4'-Fluorouridine mitigates lethal infection with pandemic human and highly pathogenic avian influenza viruses.

Influenza outbreaks are associated with substantial morbidity, mortality and economic burden. Next generation antivirals are needed to treat seasonal infections and prepare against zoonotic spillover of avian influenza viruses with pandemic potential. Having previously identified oral efficacy of the nucleoside analog 4'-Fluorouridine (4'-FlU, EIDD-2749) against SARS-CoV-2 and respiratory syncytial virus (RSV), we explored activity of the compound against seasonal and highly pathogenic influenza (HPAI) viruses in cell culture, human airway epithelium (HAE) models, and/or two animal models, ferrets and mice, that assess IAV transmission and lethal viral pneumonia, respectively. 4'-FlU inhibited a panel of relevant influenza A and B viruses with nanomolar to sub-micromolar potency in HAE cells. In vitro polymerase assays revealed immediate chain termination of IAV polymerase after 4'-FlU incorporation, in contrast to delayed chain termination of SARS-CoV-2 and RSV polymerase. Once-daily oral treatment of ferrets with 2 mg/kg 4'-FlU initiated 12 hours after infection rapidly stopped virus shedding and prevented transmission to untreated sentinels. Treatment of mice infected with a lethal inoculum of pandemic A/CA/07/2009 (H1N1)pdm09 (pdmCa09) with 4'-FlU alleviated pneumonia. Three doses mediated complete survival when treatment was initiated up to 60 hours after infection, indicating a broad time window for effective intervention. Therapeutic oral 4'-FlU ensured survival of animals infected with HPAI A/VN/12/2003 (H5N1) and of immunocompromised mice infected with pdmCa09. Recoverees were protected against homologous reinfection. This study defines the mechanistic foundation for high sensitivity of influenza viruses to 4'-FlU and supports 4'-FlU as developmental candidate for the treatment of seasonal and pandemic influenza.

The Role of Toll-like Receptors (TLRs) and Their Related Signaling Pathways in Viral Infection and Inflammation.

Toll-like receptors (TLRs) belong to a powerful system for the recognition and elimination of pathogen-associated molecular patterns (PAMPs) from bacteria, viruses, and other pathogens [...].

MEK inhibitors as novel host-targeted antivirals with a dual-benefit mode of action against hyperinflammatory respiratory viral diseases.

Acute hyperinflammatory virus infections, such as influenza or coronavirus disease-19, are still a major health burden worldwide. In these diseases, a massive overproduction of pro-inflammatory cytokines and chemokines (cytokine storm syndrome) determine the severity of the disease, especially in late stages. Direct-acting antivirals against these pathogens have to be administered very early after infection to be effective and may induce viral resistance. Here, we summarize data on a host-targeted strategy using inhibitors of the cellular Raf/MEK/ERK kinase cascade that not only block replication of different RNA viruses but also suppress the hyperinflammatory cytokine response upon infection. In the first phase-II clinical trial of that approach, the MEK inhibitor Zapnometinib shows evidence of clinical benefit.

Pharmacokinetics, absorption, distribution, metabolism and excretion of the MEK inhibitor zapnometinib in rats.

Zapnometinib is a MEK inhibitor currently under clinical development for the treatment of COVID-19 and influenza. Zapnometinib has both antiviral and immunomodulatory effects. Information concerning the absorption, distribution, metabolism, and excretion of the compound following single oral doses of 30 mg/kg [14C]-zapnometinib to rats was required to support pharmacology and toxicology studies in animals and clinical studies in man. As part of the development and safety assessment of this substance, zapnometinib was radioactively labeled and used for the investigation of time-dependent plasma concentrations, the rates and routes of excretion, the extent and time-course of compound distribution in body tissues, the metabolite profiles in plasma, urine and feces and the chemical nature of its metabolites. The present study reveals a rapid but low absorption of zapnometinib from the gastrointestinal tract, with more than 90% of the compound being excreted within 48 h, mainly via feces. Whole body autoradiography confirms that zapnometinib was rapidly and widely distributed, with greatest concentrations in the circulatory and visceral tissues. Maximum plasma and tissue concentrations occurred between two and 8 h post dose. Penetration into the brain was low, and elimination from most tissues almost complete after 168 h. Metabolic profiles showed that the main clearance routes were metabolism via oxidative reactions and glucuronidation. These results further strengthen the knowledge of zapnometinib with respect to the clinical development of the drug.

