugs and determined that sertraline (SRT) exhibits potent antiviral activity against infection of SARS-CoV-2 pseudovirus (PsV) and authentic virus in vitro. It effectively inhibits SARS-CoV-2 spike (S)-mediated cell-cell fusion. SRT targets the early stage of viral entry. It can bind to the S1 subunit of the S protein, especially the receptor binding domain (RBD), thus blocking S-hACE2 interaction and interfering with the proteolysis process of S protein. SRT is also effective against infection with SARS-CoV-2 PsV variants, including the newly emerging Omicron. The combination of SRT and other antivirals exhibits a strong synergistic effect against infection of SARS-CoV-2 PsV. The antiviral activity of SRT is independent of serotonin transporter expression. Moreover, SRT effectively inhibits infection of SARS-CoV-2 PsV and alleviates the inflammation process and lung pathological alterations in transduced mice in vivo. Therefore, SRT shows promise as a treatment option for COVID-19. [ABSTRACT FROM AUTHOR]

Bulevirtide (BLV) has recently been conditionally approved for the treatment of chronic hepatitis delta (CHD) in Europe, but its effectiveness and safety in patients with compensated cirrhosis and clinically significant portal hypertension (CSPH) are unknown. Consecutive patients with HDV-related compensated cirrhosis and CSPH who started BLV 2 mg/day were enrolled in this single-center study. Clinical/virological characteristics were collected at baseline, weeks 4, 8 and every 8 weeks thereafter. HDV RNA was quantified by Robogene 2.0 (lower limit of detection 6 IU/ml). Eighteen Caucasian patients with compensated cirrhosis and CSPH under nucleos(t)ide analogue treatment were enrolled: median (IQR) age was 48 (29-77) years, and 67% were male. Median (IQR) platelet count was 70 (37-227) x103/μl, liver stiffness measurement (LSM) 16.4 (7.8-57.8) kPa, alanine aminotransferase (ALT) 106 (32-222) U/L, HBsAg 3.7 (2.5-4.3) log IU/ml, HDV RNA 4.9 (3.3-6.6) log IU/ml. During 48 weeks of BLV monotherapy, HDV RNA declined by 3.1 (0.2-4.3) log IU/ml (p < 0.001 vs. baseline), becoming undetectable in 5 patients (23%). A virological response was observed in 14 (78%) patients while a non-response was observed in 2 (11%). ALT decreased to 35 (15-86) U/L (p < 0.001 vs. baseline), normalizing in 83% of patients. A combined response was observed in 67% of patients. Aspartate aminotransferase and gamma-glutamyltransferase levels significantly improved. Concerning liver function parameters, albumin values significantly increased and bilirubin remained stable. LSM significantly improved in patients with virological response, while platelet count was unchanged. None of the patients developed decompensating events or hepatocellular carcinoma. BLV was well tolerated, no patient discontinued treatment and the increase in bile acids was fully asymptomatic. A 48-week course of BLV 2 mg/day monotherapy is safe and effective even for difficult-to treat patients with HDV-related compensated cirrhosis and CSPH. Hepatitis delta virus (HDV) is associated with the most severe form of viral hepatitis. A new treatment for HDV called bulevirtide has recently received conditional approval for patients with chronic HDV infection. However, its safety and effectiveness in patients with more advanced liver disease is not known. Herein, we show that it is safe and effective in patients with HDV-related cirrhosis and clinically significant portal hypertension. [Display omitted] • 48 weeks of bulevirtide 2 mg/day monotherapy was safe in patients with compensated cirrhosis and clinically significant portal hypertension. • Virological, biochemical, and combined responses were achieved in 78%, 83% and 67% of patients, respectively. • BLV treatment led to a significant improvement in most biochemical variables and an increase in liver function parameters. • Treatment was safe and well tolerated, an asymptomatic increase of bile acids was observed. [ABSTRACT FROM AUTHOR]

Ebola virus (EBOV) infection leads to staggeringly high mortality rate. Effective and low-cost treatments are urgently needed to control frequent EBOV outbreaks in Africa. In this study, we report that a natural compound called berbamine hydrochloride strongly inhibits EBOV replication in vitro and in vivo. Our work further showed that berbamine hydrochloride acts by directly binding to the cleaved EBOV glycoprotein (GPcl), disrupting GPcl interaction with viral receptor Niemann-Pick C1, thus blocking the fusion of viral and cellular membranes. Our data support the probability of developing anti-EBOV small molecule drugs by targeting viral GPcl. More importantly, since berbamine hydrochloride has been used in clinic to treat leukopenia, it holds great promise of being quickly repurposed as an anti-EBOV drug. Through structure-based virtual screening, this study reported that a natural compound berbamine hydrochloride can disrupt GPcl–NPC1 interaction, and strongly inhibited EBOV replication in vitro and in vivo. [Display omitted] [ABSTRACT FROM AUTHOR]

