WO2024042050A1 - Use of mek1/2 inhibitors to synergistically potentiate the antiviral effect of direct-acting anti-coronavirus drugs - Google Patents

Abstract

The present invention relates to a MEK inhibitor or a pharmaceutically acceptable salt, a metabolite or derivate thereof for use in a method for the treatment and/or prophylaxis of a coronavirus associated disease in a subject, wherein the MEK inhibitor is administered in combination with one or more direct acting antiviral (DAA), and to a pharmaceutical composition comprising a MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof and a DAA selected from the group consisting of a nucleoside analogue, nucleoside reverse transcriptase inhibitor, RNA polymerase inhibitor, polymerase acidic endonuclease inhibitor, and a protease inhibitor for use as a medicament, as well as a kits-of-parts of a MEK inhibitor or a pharmaceutically acceptable salt, a metabolite or derivate thereof and one or more DAA.

Description

USE OF MEK1/2 INHIBITORS SYNERGISTICALLY POTENTIATES THE ANTIVIRAL

EFFECT OF DIRECT-ACTING ANTI-CORONAVIRUS DRUGS

FIELD OF THE INVENTION

The present invention relates to the use of MEK1/2 inhibitors in combination with a direct acting antiviral for the treatment and/or prophylaxis of a coronavirus associated disease in a subject.

BACKGROUND

Since its emergence in 2019, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has rapidly spread all over the world, causing millions of cases of the coronavirus disease 2019 (COVID- 19) [1 Chams, N.; Chams, S.; Badran, R.; Shams, A.; Araji, A.; Raad, M.; Mukhopadhyay, S.; Stroberg, E.; Duval, E.J.; Barton, L.M.; et al. COVID-19: A Multidisciplinary Review. Front. Public Heal. 2020, 8, 1-20, doi:10.3389/fpubh.2020.00383.]. While in many cases infections are asymptomatic or manifest through mild upper respiratory symptoms, there is a significant number of cases exhibiting symptomatic pneumonia or severe acute respiratory distress syndrome (ARDS), requiring hospitalization and even intensive medical care [2 Stasi, C.; Fallani, S.; Voller, F.; Silvestri, C. Treatment for COVID-19: An Overview. Eur. J. Pharmacol. 2020.]. The ongoing pandemic not only poses a major health burden to humans worldwide but in addition strains national health systems on a global scale. The worldwide vaccination campaign improves the protection against COVID-19 for the vaccinated individuals and is a very effective prophylactic anti-SARS-CoV-2 measure [3 Chen, Y.T. The Effect of Vaccination Rates on the Infection of Covid-19 under the Vaccination Rate below the Herd Immunity Threshold. Int. J. Environ. Res. Public Health 2021, 18, doi:10.3390/ijerphl8147491.]. Nevertheless, vaccination efficiency is limited by the availability of vaccine doses per country, the willingness of the population to get vaccinated and possible emerging escape mutations [3-5 Chen, Y.T. The Effect of Vaccination Rates on the Infection of Covid-19 under the Vaccination Rate below the Herd Immunity Threshold. Int. J. Environ. Res. Public Health 2021, 18, doi:10.3390/ijerphl8147491. - Wall, E.C.; Wu, M.; Harvey, R.; Kelly, G.; Warchai, S.; Sawyer, C.; Daniels, R.; Hobson, P.; Hatipoglu, E.; Ngai, Y.; et al. Neutralising Antibody Activity against SARS-CoV- 2 VOCs B.l.617.2 and B.1.351 by BNT162b2 Vaccination. Lancet 2021, 397, 2331-2333, doi:10.1016/S0140-6736(21)01290-3. - Kustin, T.; Harel, N.; Finkel, U.; Perchik, S.; Harari, S.; Tahor, M.; Caspi, I.; Levy, R.; Leshchinsky, M.; Ken Dror, S.; et al. Evidence for Increased Breakthrough Rates of SARS-CoV-2 Variants of Concern in BNT162b2-MRNA-Vaccinated Individuals. Nat. Med. 2021, 27, 1379-1384, doi:10.1038/s41591-021-01413-7.]. Besides immunization as a prophylactic countermeasure, antiviral and immunomodulatory agents are of great therapeutic importance, especially for the treatment of patients suffering from severe COVID-19 [6 Fierabracci, A.; Arena, A.; Rossi, P. COVID-19: A Review on Diagnosis, Treatment, and Prophylaxis. Int. J. Mol. Sci. 2020, 21, 1- 16, doi:10.3390/ijms21145145.]. Antiviral drugs can impede the viral spread within the patient, while immunomodulatory agents alleviate symptoms of severe COVID-19 [2 Stasi, C.; Fallani, S.; Voller, F.; Silvestri, C. Treatment for COVID-19: An Overview. Eur. J. Pharmacol. 2020.]. Because the development of novel antiviral drugs is time consuming, already clinically licensed drugs are tested for their efficacy against COVID-19. Those substances can either target viral components or cellular mechanisms that are exploited by the pathogen during a viral infection. Direct acting antivirals (DAA) can be very efficient but have to be applied very early on during infection. Furthermore, DAA treatment bares the risk of provoking resistance introducing mutations, e.g. such as the rapid emergence of resistant influenza viruses against the M2 ion channel inhibitor Amantadine [7 Ludwig, S.; Wolff, T.; Ehrhardt, C.; Wurzer, W.J.; Reinhardt, J.; Planz, O.; Pleschka, S. MEK Inhibition Impairs Influenza B Virus Propagation without Emergence of Resistant Variants. FEBS Lett. 2004, 561, 37-43, doi:10.1016/50014-5793(04)00108-5.]. In comparison, host targeted antivirals (HTA) are less prone to force mutations but might exhibit a lower inhibitory effect on the virus. During the ongoing pandemic, several new virus variants emerged. Hence, drugs possessing antiviral properties against a broad spectrum of SARS-CoV-2 variants would be favorable, as they might also have potent inhibitory effects against new and upcoming variants.