Pharmacokinetics, Pharmacodynamics and Antiviral Efficacy of the MEK Inhibitor Zapnometinib in Animal Models and in Humans.

The mitogen-activated protein kinase (MEK) inhibitor zapnometinib is in development to treat acute viral infections like COVID-19 and influenza. While the antiviral efficacy of zapnometinib is well documented, further data on target engagement/pharmacodynamics (PD) and pharmacokinetics (PK) are needed. Here, we report zapnometinib PK and PD parameters in mice, hamsters, dogs, and healthy human volunteers. Mice received 25 mg/kg/day zapnometinib (12.5 mg/kg p. o. twice daily, 8 h interval). Syrian hamsters received 30 mg/kg (15 mg/kg twice daily) or 60 mg/kg/day once daily. Beagle dogs were administered 300 mg/kg/day, and healthy human volunteers were administered 100, 300, 600 and 900 mg zapnometinib (once daily p. o.). Regardless of species or formulation, zapnometinib maximum plasma concentration (Cmax) was reached between 2-4 h after administration with an elimination half-life of 4-5 h in dogs, 8 h in mice or hamsters and 19 h in human subjects. Doses were sufficient to cause up to 80% MEK inhibition. Across all species approximately 10 µg/ml zapnometinib was appropriate to inhibit 50% of peripheral blood mononuclear cells (PBMC) MEK activity. In mice, a 50%-80% reduction of MEK activity was sufficient to reduce influenza virus titer in the lungs by more than 90%. In general, while >50% MEK inhibition was reached in vivo at most doses, 80% inhibition in PBMCs required significantly higher doses and appeared to be the practical maximal level obtained in vivo. However, the period of reduced phosphorylated extracellular-signal regulated kinase (pERK), a measure of MEK inhibition, was maintained even after elimination of zapnometinib from plasma, suggesting a sustained effect on MEK consistent with regulatory effects or a slow off-rate. These data suggest a target plasma Cmax of at least 10 µg/ml zapnometinib in further clinical studies.

The MEK1/2 Inhibitor ATR-002 (Zapnometinib) Synergistically Potentiates the Antiviral Effect of Direct-Acting Anti-SARS-CoV-2 Drugs.

The coronavirus disease 2019 (COVID-19) represents a global public health burden. In addition to vaccination, safe and efficient antiviral treatment strategies to restrict the viral spread within the patient are urgently needed. An alternative approach to a single-drug therapy is the combinatory use of virus- and host-targeted antivirals, leading to a synergistic boost of the drugs' impact. In this study, we investigated the property of the MEK1/2 inhibitor ATR-002's (zapnometinib) ability to potentiate the effect of direct-acting antivirals (DAA) against SARS-CoV-2 on viral replication. Treatment combinations of ATR-002 with nucleoside inhibitors Molnupiravir and Remdesivir or 3C-like protease inhibitors Nirmatrelvir and Ritonavir, the ingredients of the drug Paxlovid, were examined in Calu-3 cells to evaluate the advantage of their combinatory use against a SARS-CoV-2 infection. Synergistic effects could be observed for all tested combinations of ATR-002 with DAAs, as calculated by four different reference models in a concentration range that was very well-tolerated by the cells. Our results show that ATR-002 has the potential to act synergistically in combination with direct-acting antivirals, allowing for a reduction in the effective concentrations of the individual drugs and reducing side effects.

Could a Lower Toll-like Receptor (TLR) and NF-κB Activation Due to a Changed Charge Distribution in the Spike Protein Be the Reason for the Lower Pathogenicity of Omicron?

The novel SARS-CoV-2 Omicron variant B.1.1.529, which emerged in late 2021, is currently active worldwide, replacing other variants, including the Delta variant, due to an enormously increased infectivity. Multiple substitutions and deletions in the N-terminal domain (NTD) and the receptor binding domain (RBD) in the spike protein collaborate with the observed increased infectivity and evasion from therapeutic monoclonal antibodies and vaccine-induced neutralizing antibodies after primary/secondary immunization. In contrast, although three mutations near the S1/S2 furin cleavage site were predicted to favor cleavage, observed cleavage efficacy is substantially lower than in the Delta variant and also lower compared to the wild-type virus correlating with significantly lower TMPRSS2-dependent replication in the lungs, and lower cellular syncytium formation. In contrast, the Omicron variant shows high TMPRSS2-independent replication in the upper airway organs, but lower pathogenicity in animal studies and clinics. Based on recent data, we present here a hypothesis proposing that the changed charge distribution in the Omicron's spike protein could lead to lower activation of Toll-like receptors (TLRs) in innate immune cells, resulting in lower NF-κB activation, furin expression, and viral replication in the lungs, and lower immune hyper-activation.

