Molekulare Pinzetten als Breitbandinhibitor viraler Infektionen.

The SARS-CoV-2 pandemic once again highlighted the constant threat posed by viruses. Specific therapeutics are highly warranted, but their development is time consuming and cost intensive. Broad-spectrum antivirals provide a promising option for fast application to treat circulating or newly emerged viruses. Here, we introduce molecular tweezers as broad-spectrum antivirals, which abrogate viral infection by directly targeting the viral membrane. Furthermore, we discuss the current stage of tweezer development to fight SARS-CoV-2 and other respiratory viruses.

Serum neutralizing capacity and T-cell response against the omicron BA.1 variant in seropositive children and their parents one year after SARS-CoV-2 infection.

Introduction: Durability of immune protection against reinfection with SARS-CoV-2 remains enigmatic, especially in the pediatric population and in the context of immune-evading variants of concern. Obviously, this knowledge is required for measures to contain the spread of infection and in selecting rational preventive measures. Methods: Here, we investigated the serum neutralization capacity of 36 seropositive adults and 34 children approximately one year after infection with the ancestral Wuhan strain of SARS-CoV-2 by using a pseudovirus neutralization assay. Results: We found that 88.9% of seropositive adult (32/36) and 94.1% of seropositive children (32/34) convalescents retained the neutralizing activity against the SARS-CoV-2 Wuhan strain (WT). Although, the neutralization effect against Omicron BA.1 (B.1.1.529.1) was significantly lower, 70.6% (24/34) of children and 41.7% (15/36) of adults possessed BA.1 cross-neutralizing antibodies. The spike 1 (S1)-specific T cell recall capacity using an activation-induced marker assay was analyzed in 18 adults and 16 children. All participants had detectable S1-specific CD4 T cells against WT, and 72.2% (13/18) adults and 81,3% (13/16) children had detectable S1 WT-specific CD8 T cells. CD4 cross-reactivity against BA.1 was demonstrated in all investigated adults (18/18), and 66.7% (12/18) adult participants had also detectable specific CD8 BA.1 T cells while we detected BA.1 S1 reactive CD4 and CD8 T cells in 81.3% (13/16) children. Discussion: Together, our findings demonstrate that infection with the ancestral strain of SARS-CoV-2 in children as well as in adults induces robust serological as well as T cell memory responses that persist over at least 12 months. This suggests persistent immunological memory and partial cross-reactivity against Omicron BA.1.

Molecular Tweezers: Supramolecular Hosts with Broad-Spectrum Biological Applications.

Lysine-selective molecular tweezers (MTs) are supramolecular host molecules displaying a remarkably broad spectrum of biologic activities. MTs act as inhibitors of the self-assembly and toxicity of amyloidogenic proteins using a unique mechanism. They destroy viral membranes and inhibit infection by enveloped viruses, such as HIV-1 and SARS-CoV-2, by mechanisms unrelated to their action on protein self-assembly. They also disrupt biofilm of Gram-positive bacteria. The efficacy and safety of MTs have been demonstrated in vitro, in cell culture, and in vivo, suggesting that these versatile compounds are attractive therapeutic candidates for various diseases, infections, and injuries. A lead compound called CLR01 has been shown to inhibit the aggregation of various amyloidogenic proteins, facilitate their clearance in vivo, prevent infection by multiple viruses, display potent anti-biofilm activity, and have a high safety margin in animal models. The inhibitory effect of CLR01 against amyloidogenic proteins is highly specific to abnormal self-assembly of amyloidogenic proteins with no disruption of normal mammalian biologic processes at the doses needed for inhibition. Therapeutic effects of CLR01 have been demonstrated in animal models of proteinopathies, lysosomal-storage diseases, and spinal-cord injury. Here we review the activity and mechanisms of action of these intriguing compounds and discuss future research directions. SIGNIFICANCE STATEMENT: Molecular tweezers are supramolecular host molecules with broad biological applications, including inhibition of abnormal protein aggregation, facilitation of lysosomal clearance of toxic aggregates, disruption of viral membranes, and interference of biofilm formation by Gram-positive bacteria. This review discusses the molecular and cellular mechanisms of action of the molecular tweezers, including the discovery of distinct mechanisms acting in vitro and in vivo, and the application of these compounds in multiple preclinical disease models.