In view of this potential beneficial but also negative drawbacks of the current anti-viral drugs and in view of the demand to provide anti-viral drugs against a broad spectrum of coronavirus variants, there is a need for new compounds and compositions for use in the treatment and/or prophylaxis of a coronavirus associated disease.

SUMMARY OF THE INVENTION

The solution to the above problem provided herein is the provision of a MEK inhibitor or a pharmaceutically acceptable salt, a metabolite or derivate thereof for use in a method for the treatment and/or prophylaxis of a coronavirus associated disease in a subject, wherein the MEK inhibitor is administered in combination with one or more direct acting antivirals (DAA), wherein the DAA is selected from the group consisting of a nucleoside analogue, nucleoside reverse transcriptase inhibitor, RNA polymerase inhibitor, and a protease inhibitor.

Surprisingly, it was found that the use of a MEK inhibitor in combination with a DAA provides a synergistic antiviral effect. In fact, the effects of the combinatorial use of the MEK inhibitor and the DAA are far above the sum of the monotherapies, which clearly indicates a synergistic mode of action. Said synergistic antiviral effect is unexpected, since the combination of the DAA Remdesivir with other DAAs can act also antagonistically [43, 44 Sun, W.; He, S.; Martinez-Romero, C.; Kouznetsova, J.; Tawa, G.; Xu, M.; Shinn, P.; Fisher, E.; Long, Y.; Motabar, O.; et al. Synergistic Drug Combination Effectively Blocks Ebola Virus Infection. Physiol. Behav. 2016, 176, 139-148, doi: 10.1016/j. antiviral.2016.11.017. Synergistic. - Bafna, K.; White, K.; Harish, B.; Rosales, R.; Ramelot, T.A.; Acton, T.B.; Moreno, E.; Kehrer, T.; Miorin, L; Royer, C.A.; et al. Hepatitis C Virus Drugs That Inhibit SARS-CoV-2 Papain-like Protease Synergize with Remdesivir to Suppress Viral Replication in Cell Culture. Cell Rep. 2021, 35, doi:10.1016/j. celrep.2021.109133.]. Further, said synergistic antiviral effect is also unexpected, since a 50 % reduction in the ERK1/2 activation achieved with the MEK inhibitor PD-0184264, which alone does not show an anti-viral effect [11 Schreiber, A.; Viemann, D.; Schoning, J.; Schloer, S.; Mecate Zambrano, A.; Brunotte, L.; Faist, A.; Schofbanker, M.; Hrincius, E.; Hoffmann, H.; et al. The MEKl/2-lnhibitor ATR-002 Efficiently Blocks SARS-CoV-2 Propagation and Alleviates pro-inflammatory Cytokine/Chemokine Responses. Cell. Mol. Life Sci. 2022, 79, doi:10.1007/s00018-021-04085-l.] is sufficient to inhibit viral replication when combined with a DAA.

Therefore, a first subject-matter of the present invention is a MEK inhibitor or a pharmaceutically acceptable salt, a metabolite or derivate thereof for use in a method for the treatment and/or prophylaxis of a coronavirus associated disease in a subject, wherein the MEK inhibitor is administered in combination with one or more direct acting antiviral (DAA), wherein the DAA is selected from the group consisting of a nucleoside analogue, nucleoside reverse transcriptase inhibitor, RNA polymerase inhibitor, and a protease inhibitor.

A second subject-matter of the present invention is a pharmaceutical composition comprising a MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof and a DAA selected from the group consisting of a nucleoside analogue, nucleoside reverse transcriptase inhibitor, RNA polymerase inhibitor, and a protease inhibitor.

A third subject-matter of the present invention is a pharmaceutical composition comprising a MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof and a DAA selected from the group consisting of a nucleoside analogue, nucleoside reverse transcriptase inhibitor, RNA polymerase inhibitor, and a protease inhibitor for use as a medicament. A fourth subject-matter of the invention is a kit-of-parts of a MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof with one or more DAA, wherein the MEK inhibitor is selected from the group consisting of PD-0184264, CI-1040, GSK-1120212, GDC-0973, Binimetinib, Selumetinib, PLX-4032, AZD6244, AZD8330, AS-703026, RDEA-119, RO-5126766, RO-4987655, PD- 0325901, TAK-733, AS703026, PD98059 and PD184352, or a metabolite or derivate thereof, and wherein the DAA is selected from the group consisting of a nucleoside analogue, nucleoside reverse transcriptase inhibitor, RNA polymerase inhibitor, and a protease inhibitor, wherein the nucleoside analogue is selected from the group consisting of Molnupiravir and its prodrugs, Ribavirin, GS-441524 monophosphate, GS-441524 triphosphate, beta-D-N4-hydroxycytidine (NHC, EIDD-1931, EID 2801 prodrug) and Sofosbuvir; wherein the nucleoside reverse transcriptase inhibitor is selected from the group consisting of Azvudine (RO-0622), Emtricitabine, and Tenofovir; wherein the RNA polymerase inhibitor is selected from Remdesivir (GS-5734), Favipiravir, and Galidesivir (BCX4430); and wherein the protease inhibitor is selected from the group consisting of Nirmatrelvir, Ritonavir, the combination of Nirmatrelvir and Ritonavir, Darunavir, Danoprevir, Lopinavir, Nafamostat, and TMC- 310911 (ASC-09).

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: shows diagrams depicting the inhibitory effect of ATR-002, MPV, RDV, NTV and RTV on the SARS-CoV-2 replication.

Figure 2: shows diagrams depicting the anti-viral effect of the combinational treatments in Calu-3 cells.

Figure 3: shows landscape visualization depicting synergistic evaluation of ATR-002 and with DAAs in Calu-3 cells.

Figure 4: shows diagrams depicting synergistic effects of MPV, RDV, NTV and RTV in combination with ATR-002 for different SARS-CoV-2 VOCs in Calu-3 cells.

Figure 5: shows diagrams depicting the determination of the cell viability after single and combinational drug treatment.

