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.

Highly pathogenic avian influenza A viruses (HPAIV) pose a significant threat to both animal and human health. These viruses have the potential to cause severe respiratory and systemic infections in birds and several mammalian species. The recent global outbreak of the H5N1 clade 2.3.4.4b spread in wild and domestic birds is now considered to be a panzoonosis. Spillover events in dairy cattle farms in the U.S. have highlighted the urgent need for effective antiviral therapies, especially in view of human infections. This study investigates the selective MEK1/2 inhibitor zapnometinib (ZMN) as a potential antiviral agent against HPAIVs. Our in vitro experiments demonstrate that ZMN significantly impairs viral replication across multiple HPAIV strains, including H5N1 clade 2.3.4.4b in cell lines and primary bronchial epithelial cells. The mechanism of action is based on the nuclear retention of newly produced viral ribonucleoprotein complexes (vRNP), when the MEK/ERK/RSK1 kinase cascade is inhibited. We furthermore could show, that ZMN not only acts antiviral in a standalone treatment but has synergistic potential when used in combination with direct-acting antivirals like oseltamivir or baloxavir. Therefore, ZMN treatment offers a promising strategy for future antiviral development.

Identifying novel antiviral treatment strategies to expand the existing repertoire of available small-molecule drugs is an important task in addressing both current and emerging viral diseases. Host-targeting antivirals (HTA) and direct-acting antivirals (DAA) represent two classes of such small-molecule drugs. While both classes of drugs are active in a stand-alone treatment, it may be a promising approach to increase the benefits of both by using them in combination which may lead to a synergistic amplification of the antiviral effects. In this study, we show that SARS-CoV-2 is sensitive to the inhibition of the cellular 90 kDa ribosomal S6 kinase (RSK) family, which are direct downstream effectors of the Raf/MEK/ERK signaling pathway. Specific RSK inhibitors (BI-D1870, BRD 7389) showed anti-SARS-CoV-2 properties in single and combination treatment. We could evaluate synergistic properties with the DAAs Remdesivir (RDV) and Nirmatrelvir (NTV). Serial passaging of δ-B.1.617.2 variant under permanent drug pressure did not alter the susceptibility to the RSK inhibitors, while attenuated responsiveness was found for the DAAs. Interestingly, this diminished drug sensitivity did not occur when BRD 7389 and NTV were combined. Furthermore, we demonstrated that combining RSK inhibitors with DAAs not only helps prevent the development of reduced drug susceptibility but may also synergistically compensate for diminished DAA sensitivity, a phenotype that was not found for combined DAA (NTV + RDV) treatments. These results are indicative of the potency and benefit of a combination treatment with DAAs and HTAs, which may likely be the basis for safe and long-lasting novel antiviral therapeutic approaches.