With the aim of rationally devising a refined and potent HIV-1 blocker, the cDNA of CCL5 5p12 5m, an extremely potent CCR5 antagonist, was fused to that of C37, a gp41-targeted fusion inhibitor. The resulting CCL5 5p12 5m-C37 fusion protein was expressed in E. coli and proved to be capable of inhibiting R5 HIV-1 strains with low to sub-picomolar IC50, maintaining its antagonism toward CCR5. In addition, CCL5 5p12 5m-C37 inhibits R5/X4 and X4 HIV-1 strains in the picomolar concentration range. The combination of CCL5 5p12 5m-C37 with tenofovir (TDF) exhibited a synergic effect, promoting this antiviral cocktail. Interestingly, a CCR5-targeted combination of maraviroc (MVC) with CCL5 5p12 5m-C37 led to a synergic effect that could be explained by an extensive engagement of different CCR5 conformational populations. Within the mechanism of HIV-1 entry, the CCL5 5p12 5m-C37 chimera may fit as a powerful blocker in several instances. In its possible consideration for systemic therapy or pre-exposure prophylaxis, this protein design represents an interesting lead in the combat of HIV-1 infection. [ABSTRACT FROM AUTHOR]

Chronic hepatitis D (CHD) is the most severe form of viral hepatitis, characterised by the greatest increase in risk of cirrhosis, hepatic decompensation and hepatocellular carcinoma. Pegylated-interferon-α (pegIFNα), the only off-label therapeutic option, has been available for the last 30 years but is associated with suboptimal response rates and poor tolerability. Among the new treatment strategies under clinical evaluation, the entry inhibitor bulevirtide (BLV) is the only one that has received conditional approval from the European Medicines Agency (EMA); approval was granted in July 2020 for the treatment of adult patients with compensated CHD at a dose of 2 mg daily. Phase II studies and the week 24 interim analysis of a phase III study demonstrated the efficacy and safety of this treatment as a monotherapy or combined with pegIFNα. This favourable profile has been confirmed by recent real-world studies performed in Europe. As a long-term monotherapy, BLV has been successfully used to treat patients with advanced compensated cirrhosis. These encouraging yet preliminary findings must be viewed with caution as many critical issues related to this new antiviral strategy are still poorly understood, as summarised in this review. While waiting for new anti-HBV and anti-HDV drugs to become available for combination studies, BLV treatment is currently the only available anti-HDV therapeutic option that might improve the long-term prognosis of difficult-to-manage patients with CHD. [ABSTRACT FROM AUTHOR]

In July 2020, the entry inhibitor bulevirtide was approved in the European Union for the treatment of chronic hepatitis delta virus (HDV) infection. We describe the first 48 weeks of bulevirtide therapy in eight patients (n = 7 male, n = 1 female; n = 3 compensated cirrhosis) treated at our centre. Median ALT values declined from 82 to 34 U/L after 48 weeks. Median HDV RNA dropped from 13 380 000 to 3135 copies/ml. One patient showed no significant response and was discontinued at week 16. Overall, we observed a favourable safety profile and a marked biochemical and virological response in the majority of our patients. [ABSTRACT FROM AUTHOR]

LCB1 is a 56-mer miniprotein computationally designed to target the spike (S) receptor-binding motif of SARS-CoV-2 with potent in vitro and in vivo inhibitory activities. However, the rapid emergence and epidemic of viral variants have greatly impacted the effectiveness of S protein-targeting vaccines and antivirals. In this study, we chemically synthesized a peptidebased LCB1 inhibitor and characterized the resistance profile and underlying mechanism of SARS-CoV-2 variants. Among five variants of concern (VOCs), we found that pseudoviruses of Beta, Gamma, and Omicron were highly resistant to the LCB1 inhibition, whereas the pseudoviruses of Alpha and Delta as well as the variant of interest (VOI) Lambda only caused mild resistance. By generating a group of mutant viruses carrying single or combination mutations, we verified that K417N and N501Y substitutions in RBD critically determined the high resistance phenotype of VOCs. Furthermore, a large panel of 85 pseudoviruses with naturally occurring RBD point-mutations were generated and applied to LCB1, which identified that E406Q, K417N, and L455F conferred high-levels of resistance, when Y505W caused a -6-fold resistance fold-change. We also showed that the resistance mutations could greatly weaken the binding affinity of LCB1 to RBD and thus attenuated its blocking capacity on the interaction between RBD and the cell receptor ACE2. In conclusion, our data have provided crucial information for understanding the mechanism of SARS-CoV-2 resistance to LCB1 and will guide the design strategy of novel LCB1-based antivirals against divergent VOCs and evolutionary mutants. [ABSTRACT FROM AUTHOR]