Figure 6: shows a diagram depicting drug related inhibitory effect on the SARS-CoV-2 replication. This is related to Figure 1. Figure 7: shows diagrams depicting the determination of the ATR-002 + MPV synergism. This is related to Figure 3.

Figure 8: shows diagrams depicting the determination of the ATR-002 + RDV synergism. This is related to Figure 3.

Figure 9: shows diagrams depicting the determination of the ATR-002 + NTV synergism. This is related to Figure 3.

Figure 10: shows diagrams depicting the determination of the ATR-002 + RTV synergism. This is related to Figure 3.

Figure 11: shows diagrams depicting synergistic but also antagonistic effects of certain combinations of RDV + NTV. This is related to Figure 3.

Figure 12: shows diagrams depicting the synergistic effects of different drug combinations on the replication of SARS-CoV-2 VOCs. This is related to Figure 4.

Figure 13: shows a table with the highest synergy score values, calculated for the drug combinations. MPV 0.047 pM / ATR-002 12.05 pM, RDV 0.034 pM / ATR-002 12.05 pM, NTV 0.006 pM / ATR-002 12.05 pM, RTV 0.057 pM / ATR-002 12.05 pM and RDV / NTV (Bliss, Loewe, ZIP: RDV 0.034 pM + NTV 0.095 pM; HSA: RDV 0.199 pM + NTV 0.095 pM). This is related to Figure 3-7 j.

Figure 14: shows a table with the drug combination sensitivity score (CSS).

Figure 15: shows an exemplary print out of the web page https://cov-lineages.org/lineage_list.html, which provides an overview of the current SARS-CoV-2 variants.

DETAILED DESCRIPTION

The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

The present invention relates to a MEK inhibitor or a pharmaceutically acceptable salt, a metabolite or derivate thereof for use in a method for the treatment and/or prophylaxis of a coronavirus associated disease in a subject, wherein the MEK inhibitor is administered in combination with one or more direct acting antiviral (DAA), wherein the DAA is selected from the group consisting of a nucleoside analogue, nucleoside reverse transcriptase inhibitor, RNA polymerase inhibitor, and a protease inhibitor.

The inventors could show, that surprisingly the combination of a MEK inhibitor and one or more DAA results in an elevated antiviral effect. Said antiviral effect is much higher compared to the addition of the single anti-viral effects of a MEK inhibitor and a DAA alone. The synergistic antiviral effect is unexpected since there is no hint to such a synergistic effect in the prior art. Further, it has to be noted that a synergistic effect cannot be achieved with the combination of two arbitrarily selected DAA. This is shown in detail in the examples below. In fact, distinct combination of two DAA even lead to a virus enhancing effect instead of an antiviral effect. Therefore, the synergistic effect of a MEK inhibitor with a DAA in the prophylaxis and treatment of a coronavirus associated disease is unexpected and surprising.

In a preferred embodiment of the invention, the MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof is selected from the group consisting of PD-0184264, CI-1040, GSK- 1120212, GDC-0973, Binimetinib, Selumetinib, PLX-4032, AZD6244, AZD8330, AS-703026, RDEA-119, RO-5126766, RO-4987655, PD-0325901, TAK-733, AS703026, PD98059 and PD184352, or a metabolite or derivate thereof.

"MEK inhibitors" inhibit the mitogenic signaling cascade Raf/MEK/ERK in cells or in a subject by inhibiting the MEK (mitogen-activated protein kinase kinase). This signaling cascade is hijacked by many viruses, in particular influenza viruses, to boost viral replication. Specific blockade of the Raf/MEK/ERK pathway at the bottleneck MEK therefore impairs growth of viruses, in particular influenza viruses. Additionally, MEK inhibitors show low toxicity and little adverse side effects in humans. There is also no tendency to induce viral resistance (Ludwig, 2009).

In a particular preferred embodiment, the MEK inhibitor is PD-0184264 or a metabolite or derivate thereof. PD-0184264 is one of several metabolites of CI-1040 (Wabnitz et al., 2004, LoRusso et al., 2005). PD- 0184264 - structure 1 below - has been shown to possess antiviral effects.

1

PD-0184264 is also known as zapnometinib and is also designated as ATR-002. PD-0184264 is an active metabolite of CI-1040.

In accordance with the present invention, the term "metabolite" has to be understood as any naturally occurring metabolic product which may arise.

In accordance with the present invention, the term "derivate" has to be understood as any compound which is derived from the core compound by any reaction, such as a chemical or a biological reaction. In accordance with the present invention, a derivate may encompass any functional active derivate. A person skilled in the art is able to identify such a functional active derivate for example by testing any candidate derivate in a suitable assay.

In accordance with the present invention, the DAA is preferably selected from the group consisting of a nucleoside analogue, nucleoside reverse transcriptase inhibitor, RNA polymerase inhibitor, polymerase acidic endonuclease inhibitor, and a protease inhibitor.

Direct acting antivirals (DAA) are of particular interest for the treatment of coronavirus and in particular corona virus associated diseases. The review of Artese et al., 2020 (Artese et al., 2020, Drug Resistance Updates 53 (2020) 1000721) provides an overview of DAA and also discloses the distinct inhibitor classes nucleoside analogue, nucleoside reverse transcriptase inhibitor, RNA polymerase inhibitor, and a protease inhibitor. The direct acting antivirals, DAA, target viral components or viral enzymatic activities that are important for viral propagation of the coronavirus during the viral infection cycle.

The term "DAA", "direct acting antiviral" are substances with antiviral effect which are generally known to a person skilled in the art. Several types of DAA are disclosed and established in the prior art, for example in the above-identified review article of Artese et al. DAA are in particular nucleoside analogue, nucleoside reverse transcriptase inhibitor, RNA polymerase inhibitor, and a protease inhibitor. Said types of DAA are defined by their inhibitory effects on distinct targets and their resulting mechanism of action.