Native and activated antithrombin inhibits TMPRSS2 activity and SARS-CoV-2 infection.

Host cell proteases such as TMPRSS2 are critical determinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tropism and pathogenesis. Here, we show that antithrombin (AT), an endogenous serine protease inhibitor regulating coagulation, is a broad-spectrum inhibitor of coronavirus infection. Molecular docking and enzyme activity assays demonstrate that AT binds and inhibits TMPRSS2, a serine protease that primes the Spike proteins of coronaviruses for subsequent fusion. Consequently, AT blocks entry driven by the Spikes of SARS-CoV, MERS-CoV, hCoV-229E, SARS-CoV-2 and its variants of concern including Omicron, and suppresses lung cell infection with genuine SARS-CoV-2. Thus, AT is an endogenous inhibitor of SARS-CoV-2 that may be involved in COVID-19 pathogenesis. We further demonstrate that activation of AT by anticoagulants, such as heparin or fondaparinux, increases the anti-TMPRSS2 and anti-SARS-CoV-2 activity of AT, suggesting that repurposing of native and activated AT for COVID-19 treatment should be explored.

Advanced Molecular Tweezers with Lipid Anchors against SARS-CoV-2 and Other Respiratory Viruses.

The COVID-19 pandemic caused by SARS-CoV-2 presents a global health emergency. Therapeutic options against SARS-CoV-2 are still very limited but urgently required. Molecular tweezers are supramolecular agents that destabilize the envelope of viruses resulting in a loss of viral infectivity. Here, we show that first-generation tweezers, CLR01 and CLR05, disrupt the SARS-CoV-2 envelope and abrogate viral infectivity. To increase the antiviral activity, a series of 34 advanced molecular tweezers were synthesized by insertion of aliphatic or aromatic ester groups on the phosphate moieties of the parent molecule CLR01. A structure-activity relationship study enabled the identification of tweezers with a markedly enhanced ability to destroy lipid bilayers and to suppress SARS-CoV-2 infection. Selected tweezer derivatives retain activity in airway mucus and inactivate the SARS-CoV-2 wildtype and variants of concern as well as respiratory syncytial, influenza, and measles viruses. Moreover, inhibitory activity of advanced tweezers against respiratory syncytial virus and SARS-CoV-2 was confirmed in mice. Thus, potentiated tweezers are broad-spectrum antiviral agents with great prospects for clinical development to combat highly pathogenic viruses.

BNT162b2 booster after heterologous prime-boost vaccination induces potent neutralizing antibodies and T cell reactivity against SARS-CoV-2 Omicron BA.1 in young adults.

In light of the decreasing immune protection against symptomatic SARS-CoV-2 infection after initial vaccinations and the now dominant immune-evasive Omicron variants, 'booster' vaccinations are regularly performed to restore immune responses. Many individuals have received a primary heterologous prime-boost vaccination with long intervals between vaccinations, but the resulting long-term immunity and the effects of a subsequent 'booster', particularly against Omicron BA.1, have not been defined. We followed a cohort of 23 young adults, who received a primary heterologous ChAdOx1 nCoV-19 BNT162b2 prime-boost vaccination, over a 7-month period and analysed how they responded to a BNT162b2 'booster'. We show that already after the primary heterologous vaccination, neutralization titers against Omicron BA.1 are recognizable but that humoral and cellular immunity wanes over the course of half a year. Residual responsive memory T cells recognized spike epitopes of the early SARS-CoV-2 B.1 strain as well as the Delta and BA.1 variants of concern (VOCs). However, the remaining antibody titers hardly neutralized these VOCs. The 'booster' vaccination was well tolerated and elicited both high antibody titers and increased memory T cell responses against SARS-CoV-2 including BA.1. Strikingly, in this young heterologously vaccinated cohort the neutralizing activity after the 'booster' was almost as potent against BA.1 as against the early B.1 strain. Our results suggest that a 'booster' after heterologous vaccination results in effective immune maturation and potent protection against the Omicron BA.1 variant in young adults.

Peptidomimetic inhibitors of TMPRSS2 block SARS-CoV-2 infection in cell culture.