Pharmacokinetics, Pharmacodynamics and Antiviral Efficacy of the MEK Inhibitor Zapnometinib in Animal Models and in Humans

The mitogen-activated protein kinase (MEK) inhibitor zapnometinib is in development to treat acute viral infections like COVID-19 and influenza. While the antiviral efficacy of zapnometinib is well documented, further data on target engagement/pharmacodynamics (PD) and pharmacokinetics (PK) are needed. Here, we report zapnometinib PK and PD parameters in mice, hamsters, dogs, and healthy human volunteers. Mice received 25 mg/kg/day zapnometinib (12.5 mg/kg p. o. twice daily, 8 h interval). Syrian hamsters received 30 mg/kg (15 mg/kg twice daily) or 60 mg/kg/day once daily. Beagle dogs were administered 300 mg/kg/day, and healthy human volunteers were administered 100, 300, 600 and 900 mg zapnometinib (once daily p. o.). Regardless of species or formulation, zapnometinib maximum plasma concentration (Cmax) was reached between 2–4 h after administration with an elimination half-life of 4–5 h in dogs, 8 h in mice or hamsters and 19 h in human subjects. Doses were sufficient to cause up to 80% MEK inhibition. Across all species approximately 10 μg/ml zapnometinib was appropriate to inhibit 50% of peripheral blood mononuclear cells (PBMC) MEK activity. In mice, a 50%–80% reduction of MEK activity was sufficient to reduce influenza virus titer in the lungs by more than 90%. In general, while >50% MEK inhibition was reached in vivo at most doses, 80% inhibition in PBMCs required significantly higher doses and appeared to be the practical maximal level obtained in vivo. However, the period of reduced phosphorylated extracellular-signal regulated kinase (pERK), a measure of MEK inhibition, was maintained even after elimination of zapnometinib from plasma, suggesting a sustained effect on MEK consistent with regulatory effects or a slow off-rate. These data suggest a target plasma Cmax of at least 10 μg/ml zapnometinib in further clinical studies.

The MEK1/2-inhibitor ATR-002 efficiently blocks SARS-CoV-2 propagation and alleviates pro-inflammatory cytokine/chemokine responses.

Coronavirus disease 2019 (COVID-19), the illness caused by a novel coronavirus now called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 260 million confirmed infections and 5 million deaths to date. While vaccination is a powerful tool to control pandemic spread, medication to relieve COVID-19-associated symptoms and alleviate disease progression especially in high-risk patients is still lacking. In this study, we explore the suitability of the rapid accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway as a druggable target in the treatment of SARS-CoV-2 infections. We find that SARS-CoV-2 transiently activates Raf/MEK/ERK signaling in the very early infection phase and that ERK1/2 knockdown limits virus replication in cell culture models. We demonstrate that ATR-002, a specific inhibitor of the upstream MEK1/2 kinases which is currently evaluated in clinical trials as an anti-influenza drug, displays strong anti-SARS-CoV-2 activity in cell lines as well as in primary air-liquid-interphase epithelial cell (ALI) cultures, with a safe and selective treatment window. We also observe that ATR-002 treatment impairs the SARS-CoV-2-induced expression of pro-inflammatory cytokines, and thus might prevent COVID-19-associated hyperinflammation, a key player in COVID-19 progression. Thus, our data suggest that the Raf/MEK/ERK signaling cascade may represent a target for therapeutic intervention strategies against SARS-CoV-2 infections and that ATR-002 is a promising candidate for further drug evaluation.