Accordingly, a nucleoside analogue is defined as nucleosides which contain a nucleic acid analogue and a sugar. Nucleoside analogues exert the effect to prevent viral replication in infected cells.

A nucleoside reverse transcriptase inhibitor is able to inhibit activity of reverse transcriptase, a viral DNA polymerase that is required for replication of retroviruses. The nucleoside reverse transcriptase inhibitor blocks the enzymatic function of the reverse transcriptase and prevents completion of synthesis of the double-stranded viral DNA, thus preventing the retrovirus from multiplying.

The group of RNA polymerase inhibitor, such as Remdesivir, inhibits the action of RNA polymerase by incorporating into RNA, which exerts that additional nucleotides cannot be added and RNA transcription is terminated.

Protease inhibitors are a well-known class of molecules that inhibit the function of proteases, which are enzymes that degrade proteins.

A person skilled in the art is familiar with the possibility to determine suitable compounds which exert the desired effect of a DAA, such as a nucleoside analogue, a nucleoside reverse transcriptase inhibitor, an RNA polymerase inhibitor or a protease inhibitor. Thus, suitable assays are available for determining the desired effect of the distinct DAA.

It could be in particular shown that the MEK inhibitor PD-0184264, also known as zapnometinib or ATR-002, exerts a strong synergistic effect with different DAAs. In particular DAAs selected from the group of nucleoside analogues, RNA polymerase inhibitors and protease inhibitors provide in combination with PD-0184264 an elevated overall combinatorial effect that by far exceeds the sum of the respective single use of the MEK inhibitor and the DAA alone.

In a further preferred embodiment of the invention, is the nucleoside analogue selected from the group consisting of Molnupiravir and its prodrugs, Ribavirin, GS-441524 monophosphate, GS-441524 triphosphate, beta-D-N4-hydroxycytidine (NHC, EIDD-1931, EID 2801 prodrug) and Sofosbuvir.

According to the present invention, the nucleoside reverse transcriptase inhibitor is preferably selected from the group consisting of Azvudine (RO-0622), Emtricitabine, and Tenofovir.

In a further preferred embodiment of the invention, the RNA polymerase inhibitor is selected from Remdesivir (GS-5734), Favipiravir, and Galidesivir (BCX4430).

In accordance with the present invention, the protease inhibitor is preferably selected from the group consisting of Nirmatrelvir, Ritonavir, the combination of Nirmatrelvir and Ritonavir, Darunavir, Danoprevir, Lopinavir, Nafamostat, and TMC-310911 (ASC-09).

Strong synergistic effects of the combination of the MEK inhibitor PD-0184264 are shown with distinct groups of DAAs. Example 2 and Figures 2 and 3 as well as Figures 7, 8, 9, and 10 demonstrate the synergistic combinatorial effect of PD-0184264 with Molnupiravir, a nucleoside analogue, with Remdesivir, an RNA polymerase inhibitor, and with the protease inhibitors Nirmatrelvir and Ritonavir. Accordingly, the combination of a MEK inhibitor with a DAA allows a synergistic effect. However, said synergistic effect is not an effect which can be achieved automatically. In fact, it could be shown that the combination of Remdesivir with distinct lower concentrations of Nirmatrelvir does not provide any synergistic effect at all. In contrast the combination of the lower concentrated Nirmatrelvir provided even an antagonistic effect as shown in Figures 2e, 3e and 11 and Table in Figure 14.

In a further preferred embodiment of the invention, the MEK inhibitor is PD-0184264 and the nucleoside analog is Molnupiravir and the RNA polymerase inhibitor is Remdesivir (GS-5734).

Thus, in a preferred embodiment of the invention, the MEK inhibitor is PD-0184264 and the nucleoside analog is Molnupiravir. Accordingly, in a preferred embodiment of the invention, the MEK inhibitor is PD-0184264 and the RNA polymerase inhibitor is Remdesivir (GS-5734).

Remdesivir is a prodrug that is intended to allow intracellular delivery of GS-441524 monophosphate and subsequent biotransformation into GS-441524 triphosphate, a ribonucleotide analogue inhibitor of viral RNA polymerase. Remdesivir has the Chemical structure below:

Remdesivir, sold under the brand name Veklury, is a broad-spectrum antiviral medication developed by the biopharmaceutical company Gilead Sciences. It is administered via injection into a vein. During the COVID-19 pandemic, Remdesivir was approved or authorized for emergency use to treat COVID-19 in around 50 countries. Updated guidelines from the World Health Organization in November 2020 include a conditional recommendation against the use of remdesivir for the treatment of COVID-19. Remdesivir was originally developed to treat hepatitis C, and was subsequently investigated for Ebola virus disease and Marburg virus infections before being studied as a post-infection treatment for COVID-19.

In accordance with the present invention, the MEK inhibitor is preferably PD-0184264 and the protease inhibitor is preferably Nirmatrelvir.

The combination of PD-0184264 has been shown to possess an advantageous synergistic effect. This is shown in particular in Example 2 and Figures 2 and 3 as well as Figures 7, 8, 9, and 10.

In a preferred embodiment of the invention, the MEK inhibitor is PD-0184264 and the protease inhibitor is Ritonavir.

In a preferred embodiment of the invention, the coronavirus associated disease is a disease comprising respiratory insufficiency, fever or chills, cough, shortness of breath or difficulty breathing, Fatigue, muscle or body aches, headache, new loss of taste or smell, sore throat, Congestion or runny nose, nausea or vomiting, diarrhea, cytokine storm, hyperinflammatory processes, or the coronavirus disease 2019 (COVID-19), wherein COVID-19 is selected from the group consisting of Stage I COVID- 19, Stage II COVID-19, Stage III COVID-19, and a COVID-19 cytokine storm, such as a hyperinflammatory cytokine storm associated with COVID-19.