The transmembrane serine protease 2 (TMPRSS2) primes the SARS-CoV-2 Spike (S) protein for host cell entry and represents a promising target for COVID-19 therapy. Here we describe the in silico development and in vitro characterization of peptidomimetic TMPRSS2 inhibitors. Molecular docking studies identified peptidomimetic binders of the TMPRSS2 catalytic site, which were synthesized and coupled to an electrophilic serine trap. The compounds inhibit TMPRSS2 while demonstrating good off-target selectivity against selected coagulation proteases. Lead candidates are stable in blood serum and plasma for at least ten days. Finally, we show that selected peptidomimetics inhibit SARS-CoV-2 Spike-driven pseudovirus entry and authentic SARS-CoV-2 infection with comparable efficacy as camostat mesylate. The peptidomimetic TMPRSS2 inhibitors also prevent entry of recent SARS-CoV-2 variants of concern Delta and Omicron BA.1. In sum, our study reports antivirally active and stable TMPRSS2 inhibitors with prospects for further preclinical and clinical development as antiviral agents against SARS-CoV-2 and other TMPRSS2-dependent viruses.

Inhibitors of Activin Receptor-like Kinase 5 Interfere with SARS-CoV-2 S-Protein Processing and Spike-Mediated Cell Fusion via Attenuation of Furin Expression.

Screening of a protein kinase inhibitor library identified SB431542, targeting activin receptor-like kinase 5 (ALK5), as a compound interfering with SARS-CoV-2 replication. Since ALK5 is implicated in transforming growth factor ß (TGF-ß) signaling and regulation of the cellular endoprotease furin, we pursued this research to clarify the role of this protein kinase for SARS-CoV-2 infection. We show that TGF-ß1 induces the expression of furin in a broad spectrum of cells including Huh-7 and Calu-3 that are permissive for SARS-CoV-2. The inhibition of ALK5 by incubation with SB431542 revealed a dose-dependent downregulation of both basal and TGF-ß1 induced furin expression. Furthermore, we demonstrate that the ALK5 inhibitors SB431542 and Vactosertib negatively affect the proteolytic processing of the SARS-CoV-2 Spike protein and significantly reduce spike-mediated cell-cell fusion. This correlated with an inhibitory effect of ALK5 inhibition on the production of infectious SARS-CoV-2. Altogether, our study shows that interference with ALK5 signaling attenuates SARS-CoV-2 infectivity and cell-cell spread via downregulation of furin which is most pronounced upon TGF-ß stimulation. Since a TGF-ß dominated cytokine storm is a hallmark of severe COVID-19, ALK5 inhibitors undergoing clinical trials might represent a potential therapy option for COVID-19.

Immunodetection assays for the quantification of seasonal common cold coronaviruses OC43, NL63, or 229E infection confirm nirmatrelvir as broad coronavirus inhibitor.

Besides pandemic SARS-CoV-2, also endemic seasonal human common cold coronaviruses (hCoVs) have a significant impact on human health and economy. Studies on hCoVs and the identification of antivirals are therefore crucial to improve human well-being. However, hCoVs have long been neglected and the methodology to study virus infection, replication and inhibition warrants being updated. We here evaluated the established plaque-based assays to determine viral titers and cell-to-cell spread and developed protocols for the immunodetection of the viral nucleocapsid protein by flow cytometry and in-cell ELISA to study infection rates at early time points. The developed protocols allow detection of hCoV-229E infection after 2, and hCoV-NL63 and -OC43 infection after 3 days at a single cell level or in a 96 well microtiter format, in large sample numbers without being laborious or expensive. Both assays can be applied to assess the susceptibility of cells to hCoV infection and replication, and to determine the efficacy of antiviral compounds as well as neutralizing antibodies in a sensitive and quantitative manner. Application revealed that clinically applied SARS-CoV-2 targeting monoclonal antibodies are inactive against hCoVs, but that the viral polymerase targeting antivirals remdesivir and molnupiravir are broadly active also against all three hCoVs. Further, the in-cell ELISA provided evidence that nirmatrelvir, previously shown to broadly inhibit coronavirus proteases, also prevents replication of authentic hCoVs. Importantly, the protocols described here can be easily adapted to other coronavirus strains and species as well as viruses of other families within a short time. This will facilitate future research on known and emerging (corona)viruses, support the identification of antivirals and increase the preparedness for future virus outbreaks.