Introduction: Entry inhibitors are a relatively new class of antiretroviral therapy and are typically indicated in heavily treatment experienced individuals living with HIV. Despite this, there is no formal definition of 'heavily treatment experienced'. Interpretation of this term generally includes acknowledgement of multidrug resistance and reflects the fact that patients in need of further treatment options may have experienced multiple lines of therapy. However, it fails to recognize treatment limiting factors including contraindications, age‐associated comorbidities, and difficulty adhering to regimens. Methods: This manuscript follows a roundtable discussion and aims to identify the unmet needs of those living with HIV who are in need of further treatment options, to broaden the definition of heavily treatment experienced and to clarify the use of newer agents, with an emphasis on the potential role of entry inhibitors, in this population. Results/Conclusions: Within the entry inhibitor class, mechanisms of action differ between agents; resistance to one subclass does not confer resistance to others. Combinations of entry inhibitors should be considered in the same regimen, and if lack of response is seen to one entry inhibitor another can be tried. When selecting an entry inhibitor, physicians should account for patient preferences and needs as well as agent‐specific clinical characteristics. Absence of documented multidrug resistance should not exclude an individual from treatment with an entry inhibitor; entry inhibitors are a valuable treatment option for all individuals who are treatment limited or treatment exhausted. We should advocate for additional clinical trials that help define the role of entry inhibitors in people with exhausted/limited ART options other than drug resistance. [ABSTRACT FROM AUTHOR]

Abstract Background We previously developed drug-like peptide triazoles (PTs) that target HIV-1 Envelope (Env) gp120, potently inhibit viral entry, and irreversibly inactivate virions. Here, we investigated potential mechanisms of viral escape from this promising class of HIV-1 entry inhibitors. Results HIV-1 resistance to cyclic (AAR029b) and linear (KR13) PTs was obtained by dose escalation in viral passaging experiments. High-level resistance for both inhibitors developed slowly (relative to escape from gp41-targeted C-peptide inhibitor C37) by acquiring mutations in gp120 both within (Val255) and distant to (Ser143) the putative PT binding site. The similarity in the resistance profiles for AAR029b and KR13 suggests that the shared IXW pharmacophore provided the primary pressure for HIV-1 escape. In single-round infectivity studies employing recombinant virus, V255I/S143N double escape mutants reduced PT antiviral potency by 150- to 3900-fold. Curiously, the combined mutations had a much smaller impact on PT binding affinity for monomeric gp120 (four to ninefold). This binding disruption was entirely due to the V255I mutation, which generated few steric clashes with PT in molecular docking. However, this minor effect on PT affinity belied large, offsetting changes to association enthalpy and entropy. The escape mutations had negligible effect on CD4 binding and utilization during entry, but significantly altered both binding thermodynamics and inhibitory potency of the conformationally-specific, anti-CD4i antibody 17b. Moreover, the escape mutations substantially decreased gp120 shedding induced by either soluble CD4 or AAR029b. Conclusions Together, the data suggest that the escape mutations significantly modified the energetic landscape of Env’s prefusogenic state, altering conformational dynamics to hinder PT-induced irreversible inactivation of Env. This work therein reveals a unique mode of virus escape for HIV-1, namely, resistance by altering the intrinsic conformational dynamics of the Env trimer.

The utility of human neuroblastoma cell lines as in vitro model to study neuro-invasiveness and neuro-virulence of SARS-CoV-2 has been demonstrated by our laboratory and others. The aim of this report is to further characterize the associated cellular responses caused by a pre-alpha SARS-CoV-2 strain on differentiated SH-SY5Y and to prevent its cytopathic effect by using a set of entry inhibitors. The susceptibility of SH-SY5Y to SARS-CoV-2 was confirmed at high multiplicity-of-infection, without viral replication or release. Infection caused a reduction in the length of neuritic processes, occurrence of plasma membrane blebs, cell clustering, and changes in lipid droplets electron density. No changes in the expression of cytoskeletal proteins, such as tubulins or tau, could explain neurite shortening. To counteract the toxic effect on neurites, entry inhibitors targeting TMPRSS2, ACE2, NRP1 receptors, and Spike RBD were co-incubated with the viral inoculum. The neurite shortening could be prevented by the highest concentration of camostat mesylate, anti-RBD antibody, and NRP1 inhibitor, but not by soluble ACE2. According to the degree of entry inhibition, the average amount of intracellular viral RNA was negatively correlated to neurite length. This study demonstrated that targeting specific SARS-CoV-2 host receptors could reverse its neurocytopathic effect on SH-SY5Y.