In accordance with the present invention, the term "coronavirus associated disease" has to be understood as any symptom or disorder which arises directly or indirectly in a subject infected or exposed to a coronavirus. Distinct examples of such symptoms of a coronavirus associated disease are disclosed for example by the Centers for Disease Control and Prevention (CDC) (https://www.cdc.gov/coronavirus/2019-ncov/symptoms-testing/symptoms.html).

The stages of COVID-19 have been summarized by Hasan et al., in a paper entitled "COVID-19 Illness in Native and Immunosuppressed States: A Clinical-Therapeutic Staging Proposal" ((2020). J Heart Lung Transplant. 2020 Mar 20) summarized below.

The initial stage, termed Stage I, is a mild infection and occurs at the time of inoculation and early establishment of disease. For most people, this involves an incubation period associated with mild and often non-specific symptoms for some days such as malaise, fever, and a dry cough. In patients who can keep the virus limited to this stage of COVID-19, prognosis and recovery is excellent. Treatment at this stage is primarily targeted towards symptomatic relief. Should an antiviral therapy be proven beneficial, targeting selected patients during this stage may reduce duration of symptoms, minimize contagiousness, and prevent progression of severity.

In the second stage, termed Stage II, of an established pulmonary disease, viral multiplication and localized inflammation in the lung is the norm. Stage II includes pulmonary involvement, termed Stage Ila, without and Stage lib with hypoxia. During this stage, patients develop a viral pneumonia, with cough, fever and possibly hypoxia. Over the course of the disease, dyspnea occurs after a median of 13 days after the first onset of symptoms (range 9-16.5 days). Dyspnea is a sign of serious disease of the airway, lungs, or heart and is characterized by difficult or labored breathing and shortness of breath. In the case of COVID-19, imaging with chest X-ray or computerized tomography reveals bilateral infiltrates or ground glass opacities. It is at this stage that most patients with COVID- 19 need to be hospitalized for close observation and management. Treatment primarily consists of supportive measures and antiviral therapies, once available. It is possible that patients will nevertheless progress to Stage III to require mechanical ventilation in an intensive care unit (ICU). In Stage II COVID-19, markers of systemic inflammation may be elevated, but not remarkably so. In early studies performed on the first group of patients in Wuhan, China, it was found that upon entry into the hospital, plasma IL10, IL1RA, IL7, IL8, IL9, IL10, basic FGF, GCSF, GMCSF, IFNy, IP10, MCP1, MIPla, MIP10, PDGF, TNF-a, and VEGF concentrations were higher than in healthy adults. In addition, it was found that patients in ICU had plasma concentrations of IL2, IL7, IL10, GCSF, IP10, MCP1, MIPla, and TNF-a that were higher than in patients upon admission to the hospital (Huang et al.; The Lancet; Vol 395; pp.: 497-506 February 15, 2020), indicating that an increase in these cytokines marks the transition from COVID-19 stage II to stage III. This transition marked by sudden and rapidly progressing clinical deterioration that is called the "COVID-19 cytokine storm" (also termed "cytokine storm" herein). As described in detail in Jamilloux et al. (Autoimmunity Reviews 2020), here markers of systemic inflammation are elevated, such as IL-13, IL-Ra, IL-6, TNF-a and slL2Ra. This corresponds to what was shown by Huang et al. discussed above.

A minority of COVID-19 patients will experience a COVID-19 cytokine storm and transition into the third and most severe stage of illness, termed Stage III, which manifests as an extra-pulmonary systemic hyperinflammation syndrome. Overall, the prognosis and recovery from this critical stage of illness is poor.

The term "cytokine storm" is used within its regular meaning as used in the art (see, in this respect, Jamilloux et al, 2020) to mean a cytokine storm that may occur in subjects that have been infected with a human-pathogenic coronavirus, in particular SARS-CoV-2. Such a cytokine storm is marked by rapid clinical deterioration and an increase in pro-inflammatory cytokines marks the transition from Stage II to Stage III COVID-19. Specifically, both Huang et al. and Jamilloux et al. note that a sudden increase in IL-10 and/or TNF-a was observed, possibly together with an increase in two or more, three or more, four or more, five or more or all six of TNF-a, IL-1R, IP-10, IL-8, MCP-1, and MIP-10. In one aspect, the MEK inhibitor is used to reduce the level of IL-10 and/or TNF-a in the subject, preferably reducing the level of one or more, two or more, three or more, four or more, five or more or all six of TNF-a, IL-1R, IP-10, IL-6, IL-8, MCP-1, MIP-la and MIP-10 in a subject.

The inflammatory cytokines and chemokines cited above are well known in the art. Specifically, the terms TNF-a, IL-1R, IP-10, IL-8, MCP-1, and MIP-10 refer to the human protein sequences known in UNIPROT and GENEBANK under the following accession numbers:

Gene GeneBank accession Uniprot accession species

TNF-a NM

^_ 000594 P01375 Human

IL-13 NM_000576 P01584 IP-10 NM_001565 P02778

IL-8 NM_000584 P10145

MCP-1 NM_002982 P13500

MIP-1P NM_002984 P13236

The direct acting antivirals, DAA, target viral components or viral enzymatic activities that are important for viral propagation of the coronavirus during the viral infection cycle. Thus, the use of DAA provides an antiviral response in particular at an early phase of the viral infection. The MEK inhibitor, in particular PD-0184264, possess a dual effect on the virus infection and viral disease. On the one hand side, the MEK inhibitor is able to interfere with the replication process of the coronavirus and thereby leads to the reduction of coronavirus titers. On the other hand side, the MEK inhibitor is able to downmodulate virus-induced expression of cytokines and chemokines that are causative for the hyperinflammatory cytokine storm, in particular hyperinflammatory cytokine storms at late stages of the hyperinflammatory viral diseases, one example of a coronavirus associated disease according to the present invention. Such hyperinflammatory viral diseases can for example arise in the course of COVID-19. Thus, the combination of a MEK inhibitor and one or more DAA according to the present invention is useful for to provide an effective treatment and/or prevention of early infection symptoms as well as later stages of coronavirus associated disease, such as an hyperinflammatory cytokine storm. In view of the effects of DAA and the MEK inhibitor, in particular the MEK inhibitor PD-0184264, the whole course of a coronavirus associated disease in a subject can be effectively treated and/or prevented.