COVID-19: Mechanistic Model of the African Paradox Supports the Central Role of the NF-κB Pathway.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has expanded into a global pandemic, with more than 220 million affected persons and almost 4.6 million deaths by 8 September 2021. In particular, Europe and the Americas have been heavily affected by high infection and death rates. In contrast, much lower infection rates and mortality have been reported generally in Africa, particularly in the sub-Saharan region (with the exception of the Southern Africa region). There are different hypotheses for this African paradox, including less testing, the young age of the population, genetic disposition, and behavioral and epidemiological factors. In the present review, we address different immunological factors and their correlation with genetic factors, pre-existing immune status, and differences in cytokine induction patterns. We also focus on epidemiological factors, such as specific medication coverage, helminth distribution, and malaria endemics in the sub-Saharan region. An analysis combining different factors is presented that highlights the central role of the NF-κB signaling pathway in the African paradox. Importantly, insights into the interplay of different factors with the underlying immune pathological mechanisms for COVID-19 can provide a better understanding of the disease and the development of new targets for more efficient treatment strategies.

Designing a SARS-CoV-2 T-Cell-Inducing Vaccine for High-Risk Patient Groups.

We describe the results of two vaccinations of a self-experimenting healthy volunteer with SARS-CoV-2-derived peptides performed in March and April 2020, respectively. The first set of peptides contained eight peptides predicted to bind to the individual's HLA molecules. The second set consisted of ten peptides predicted to bind promiscuously to several HLA-DR allotypes. The vaccine formulation contained the new TLR 1/2 agonist XS15 and was administered as an emulsion in Montanide as a single subcutaneous injection. Peripheral blood mononuclear cells isolated from blood drawn before and after vaccinations were assessed using Interferon-γ ELISpot assays and intracellular cytokine staining. We detected vaccine-induced CD4 T cell responses against six out of 11 peptides predicted to bind to HLA-DR after 19 days, following vaccination, for one peptide already at day 12. We used these results to support the design of a T-cell-inducing vaccine for application in high-risk patients, with weakened lymphocyte performance. Meanwhile, an according vaccine, incorporating T cell epitopes predominant in convalescents, is undergoing clinical trial testing.

Inactivation of SARS-CoV-2 through Treatment with the Mouth Rinsing Solutions ViruProX® and BacterX® Pro.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic effects daily dental work. Therefore, infection control measures are necessary to prevent infection of dental personnel during dental treatments. The use of a preprocedural mouth rinse with chlorhexidine (CHX), cetylpyridinium chloride (CPC), or hydrogen peroxide (H2O2) solution for 30-60 s may reduce the viral load and may protect the personnel in a dental practice. In the present study the virucidal effect of the mouth rinsing solutions ViruProX® with 0.05% CPC and 1.5% H2O2 and BacterX® pro containing 0.1% CHX, 0.05% CPC, and 0.005% sodium fluoride (F-) was investigated in vitro. The mouth rinsing solutions successfully inactivated infectious SARS-CoV-2 particles, the causative agent of coronavirus disease 2019 (COVID-19), within 30 s. To determine the effective components, CHX, CPC, H2O2, and a combination of CHX and CPC, were tested against SARS-CoV-2 in addition. While a combination of CPC and CHX as well as CPC alone led to a significant reduction of infectious viral particles, H2O2 and CHX alone had no virucidal effect against SARS-CoV-2. It can be assumed that preprocedural rinsing of the mouth with ViruProX® or BacterX® pro will reduce the viral load in the oral cavity and could thus lower the transmission of SARS-CoV-2 in dental practice.

NF-κB Pathway as a Potential Target for Treatment of Critical Stage COVID-19 Patients.

Patients infected with SARS-CoV-2 show a wide spectrum of clinical manifestations ranging from mild febrile illness and cough up to acute respiratory distress syndrome, multiple organ failure, and death. Data from patients with severe clinical manifestations compared to patients with mild symptoms indicate that highly dysregulated exuberant inflammatory responses correlate with severity of disease and lethality. Epithelial-immune cell interactions and elevated cytokine and chemokine levels, i.e. cytokine storm, seem to play a central role in severity and lethality in COVID-19. The present perspective places a central cellular pro-inflammatory signal pathway, NF-κB, in the context of recently published data for COVID-19 and provides a hypothesis for a therapeutic approach aiming at the simultaneous inhibition of whole cascades of pro-inflammatory cytokines and chemokines. The simultaneous inhibition of multiple cytokines/chemokines is expected to have much higher therapeutic potential as compared to single target approaches to prevent cascade (i.e. redundant, triggering, amplifying, and synergistic) effects of multiple induced cytokines and chemokines in critical stage COVID-19 patients.