Novel coronavirus disease 2019 (COVID-19), a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has brought an unprecedented public health crisis and continues to threaten humanity due to the persistent emergence of new variants. Therefore, developing more effective and broad-spectrum therapeutic and prophylactic drugs against infection by SARS-CoV-2 and its variants, as well as future emerging CoVs, is urgently needed. In this study, we screened several US FDA-approved drugs and identified phenothiazine derivatives with the ability to potently inhibit the infection of pseudotyped SARS-CoV-2 and distinct variants of concern (VOCs), including B.1.617.2 (Delta) and currently circulating Omicron sublineages XBB and BQ.1.1, as well as pseudotyped SARS-CoV and MERS-CoV. Mechanistic studies suggested that phenothiazines predominantly inhibited SARS-CoV-2 pseudovirus (PsV) infection at the early stage and potentially bound to the spike (S) protein of SARS-CoV-2, which may prevent the proteolytic cleavage of the S protein, thereby exhibiting inhibitory activity against SARS-CoV-2 infection. In summary, our findings suggest that phenothiazines can serve as a potential broad-spectrum therapeutic drug for the treatment of SARS-CoV-2 infection as well as the infection of future emerging human coronaviruses (HCoVs).

Binding to the host cell receptors CD4 and CCR5/CXCR4 triggers conformational changes in the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer that promote virus entry. CD4 binding allows the gp120 exterior Env to bind CCR5/CXCR4 and induces a short-lived prehairpin intermediate conformation in the gp41 transmembrane Env. Small-molecule CD4-mimetic compounds (CD4mcs) bind within the conserved Phe-43 cavity of gp120, near the binding site for CD4. CD4mcs like BNM-III-170 inhibit HIV-1 infection by competing with CD4 and by prematurely activating Env, leading to irreversible inactivation. In cell culture, we selected and analyzed variants of the primary HIV-1AD8 strain resistant to BNM-III-170. Two changes (S375N and I424T) in gp120 residues that flank the Phe-43 cavity each conferred an; 5-fold resistance to BNM-III-170 with minimal fitness cost. A third change (E64G) in layer 1 of the gp120 inner domain resulted in; 100-fold resistance to BNM-III-170,; 2- to 3-fold resistance to soluble CD4-Ig, and a moderate decrease in viral fitness. The gp120 changes additively or synergistically contributed to BNM-III-170 resistance. The sensitivity of the Env variants to BNM-III-170 inhibition of virus entry correlated with their sensitivity to BNM-III-170-induced Env activation and shedding of gp120. Together, the S375N and I424T changes, but not the E64G change, conferred .100-fold and 33-fold resistance to BMS-806 and BMS-529 (temsavir), respectively, potent HIV-1 entry inhibitors that block Env conformational transitions. These studies identify pathways whereby HIV-1 can develop resistance to CD4mcs and conformational blockers, two classes of entry inhibitors that target the conserved gp120 Phe-43 cavity. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant socioeconomic burden, and combating COVID-19 is imperative. Blocking the SARS-CoV-2 RBD–ACE2 interaction is a promising therapeutic approach for viral infections, as SARS-CoV-2 binds to the ACE2 receptors of host cells via the RBD of spike proteins to infiltrate these cells. We used computer-aided drug design technology and cellular experiments to screen for peptide S4 with high affinity and specificity for the human ACE2 receptor through structural analysis of SARS-CoV-2 and ACE2 interactions. Cellular experiments revealed that peptide S4 effectively inhibited SARS-CoV-2 and HCoV-NL63 viruses from infecting host cells and was safe for cells at effective concentrations. Based on these findings, peptide S4 may be a potential pharmaceutical agent for clinical application in the treatment of the ongoing SARS-CoV-2 pandemic. • We use computer-aided drug design and cellular tests to screen peptide inhibitors. • Peptide S4 is a potent coronavirus entry inhibitor. • Peptide S4 inhibits the ACE2 receptor and competes with the virus for binding. [ABSTRACT FROM AUTHOR]

Previous data suggest a lack of cross-resistance between the gp120-directed attachment inhibitor temsavir (active moiety of fostemsavir) and the CD4-directed post-attachment inhibitor ibalizumab. Recently, analysis of HIV-1 envelopes with reduced sensitivity to both inhibitors was undertaken to determine whether they shared genotypic correlates of resistance. Sequences from 2 envelopes with reduced susceptibility to both agents were mapped onto a temsavir-bound gp120 structure. Residues within 5.0 Å of the temsavir binding site were evaluated using reverse genetics. Broader applicability and contextual determinants of key substitutions were further assessed using envelopes from participants in the phase 3 BRIGHTE study. Temsavir sensitivity was measured by half-maximal inhibitory concentration (IC 50) and ibalizumab sensitivity by IC 50 and maximum percent inhibition (MPI). One envelope required substitutions of E113D and T434M for full restoration of temsavir susceptibility. Neither substitution nor their combination affected ibalizumab sensitivity. However, in the second envelope, an E202 substitution (HXB2, T202) was sufficient for observed loss of susceptibility to both inhibitors. One BRIGHTE participant with no ibalizumab exposure had an emergent K202E substitution at protocol-defined virologic failure, with reduced sensitivity to both inhibitors. Introducing T202E into previously susceptible clinical isolates reduced temsavir potency by ≥ 40-fold and ibalizumab MPI from >99% to ∼80%. Interestingly, introduction of the gp120 V5 region from a highly ibalizumab-susceptible envelope mitigated the E202 effect on ibalizumab but not temsavir. A rare HIV-1 gp120 E202 mutation reduced temsavir susceptibility, and depending on sequence context, could result in reduced susceptibility to ibalizumab. • HIV-1 envelopes with reduced sensitivity to both temsavir and ibalizumab were investigated for potential cross-resistance. • An E202 substitution was sufficient for the observed loss of sensitivity to both inhibitors. • An envelope with K202E from a BRIGHTE participant naive to ibalizumab was less susceptible to both inhibitors in vitro. • Reduced susceptibility to ibalizumab of E202-containing envelopes was context dependent on the sequence of the V5 region. • Pre-existing cross-resistance between temsavir and ibalizumab due to E202 is unlikely as the E202 polymorphism is rare. [ABSTRACT FROM AUTHOR]