In a preferred embodiment of the invention, COVID-19 is caused by a SARS-CoV-2 variant, preferably selected from the group consisting of D614G, B.l.1.7, B.1.351, Pl, P2, B.1.617, B.1.427, B.1.429, B.1.525, B.1.526, and Bl.617.2. An overview of the current SARS-CoV-2 variants is disclosed for example on the web page https://cov-lineages.org/lineage_list.html. An exemplary print out of said web page is disclosed in Figure 15.

During the ongoing pandemic, several new virus variants emerged. Hence, drugs possessing antiviral properties against a broad spectrum of SARS-CoV-2 variants are favorable, as they also have potent inhibitory effects against new and upcoming variants. The inventors already could show, that ATR- 002 effectively inhibits SARS-CoV-2 variants of concern including a-Bl.1.7, 0-B1.351 and 6-B1.617.2 [11 Schreiber, A.; Viemann, D.; Schoning, J.; Schloer, S.; Mecate Zambrano, A.; Brunotte, L; Faist, A.; Schofbanker, M.; Hrincius, E.; Hoffmann, H.; et al. The MEKl/2-lnhibitor ATR-002 Efficiently Blocks SARS-CoV-2 Propagation and Alleviates pro-inflammatory Cytokine/Chemokine Responses. Cell. Mol. Life Sci. 2022, 79, doi:10.1007/s00018-021-04085-l.]. In line with these results, the inhibitory effect of the drug combinations used in this present invention on VOCs surpassed the sum of the mono effects, comparable to the effects shown for the D614G variant, indicating a variant-independent synergistic impact on SARS-CoV-2 viruses. Therefore, MEK inhibitors, in particular ATR-002, are a beneficial supplement to boost the effect of DAA therapies. An advantage of a HTA is the reduced possibility of the emergence of resistance introducing mutations, because the virus cannot overcome the absence of the cellular mechanism.

It could be shown that the combination of a MEK inhibitor and one or more DAA according to the present invention is effective against several coronavirus variants of concern - VOC. This is shown in particular in Example 3 and Figures 4 and 12. A treatment with DAA alone may have the potential drawback concerning the possibility of provoking resistance introducing mutations into the coronavirus. In view of this, the combination of the DAA with a MEK inhibitor with strong synergistic effects possess the advantage to minimize the pressure forced escape mutations and maximize the inhibitory potency.

Thus, it is foreseen in accordance with the present invention that the MEK inhibitor in combination with one or more DAA for use in a method for the treatment and prophylaxis of a coronavirus associated disease is useful against the existing SARS-CoV-2 variants and any SARS-CoV2 variants which will develop in future.

In a preferred embodiment of the present invention, the MEK inhibitor or a pharmaceutically acceptable salt, metabolite or derivate thereof is administered contemporaneously, previously or subsequently to the DAA. Preferably, dependent on the coronavirus associated disease or the severity of the coronavirus associated disease, the DAA may be administered to a subject in need of a treatment prior to administration of a MEK inhibitor. In the case of a hyperinflammatory cytokine storm, a subject in need thereof may receive a MEK inhibitor subsequently to the administration of one or more DAA. This may be the case, if the subject requires intensive care treatment in a hospital.

In accordance with the present invention, the MEK inhibitor may be administered orally, intravenously, intrapleurally, intramuscularly, topically or via inhalation. Preferably, PD-0184264 is administered via inhalation, topically or orally.

Similarly, the DAA is administered in accordance with the present invention orally, intravenously, intrapleurally, intramuscularly, topically or via inhalation. The person skilled in the art is able to apply the suitable mode of administration for the MEK inhibitor and the DAA on the basis of common knowledge.

The "subject", which is treated by the MEK inhibitors in combination with the DAA according to the present invention, is preferably a vertebrate. In the context of the present invention the term "subject" includes an individual in need of a treatment of a coronavirus associated disease as described herein. Preferably, the subject is a patient suffering from a coronavirus associated disease or being at a risk thereof. Preferably, the patient is a vertebrate, more preferably a mammal. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice and rats. Preferably, a mammal is a human, horse, dog, cat, cow, pig, mouse, rat etc., particularly preferred, it is a human. In some embodiments, the subject is a human subject, which optionally is more than 1 year old and less than 14 years old, between the ages of 50 and 65, between the ages of 18 or 50, or older than 65 years of age. In other embodiments the subject is a human subject, which is selected from the group consisting of subjects who are at least 50 years old, subjects who reside in chronic care facilities, subjects who have chronic disorders of the pulmonary or cardiovascular system, subjects who required regular medical follow-up or hospitalization during the preceding year because of chronic metabolic diseases, renal dysfunction, hemoglobinopathies, or immunosuppression, subjects with less than 14 years of age, subjects between 6 months and 18 years of age who are receiving long-term aspirin therapy, and women who will be in the second or third trimester of pregnancy during the influenza season.

A further aspect of the invention is directed to a pharmaceutical composition comprising a MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof and one or more DAA selected from the group consisting of a nucleoside analogue, nucleoside reverse transcriptase inhibitor, RNA polymerase inhibitor, and a protease inhibitor.

Another aspect of the invention is directed to a pharmaceutical composition comprising a MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof and one or more DAA selected from the group consisting of a nucleoside analogue, nucleoside reverse transcriptase inhibitor, RNA polymerase inhibitor, and a protease inhibitor for use as a medicament.

The present invention also envisages different compositions, preferably pharmaceutical compositions. Preferably, such compositions further comprise a carrier, preferably a pharmaceutically acceptable carrier. The composition can be in the form of orally administrable suspensions or tablets, nasal sprays, preparations for inhalation devices, sterile injectable preparations - intravenously, intrapleurally, intramuscularly-, for example, as sterile injectable aqueous or oleaginous suspensions or suppositories.