Macromolecular Viral Entry Inhibitors as Broad-Spectrum First-Line Antivirals with Activity against SARS-CoV-2.

Inhibitors of viral cell entry based on poly(styrene sulfonate) and its core-shell nanoformulations based on gold nanoparticles are investigated against a panel of viruses, including clinical isolates of SARS-CoV-2. Macromolecular inhibitors are shown to exhibit the highly sought-after broad-spectrum antiviral activity, which covers most analyzed enveloped viruses and all of the variants of concern for SARS-CoV-2 tested. The inhibitory activity is quantified in vitro in appropriate cell culture models and for respiratory viral pathogens (respiratory syncytial virus and SARS-CoV-2) in mice. Results of this study comprise a significant step along the translational path of macromolecular inhibitors of virus cell entry, specifically against enveloped respiratory viruses.

Severe Acute Respiratory Syndrome Coronavirus 2 Vaccination Boosts Neutralizing Activity Against Seasonal Human Coronaviruses.

BACKGROUND: Most of the millions of people that are vaccinated against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), have previously been infected by related circulating human coronaviruses (hCoVs) causing common colds and will experience further encounters with these viruses in the future. Whether COVID-19 vaccinations impact neutralization of seasonal coronaviruses is largely unknown. METHODS: We analyzed the capacity of sera derived from 24 individuals before and after heterologous ChAdOx1 nCoV-19 BNT162b2 prime-boost vaccination to neutralize genuine OC43, NL63, and 229E hCoVs, as well as viral pseudoparticles carrying the SARS-CoV-1, SARS-CoV-2, Middle East Respiratory Syndrome (MERS)-CoV, and hCoV-OC43, hCoV-NL63, and hCoV-229E spike proteins. Genuine hCoVs or spike containing pseudovirions were incubated with different concentrations of sera and neutralization efficiencies were determined by measuring viral RNA yields, intracellular viral nucleocapsid expression, or reporter gene expression in Huh-7 cells. RESULTS: All individuals showed strong preexisting immunity against hCoV-OC43. Neutralization of hCoV-NL63 was more variable and all sera showed only modest inhibitory activity against genuine hCoV-229E. SARS-CoV-2 vaccination resulted in efficient cross-neutralization of SARS-CoV-1 but not of MERS-CoV. On average, vaccination significantly increased the neutralizing activity against genuine hCoV-OC43, hCoV-NL63, and hCoV-229E. CONCLUSIONS: Heterologous COVID-19 vaccination may confer some cross-protection against endemic seasonal coronaviruses.

IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition in vitro.

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) can restrict viral pathogens, but pro- and anti-viral activities have been reported for coronaviruses. Here, we show that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. However, endogenous IFITM expression supports efficient infection of SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral infection. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. IFITM-derived peptides and targeting antibodies inhibit SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are cofactors for efficient SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and are potential targets for therapeutic approaches.

Alpha-1 antitrypsin inhibits TMPRSS2 protease activity and SARS-CoV-2 infection.

SARS-CoV-2 is a respiratory pathogen and primarily infects the airway epithelium. As our knowledge about innate immune factors of the respiratory tract against SARS-CoV-2 is limited, we generated and screened a peptide/protein library derived from bronchoalveolar lavage for inhibitors of SARS-CoV-2 spike-driven entry. Analysis of antiviral fractions revealed the presence of α1-antitrypsin (α1AT), a highly abundant circulating serine protease inhibitor. Here, we report that α1AT inhibits SARS-CoV-2 entry at physiological concentrations and suppresses viral replication in cell lines and primary cells including human airway epithelial cultures. We further demonstrate that α1AT binds and inactivates the serine protease TMPRSS2, which enzymatically primes the SARS-CoV-2 spike protein for membrane fusion. Thus, the acute phase protein α1AT is an inhibitor of TMPRSS2 and SARS-CoV-2 entry, and may play an important role in the innate immune defense against the novel coronavirus. Our findings suggest that repurposing of α1AT-containing drugs has prospects for the therapy of COVID-19.