Despite considerable progress in developing vaccines and antivirals to combat COVID-19, the rapid mutations of the SARS-CoV-2 genome have limited the durability and efficacy of the current vaccines and therapeutic interventions. Hence, it necessitates the development of novel therapeutic approaches or repurposing existing drugs that target either viral life cycle, host factors, or both. Here, we report that SRX3177, a potent triple-activity CDK4/6-PI3K-BET inhibitor, blocks replication of the SARS-CoV-2 Omicron variant with IC 50 values at sub-micromolar concentrations without any impact on the cell proliferation of Calu-3 cells at and below its IC 50 concentration. When SRX3177 is combined with EIDD-1931 (active moiety of a small-molecule prodrug Molnupiravir) or MU-UNMC-2 (a SARS-CoV-2 entry inhibitor) at a fixed doses matrix, a synergistic effect was observed, leading to the significant reduction in the dose of the individual compounds to achieve similar inhibition of SARS-CoV-2 replication. Herein, we report that the combination of SRX3177/MPV or SRX3177/UM-UNMC-2 has the potential for further development as a combinational therapy against SARS-CoV-2 and in any future outbreak of beta coronavirus. • SRX3177 inhibits PI3K and CDK4/6 in addition to inhibiting BRD4 and BRD2. • SRX3177 blocks the interaction between the SARS-CoV-2 E protein and the BD1 domain of BRD4. • SRX3177 acts synergistically with Molnupiravir and MU-UNMC-2 to block SARS-CoV-2 replication. [ABSTRACT FROM AUTHOR]

The Na+/taurocholate co-transporting polypeptide (NTCP, gene symbol SLC10A1) is both a physiological bile acid transporter and the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Virus entry via endocytosis of the virus/NTCP complex involves co-factors, but this process is not fully understood. As part of the innate immunity, interferon-induced transmembrane proteins (IFITM) 1–3 have been characterized as virus entry-restricting factors for many viruses. The present study identified IFITM3 as a novel protein–protein interaction (PPI) partner of NTCP based on membrane yeast-two hybrid and co-immunoprecipitation experiments. Surprisingly, IFITM3 knockdown significantly reduced in vitro HBV infection rates of NTCP-expressing HuH7 cells and primary human hepatocytes (PHHs). In addition, HuH7-NTCP cells showed significantly lower HDV infection rates, whereas infection with influenza A virus was increased. HBV-derived myr-preS1 peptide binding to HuH7-NTCP cells was intact even under IFITM3 knockdown, suggesting that IFITM3-mediated HBV/HDV infection enhancement occurs in a step subsequent to the viral attachment to NTCP. In conclusion, IFITM3 was identified as a novel NTCP co-factor that significantly affects in vitro infection with HBV and HDV in NTCP-expressing hepatoma cells and PHHs. While there is clear evidence for a direct PPI between IFITM3 and NTCP, the specific mechanism by which this PPI facilitates the infection process remains to be identified in future studies. [ABSTRACT FROM AUTHOR]