Preferably, the pharmaceutical composition according to the present invention is administered to a patient which is a mammal or bird. Examples of suitable mammals include, but are not limited to, a mouse, a rat, a cow, a goat, a sheep, a pig, a dog, a cat, a horse, a guinea pig, a canine, a hamster, a mink, a seal, a whale, a camel, a chimpanzee, a rhesus monkey and a human, with a human being preferred. Examples of suitable birds include, but are not limited to, a turkey, a chicken, a goose, a duck, a teal, a mallard, a starling, a Northern pintail, a gull, a swan, Guinea fowl or water fowl to name a few. Human patients are a particular embodiment of the present invention.

The inhibitor or inhibitors are preferably administered in a therapeutically effective amount. The "therapeutically effective amount" for the MEK inhibitor and the DAA or each active compound/inhibitor envisaged according to the present invention can vary with factors including but not limited to the activity of the compound used, stability of the active compound in the patient's body, the severity of the conditions to be alleviated, the total weight of the patient treated, the route of administration, the ease of absorption, distribution, and excretion of the compound by the body, the age and sensitivity of the patient to be treated, adverse events, and the like, as will be apparent to a skilled artisan. The amount of administration can be adjusted as the various factors change over time.

In a preferred embodiment of the invention, the MEK inhibitor, preferably PD-0184264, is administered to a human subject once daily in a dose of 50 to 1000 mg, preferably 150 to 900 mg, most preferably 150, 300, 600 or 900 mg.

Preferably, the DAA is administered in a dose which is usually known to be feasible and effective for a person skilled in the art.

In particular any dose may be envisaged which is has been approved to be feasible, in particular in the context of clinical studies.

In view of the combined synergistic effect of the MEK inhibitor and the one or more DAA, lower doses may be applied for administration. A skilled person is able to adapt the dose of the MEK inhibitor and the one or more DAA accordingly. The inhibitors, methods and uses described herein are applicable to both human therapy and veterinary applications. The compounds described herein, in particular, the MEK inhibitor and the DAA, may be administered in a physiologically acceptable carrier to a subject, as known in the art. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways. A skilled person is able to use a suitable formulation and administration route on the basis of common knowledge.

In a preferred embodiment of the invention, it is foreseen that in the pharmaceutical composition the MEK inhibitor is selected from the group consisting of PD-0184264, CI-1040, GSK-1120212, GDC- 0973, Binimetinib, Selumetinib, PLX-4032, AZD6244, AZD8330, AS-703026, RDEA-119, RO-5126766, RO-4987655, PD-0325901, TAK-733, AS703026, PD98059 and PD184352, or a metabolite or derivate thereof.

Preferably, in the pharmaceutical composition according to the present invention, the nucleoside analogue is selected from the group consisting of Molnupiravir and its prodrugs, Ribavirin, GS-441524 monophosphate, GS-441524 triphosphate, beta-D-N4-hydroxycytidine (NHC, EIDD-1931, EID 2801 prodrug) and Sofosbuvir.

Further preferred, in the pharmaceutical composition according to the present invention the nucleoside reverse transcriptase inhibitor is selected from the group consisting of Azvudine (RO- 0622), Emtricitabine, and Tenofovir.

Preferably, in the pharmaceutical composition according to the present invention, the RNA polymerase inhibitor is selected from Remdesivir (GS-5734), Favipiravir, and Galidesivir (BCX4430).

Further preferred, in the pharmaceutical composition according to the present invention the protease inhibitor is selected from the group consisting of Nirmatrelvir, Ritonavir, the combination of Nirmatrelvir and Ritonavir, Darunavir, Danoprevir, Lopinavir, Nafamostat, and TMC-310911 (ASC-09).

Preferably, in the pharmaceutical composition according to the present invention is for use in the prophylaxis and/or treatment of a coronavirus associated disease.

As such, the term "treating" or "treatment" includes administration of MEK inhibitor in combination with one or more DAA preferably in the form of a medicament, to a subject suffering from a coronavirus associated disease for the purpose of ameliorating or improving symptoms

Y1 accompanying said coronavirus associated disease. A coronavirus associated disease is defined in detail above.

Furthermore, the term "prophylaxis" as used herein, refers to any medical or public health procedure whose purpose is to prevent a medical condition, in particular a coronavirus associated disease, described herein. As used herein, the terms "prevent", "prevention" and "preventing" refer to the reduction in the risk of acquiring or developing a given condition, namely a coronavirus associated disease, as described herein. Also meant by "prophylaxis" is the reduction or inhibition of the recurrence of a coinfection comprising a coronavirus associated disease in a subject.

Preferably, the pharmaceutical composition according to the present invention is for use of the prophylaxis or treatment of a coronavirus associated disease, wherein the coronavirus associated disease is a disease comprising respiratory insufficiency, fever or chills, cough, shortness of breath or difficulty breathing, Fatigue, muscle or body aches, headache, new loss of taste or smell, sore throat, Congestion or runny nose, nausea or vomiting, diarrhea, cytokine storm, hyperinflammatory processes, or the coronavirus disease 2019 (COVID-19), wherein COVID-19 is selected from the group consisting of Stage I COVID-19, Stage II COVID-19, Stage III COVID-19, and a COVID-19 cytokine storm, such as an hyperinflammatory cytokine storm associated with COVID-19.

In one embodiment, the reduction of the viral infection is a reduction in plaque forming units (PFU)/ml. The "plaque forming units" is a measure of the number of particles capable of forming plaques per unit volume, such as virus particles. It is a functional measurement rather than a measurement of the absolute quantity of particles: viral particles that are defective or which fail to infect their target cell will not produce a plaque and thus will not be counted. For example, a solution of coronavirus with a concentration of 1,000 PFU/pl indicates that 1 pl of the solution carries enough virus particles to produce 1000 infectious plaques in a cell monolayer. In the case of the present invention, a cell culture treated with a MEK inhibitor and one or more DAA shows a reduced number of plaque forming units in a culture after the treatment, when compared to a culture before the treatment with the MEK inhibitor or DAA.

