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.

Influenza A virus replication has a stronger dependency on Raf/MEK/ERK signaling pathway activity than SARS-CoV-2.

The recent COVID-19 pandemic again highlighted the urgent need for broad-spectrum antivirals, both for therapeutic use in acute viral infection and for pandemic preparedness in general. The targeting of host cell factors hijacked by viruses during their replication cycle presents one possible strategy for development of broad-spectrum antivirals. By inhibiting the Raf/MEK/ERK signaling pathway, a central kinase cascade of eukaryotic cells, which is being exploited by numerous viruses of different virus phyla, the small-molecule MEK inhibitor zapnometinib has the potential to address this need. We here performed a side-by-side comparison of the antiviral efficacy of zapnometinib against IAV and SARS-CoV-2 to determine the concentration leading to 50% of its effect on the virus (EC50) and the concentration leading to 50% reduction of ERK phosphorylation (IC50) in a comparable manner, using the same experimental conditions. Our results show that the EC50 value and IC50 value of zapnometinib are indeed lower for IAV compared to SARS-CoV-2 using one representative strain for each. The results suggest that IAV's replication has a stronger dependency on an active Raf/MEK/ERK pathway and, thus, that IAV is more susceptible to treatment with zapnometinib than SARS-CoV-2. With zapnometinib's favorable outcome in a recent phase II clinical trial in hospitalized COVID-19 patients, the present results are even more promising for an upcoming phase II clinical trial in severe influenza virus infection.

Regioselective ω-hydroxylation of medium-chain n-alkanes and primary alcohols by CYP153 enzymes from Mycobacterium marinum and Polaromonas sp. strain JS666.

The oxofunctionalization of saturated hydrocarbons is an important goal in basic and applied chemistry. Biocatalysts like cytochrome P450 enzymes can introduce oxygen into a wide variety of molecules in a very selective manner, which can be used for the synthesis of fine and bulk chemicals. Cytochrome P450 enzymes from the CYP153A subfamily have been described as alkane hydroxylases with high terminal regioselectivity. Here we report the product yields resulting from C(5)-C(12) alkane and alcohol oxidation catalyzed by CYP153A enzymes from Mycobacterium marinum (CYP153A16) and Polaromonas sp. (CYP153A P. sp.). For all reactions, byproduct formation is described in detail. Following cloning and expression in Escherichia coli, the activity of the purified monooxygenases was reconstituted with putidaredoxin (CamA) and putidaredoxin reductase (CamB). Although both enzyme systems yielded primary alcohols and α,ω-alkanediols, each one displayed a different oxidation pattern towards alkanes. For CYP153A P. sp. a predominant ω-hydroxylation activity was observed, while CYP153A16 possessed the ability to catalyze both ω-hydroxylation and α,ω-dihydroxylation reactions.

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.

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.

Adeno-associated virus-vectored influenza vaccine elicits neutralizing and Fcγ receptor-activating antibodies.

The current seasonal inactivated influenza vaccine protects only against a narrow range of virus strains as it triggers a dominant antibody response toward the hypervariable hemagglutinin (HA) head region. The discovery of rare broadly protective antibodies against conserved regions in influenza virus proteins has propelled research on distinct antigens and delivery methods to efficiently induce broad immunity toward drifted or shifted virus strains. Here, we report that adeno-associated virus (AAV) vectors expressing influenza virus HA or chimeric HA protected mice against homologous and heterologous virus challenges. Unexpectedly, immunization even with wild-type HA induced antibodies recognizing the HA-stalk and activating FcγR-dependent responses indicating that AAV-vectored expression balances HA head- and HA stalk-specific humoral responses. Immunization with AAV-HA partially protected also ferrets against a harsh virus challenge. Results from this study provide a rationale for further clinical development of AAV vectors as influenza vaccine platform, which could benefit from their approved use in human gene therapy.