Entry inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed to control the outbreak of coronavirus disease 2019 (COVID-19). This study developed a robust and straightforward assay that detected the molecular interaction between the receptor-binding domain (RBD) of viral spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor in just 10 min. A drug library of 1068 approved compounds was used to screen for SARS-CoV2 entry inhibition, and 9 active drugs were identified as specific pseudovirus entry inhibitors. A plaque reduction neutralization test using authentic SARS-CoV-2 virus in Vero E6 cells confirmed that 2 of these drugs (Etravirine and Dolutegravir) significantly inhibited the infection of SARS-CoV-2. With molecular docking, we showed that both Etravirine and Dolutegravir are preferentially bound to primary ACE2-interacting residues on the RBD domain, implying that these two drug blocks may prohibit the viral attachment of SARS-CoV-2. We compared the neutralizing activities of these entry inhibitors against different pseudoviruses carrying spike proteins from alpha, beta, gamma, and delta variants. Both Etravirine and Dolutegravir showed similar neutralizing activities against different variants, with EC50 values between 4.5 to 5.8 nM for Etravirine and 10.2 to 22.9 nM for Dolutegravir. These data implied that Etravirine and Dolutegravir may serve as general spike inhibitors against dominant viral variants of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Background & Aims: Infection with hepatitis C virus (HCV) remains a major cause of morbidity and mortality worldwide despite the recent advent of highly effective direct-acting antivirals. The envelope glycoproteins of HCV are heavily glycosylated with a high proportion of high-mannose glycans (HMGs), which serve as a shield against neutralizing antibodies and assist in the interaction with cell-entry receptors. However, there is no approved therapeutic targeting this potentially druggable biomarker. Methods: The anti-HCV activity of a fusion protein consisting of Avaren lectin and the fragment crystallizable (Fc) region of a human immunoglobulin G1 antibody, Avaren-Fc (AvFc) was evaluated through the use of in vitro neutralization assays as well as an in vivo challenge in a chimeric human liver (PXB) mouse model. Drug toxicity was assessed by histopathology, serum alanine aminotransferase, and mouse body weights. Results: AvFc was capable of neutralizing cell culture–derived HCV in a genotype-independent manner, with 50% inhibitory concentration values in the low nanomolar range. Systemic administration of AvFc in a histidine-based buffer was well tolerated; after 11 doses every other day at 25 mg/kg there were no significant changes in body or liver weights or in blood human albumin or serum alanine aminotransferase activity. Gross necropsy and liver pathology confirmed the lack of toxicity. This regimen successfully prevented genotype 1a HCV infection in all animals, although an AvFc mutant lacking HMG binding activity failed. Conclusions: These results suggest that targeting envelope HMGs is a promising therapeutic approach against HCV infection, and AvFc may provide a safe and efficacious means to prevent recurrent infection upon liver transplantation in HCV-related end-stage liver disease patients.

To develop unique small-molecule inhibitors of hepatitis C virus (HCV), thiophen urea (TU) derivatives were synthesised and screened for HCV entry inhibitory activities. Among them, seven TU compounds exhibited portent anti-viral activities against genotypes 1/2 (EC50 < 30 nM) and subsequently, they were further investigated; based on the pharmacological, metabolic, pharmacokinetic, and safety profiles, J2H-1701 was selected as the optimised lead compound as an HCV entry inhibitor. J2H-1701 possesses effective multi-genotypic antiviral activity. The docking results suggested the potential interaction of J2H-1701 with the HCV E2 glycoprotein. These results suggest that J2H-1701 can be a potential candidate drug for the development of HCV entry inhibitors.

Our previous studies have shown that cholesterol-conjugated, peptide-based pan-coronavirus (CoV) fusion inhibitors can potently inhibit human CoV infection. However, only palmitic acid (C16)-based lipopeptide drugs have been tested clinically, suggesting that the development of C16-based lipopeptide drugs is feasible. Here, we designed and synthesized a C16-modified pan-CoV fusion inhibitor, EK1-C16, and found that it potently inhibited infection by SARS-CoV-2 and its variants of concern (VOCs), including Omicron, and other human CoVs and bat SARS-related CoVs (SARSr-CoVs). These results suggest that EK1-C16 could be further developed for clinical use to prevent and treat infection by the currently circulating MERS-CoV, SARS-CoV-2 and its VOCs, as well as any future emerging or re-emerging coronaviruses. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has led to the search for new molecules with antiviral activity against SARS-CoV-2. The entry of the virus into the cell is one of the main targets for inhibiting SARS-CoV-2 infection. Natural products are an important source of new therapeutic alternatives against diseases. Pseudotyped viruses allow the study of SARS-CoV-2 viral entry inhibitors, and due to their simplicity, they allow the screening of a large number of antiviral candidates in Biosafety Level 2 facilities. We used pseudotyped HIV-1 with the D614G SARS-CoV-2 spike glycoprotein to test its ability to infect ACE2-expressing HEK 293T cells in the presence of diverse natural products, including 21 plant extracts, 7 essential oils, and 13 compounds from plants and fungi. The 50% cytotoxic concentration (CC50) was evaluated using the resazurin method. From these analyses, we determined the inhibitory activity of the extract of Stachytarpheta cayennensis, which had a half-maximal inhibitory concentration (IC50) of 91.65 µg/mL, a CC50 of 693.5 µg/mL, and a selectivity index (SI) of 7.57, indicating its potential use as an inhibitor of SARS-CoV-2 entry. Moreover, our work indicates the usefulness of the pseudotyped-virus system in the screening of SARS-CoV-2 entry inhibitors. [ABSTRACT FROM AUTHOR]