Carrageenan-containing over-the-counter nasal and oral sprays inhibit SARS-CoV-2 infection of airway epithelial cultures.

Pharmaceutical interventions are urgently needed to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and transmission. As SARS-CoV-2 infects and spreads via the nasopharyngeal airways, we analyzed the antiviral effect of selected nasal and oral sprays on virus infection in vitro. Two nose sprays showed virucidal activity but were cytotoxic precluding further analysis in cell culture. One nasal and one mouth spray suppressed SARS-CoV-2 infection of TMPRSS2-expressing Vero E6 cells and primary differentiated human airway epithelial cultures. The antiviral activity in both sprays could be attributed to polyanionic ι- and κ-carrageenans. Thus, application of carrageenan-containing nasal and mouth sprays may reduce the risk of acquiring SARS-CoV-2 infection and may limit viral spread, warranting further clinical evaluation.

SARS-CoV-2 infects and replicates in cells of the human endocrine and exocrine pancreas.

Infection-related diabetes can arise as a result of virus-associated ß-cell destruction. Clinical data suggest that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the coronavirus disease 2019 (COVID-19), impairs glucose homoeostasis, but experimental evidence that SARS-CoV-2 can infect pancreatic tissue has been lacking. In the present study, we show that SARS-CoV-2 infects cells of the human exocrine and endocrine pancreas ex vivo and in vivo. We demonstrate that human ß-cells express viral entry proteins, and SARS-CoV-2 infects and replicates in cultured human islets. Infection is associated with morphological, transcriptional and functional changes, including reduced numbers of insulin-secretory granules in ß-cells and impaired glucose-stimulated insulin secretion. In COVID-19 full-body postmortem examinations, we detected SARS-CoV-2 nucleocapsid protein in pancreatic exocrine cells, and in cells that stain positive for the ß-cell marker NKX6.1 and are in close proximity to the islets of Langerhans in all four patients investigated. Our data identify the human pancreas as a target of SARS-CoV-2 infection and suggest that ß-cell infection could contribute to the metabolic dysregulation observed in patients with COVID-19.

Drug Inhibition of SARS-CoV-2 Replication in Human Pluripotent Stem Cell-Derived Intestinal Organoids.

BACKGROUND AND AIMS: The COVID-19 pandemic has spread worldwide and poses a severe health risk. While most patients present mild symptoms, descending pneumonia can lead to severe respiratory insufficiency. Up to 50% of patients show gastrointestinal symptoms like diarrhea or nausea, intriguingly associating with prolonged symptoms and increased severity. Thus, models to understand and validate drug efficiency in the gut of COVID-19 patients are of urgent need. METHODS: Human intestinal organoids derived from pluripotent stem cells (PSC-HIOs) have led, due to their complexity in mimicking human intestinal architecture, to an unprecedented number of successful disease models including gastrointestinal infections. Here, we employed PSC-HIOs to dissect SARS-CoV-2 pathogenesis and its inhibition by remdesivir, one of the leading drugs investigated for treatment of COVID-19. RESULTS: Immunostaining for viral entry receptor ACE2 and SARS-CoV-2 spike protein priming protease TMPRSS2 showed broad expression in the gastrointestinal tract with highest levels in the intestine, the latter faithfully recapitulated by PSC-HIOs. Organoids could be readily infected with SARS-CoV-2 followed by viral spread across entire PSC-HIOs, subsequently leading to organoid deterioration. However, SARS-CoV-2 spared goblet cells lacking ACE2 expression. Importantly, we challenged PSC-HIOs for drug testing capacity. Specifically, remdesivir effectively inhibited SARS-CoV-2 infection dose-dependently at low micromolar concentration and rescued PSC-HIO morphology. CONCLUSIONS: Thus, PSC-HIOs are a valuable tool to study SARS-CoV-2 infection and to identify and validate drugs especially with potential action in the gut.

Supramolecular Mechanism of Viral Envelope Disruption by Molecular Tweezers.

Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the "phosphate tweezer" CLR01, a "carboxylate tweezer" CLR05, and a "phosphate clip" PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.