The entry inhibitor bulevirtide (BLV) received conditional approval from the EMA in July 2020 for the treatment of adult patients with compensated chronic hepatitis delta. However, the effectiveness and safety of BLV administered as monotherapy beyond 48 weeks in difficult-to-treat patients with HDV-related cirrhosis is presently unknown. Herein, we describe the first patients with HDV-related compensated cirrhosis who were treated with BLV (10 mg/day as a starting dose) for up to 3 years on a compassionate use program. Patients were also monitored for HBcrAg and HBV RNA levels, and HDV- and HBV-specific T-cell markers. In the patient who stopped BLV at week 48, after achieving a virological and biochemical response, the initial virological and biochemical rebound was followed by alanine aminotransferase normalization coupled with low HDV RNA and HBsAg levels. In the 2 patients treated continuously for 3 years, virological and biochemical responses were maintained throughout the treatment period even after dose reduction. In a patient with advanced compensated cirrhosis, liver function tests significantly improved, esophageal varices disappeared, and histological/laboratory features of autoimmune hepatitis resolved. Overall, no safety issues were recorded, as bile salt increase was asymptomatic. While serum HBV RNA levels remained undetectable in all patients, HBV core-related antigen levels showed a progressive, yet modest decline during long-term BLV treatment. No HDV-specific interferon-γ-producing T cells were detected, neither after HDV reactivation (after BLV withdrawn in Patient 1) nor during 3 years of BLV treatment. In conclusion, this report shows that continuous administration of BLV monotherapy for 3 years leads to excellent virological and clinical responses in patients with HDV-related cirrhosis who had contraindications to interferon-based therapies. [ABSTRACT FROM AUTHOR]

Lassa virus (LASV) is the causative agent of human Lassa fever which in severe cases manifests as hemorrhagic fever leading to thousands of deaths annually. However, no approved vaccines or antiviral drugs are currently available. Recently, we screened approximately 2,500 compounds using a recombinant vesicular stomatitis virus (VSV) expressing LASV glycoprotein GP (VSV-LASVGP) and identified a P-glycoprotein inhibitor as a potential LASV entry inhibitor. Here, we show that another identified candidate, hexestrol (HES), an estrogen receptor agonist, is also a LASV entry inhibitor. HES inhibited VSV-LASVGP replication with a 50% inhibitory concentration (IC 50 ) of 0.63 µM. Importantly, HES also inhibited authentic LASV replication with IC 50 values of 0.31 µM-0.61 µM. Time-of-addition and cell-based membrane fusion assays suggested that HES inhibits the membrane fusion step during virus entry. Alternative estrogen receptor agonists did not inhibit VSV-LASVGP replication, suggesting that the estrogen receptor itself is unlikely to be involved in the antiviral activity of HES. Generation of a HES-resistant mutant revealed that the phenylalanine at amino acid position 446 (F446) of LASVGP, which is located in the transmembrane region, conferred resistance to HES. Although mutation of F446 enhanced the membrane fusion activity of LASVGP, it exhibited reduced VSV-LASVGP replication, most likely due to the instability of the pre-fusion state of LASVGP. Collectively, our results demonstrated that HES is a promising anti-LASV drug that acts by inhibiting the membrane fusion step of LASV entry. This study also highlights the importance of the LASVGP transmembrane region as a target for anti-LASV drugs.IMPORTANCELassa virus (LASV), the causative agent of Lassa fever, is the most devastating mammarenavirus with respect to its impact on public health in West Africa. However, no approved antiviral drugs or vaccines are currently available. Here, we identified hexestrol (HES), an estrogen receptor agonist, as the potential antiviral candidate drug. We showed that the estrogen receptor itself is not involved in the antiviral activity. HES directly bound to LASVGP and blocked membrane fusion, thereby inhibiting LASV infection. Through the generation of a HES-resistant virus, we found that phenylalanine at position 446 (F446) within the LASVGP transmembrane region plays a crucial role in the antiviral activity of HES. The mutation at F446 caused reduced virus replication, likely due to the instability of the pre-fusion state of LASVGP. These findings highlight the potential of HES as a promising candidate for the development of antiviral compounds targeting LASV., Competing Interests: The authors declare no conflict of interest.

Zika virus (ZIKV) is one of the mosquito-borne flaviviruses that exhibits a unique tropism to nervous systems and is associated with Guillain-Barre syndrome and congenital Zika syndrome (CZS). Dengue virus (DENV) and yellow fever virus (YFV), the other two mosquito-borne flaviviruses, have also been circulating for a long time and cause severe diseases, such as dengue hemorrhagic fever and yellow fever, respectively. However, there are no safe and effective antiviral drugs approved for the treatment of infections or coinfections of these flaviviruses. Here, we found that zafirlukast, a pregnancy-safe leukotriene receptor antagonist, exhibited potent antiviral activity against infections of ZIKV strains from different lineages in different cell lines, as well as against infections of DENV-2 and YFV 17D. Mechanistic studies demonstrated that zafirlukast directly and irreversibly inactivated these flaviviruses by disrupting the integrity of the virions, leading to the loss of viral infectivity, hence inhibiting the entry step of virus infection. Considering its efficacy against flaviviruses, its safety for pregnant women, and its neuroprotective effect, zafirlukast is a promising candidate for prophylaxis and treatment of infections or coinfections of ZIKV, DENV, and YFV, even in pregnant women., Competing Interests: The authors declare no conflict of interest.

To develop unique small-molecule inhibitors of hepatitis C virus (HCV), thiophen urea (TU) derivatives were synthesised and screened for HCV entry inhibitory activities. Among them, seven TU compounds exhibited portent anti-viral activities against genotypes 1/2 (EC50 < 30 nM) and subsequently, they were further investigated; based on the pharmacological, metabolic, pharmacokinetic, and safety profiles, J2H-1701 was selected as the optimised lead compound as an HCV entry inhibitor. J2H-1701 possesses effective multi-genotypic antiviral activity. The docking results suggested the potential interaction of J2H-1701 with the HCV E2 glycoprotein. These results suggest that J2H-1701 can be a potential candidate drug for the development of HCV entry inhibitors. [ABSTRACT FROM AUTHOR]

Lujo virus (LUJV) belongs to the Old World (OW) genus Mammarenavirus (family Arenaviridae). It is categorized as a biosafety level (BSL) 4 agent. Currently, there are no U.S. Food and Drug Administration (FDA)-approved drugs or vaccines specifically for LUJV or other pathogenic OW mammarenaviruses. Here, a high-throughput screening of an FDA-approved drug library was conducted using pseudotype viruses bearing LUJV envelope glycoprotein (GPC) to identify inhibitors of LUJV entry. Three hit compounds, trametinib, manidipine, and lercanidipine, were identified as LUJV entry inhibitors in the micromolar range. Mechanistic studies revealed that trametinib inhibited LUJV GPC-mediated membrane fusion by targeting C410 [located in the transmembrane (TM) domain], while manidipine and lercanidipine inhibited LUJV entry by acting as calcium channel blockers. Meanwhile, all three hits extended their antiviral spectra to the entry of other pathogenic mammarenaviruses. Furthermore, all three could inhibit the authentic prototype mammarenavirus, lymphocytic choriomeningitis virus (LCMV), and could prevent infection at the micromolar level. This study shows that trametinib, manidipine, and lercanidipine are candidates for LUJV therapy and highlights the critical role of calcium in LUJV infection. The presented findings reinforce the notion that the key residue(s) located in the TM domain of GPC provide an entry-targeted platform for designing mammarenavirus inhibitors. [ABSTRACT FROM AUTHOR]

Background: We previously developed drug-like peptide triazoles (PTs) that target HIV-1 Envelope (Env) gp120, potently inhibit viral entry, and irreversibly inactivate virions. Here, we investigated potential mechanisms of viral escape from this promising class of HIV-1 entry inhibitors. Results: HIV-1 resistance to cyclic (AAR029b) and linear (KR13) PTs was obtained by dose escalation in viral passaging experiments. High-level resistance for both inhibitors developed slowly (relative to escape from gp41-targeted C-peptide inhibitor C37) by acquiring mutations in gp120 both within (Val255) and distant to (Ser143) the putative PT binding site. The similarity in the resistance profiles for AAR029b and KR13 suggests that the shared IXW pharmacophore provided the primary pressure for HIV-1 escape. In single-round infectivity studies employing recombinant virus, V255I/S143N double escape mutants reduced PT antiviral potency by 150- to 3900-fold. Curiously, the combined mutations had a much smaller impact on PT binding affinity for monomeric gp120 (four to ninefold). This binding disruption was entirely due to the V255I mutation, which generated few steric clashes with PT in molecular docking. However, this minor effect on PT affinity belied large, offsetting changes to association enthalpy and entropy. The escape mutations had negligible effect on CD4 binding and utilization during entry, but significantly altered both binding thermodynamics and inhibitory potency of the conformationally-specific, anti-CD4i antibody 17b. Moreover, the escape mutations substantially decreased gp120 shedding induced by either soluble CD4 or AAR029b. Conclusions: Together, the data suggest that the escape mutations significantly modified the energetic landscape of Env's prefusogenic state, altering conformational dynamics to hinder PT-induced irreversible inactivation of Env. This work therein reveals a unique mode of virus escape for HIV-1, namely, resistance by altering the intrinsic conformational dynamics of the Env trimer. [ABSTRACT FROM AUTHOR]