A possible "reduction in plaque forming units (PFU)/ml" is analyzed in the following way. First the cultured cells, which are infected with a coronavirus, are analyzed for their ability to generate plaque forming units (PFU)/ml by e.g. plating them with an immobilizing overlay for counting the viral plaques that will form. This result is then compared to the number of plaque forming units (PFU)/ml generated by cells of the same culture after the inhibitor was applied. If the number of the plaque forming units (PFU)/ml is reduced after the treatment with an inhibitor compared to the number generated before the application of the inhibitor, there is a reduction in the plaque forming units.

In general, the person skilled in the art knows these well-known techniques of analyzing viral infections. How one can measure the plaque forming units (PFU)/ml is further described in the literature (Hrincius et al. 2010 Cell Microbiol. 2010 Jun;12(6):831-43.).

For the purpose of the invention the pharmaceutical compounds of the invention comprising an active compound as defined above also includes the pharmaceutically acceptable salt(s) thereof. The phrase "pharmaceutically or cosmetically acceptable salt(s)", as used herein, means those salts of compounds of the invention that are safe and effective for the desired administration form. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

A further aspect of the invention is directed to a kit-of-parts of a MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof with one or more DAA, wherein the MEK inhibitor is selected from the group consisting of PD-0184264, CI-1040, GSK-1120212, GDC-0973, Binimetinib, Selumetinib, PLX-4032, AZD6244, AZD8330, AS-703026, RDEA-119, RO-5126766, RO-4987655, PD- 0325901, TAK-733, AS703026, PD98059 and PD184352, or a metabolite or derivate thereof, and wherein the DAA is selected from the group consisting of a nucleoside analogue, nucleoside reverse transcriptase inhibitor, RNA polymerase inhibitor and a protease inhibitor, wherein the nucleoside analogue is selected from the group consisting of Molnupiravir and its prodrugs, Ribavirin, GS-441524 monophosphate, GS-441524 triphosphate, beta-D-N4-hydroxycytidine (NHC, EIDD-1931, EID 2801 prodrug) and Sofosbuvir; wherein the nucleoside reverse transcriptase inhibitor is selected from the group consisting of Azvudine (RO-0622), Emtricitabine, and Tenofovir; wherein the RNA polymerase inhibitor is selected from Remdesivir (GS-5734), Favipiravir, and Galidesivir (BCX4430); and wherein the protease inhibitor is selected from the group consisting of Nirmatrelvir, Ritonavir, the combination of Nirmatrelvir and Ritonavir, Darunavir, Danoprevir, Lopinavir, Nafamostat, and TMC- 310911 (ASC-09).

* * * It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or sometimes when used herein with the term "having".

When used herein "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. EXAMPLES

The following examples illustrate the invention. These examples should not be construed as to limit the scope of the invention. The examples are included for purposes of illustration only.

Materials and Methods

Cell lines

The human airway epithelial cells (Calu-3) were cultured in Dulbecco's modified Eagle medium/Nutrient Mixture F12-Ham (DMEM/F12-HAM) (Sigma-Life Science) with 10 % (v/v) fetal bovine serum (FBS) (Capricorn Scientific) and 1 % (v/v) Penicillin/Streptomycin (P/S) (Sigma-Life Science). African green monkey kidney epithelial cells (VeroEG) were cultured in DMEM supplemented with 10 % (v/v) FBS. Vero76 cells stably overexpressing TMPRSS2 (Vero76-TMPRSS2, German Primate Center Goettingen) were cultured in DMEM supplemented with 10 % (v/v) FBS, 1 % (v/v) P/S and 10 pg/ml blasticidin (Roth). Cells were incubated under humidified conditions at 37 °C and 5 % CO₂.

Viruses

All SARS-CoV-2 experiments were performed in a laboratory approved for biosafety level (BSL) 3 work. Viruses were isolated as described previously [11]. To exclude genomic mutations due to cell culture propagation, virus stocks were sequenced before usage. Sequences can be accessed at GISAID.org: D614G: hCoV-19/Germany/Flll03201/2020; Munchen-01: Munchen-1/02/2020/984; a- Bl.1.7: hCoV-19/Germany/NW-RKI-l-0026/2020; 0-B1.351: hCoV-19/Germany/NW-RKI-l-0029/2020; 6-B1.617.2: hCoV-19/Germany/326763/2021

Virus propagation

SARS-CoV-2 viruses were propagated on Vero76-TMPRSS2 cells in DMEM with 2 % (v/v) FBS, 1 % (v/v) P/S, 1 % (v/v) sodium pyruvate solution (Gibco), 1 % (v/v) non-essential amino acid (NEAA) solution (Sigma-Life Science) and 1 % (v/v) HEPES (IM; pH 7.2) (Sigma-Aldrich) using a MOI of 0.01. 72 h post-infection the virus containing supernatants were collected and the virus titers were determined by plaque titration.

Virus infection

Virus dilutions were prepared in cell culture medium. lxlO⁶ Calu-3 cells seeded in 12-wells were washed once with PBS and infected for 1 h at 37°C, followed by a second PBS wash step and the incubation in cell culture medium supplemented with the different inhibitors. DMSO served as control and was used in a final concentration of 0.1 %.

Inhibitors

All inhibitors were dissolved in DMSO (Roth). ATR-002 (zapnometinib, PD 0184264) was obtained from Atriva Therapeutics GmbH. Remdesivir (Veklury") and Ritonavir (Norvir") were purchased from Tocris. Molnupiravir (Lagevrio") and Nirmatrelvir (PF-07321332) were purchased from Selleckchem.

Virus titration by plaque assay

SARS-CoV-2 solutions were diluted 10-fold in PBS supplemented with 0.01 % (w/v) CaCI₂ (Roth), 0.01 % (w/v) MgCI₂ (Roth), 0.6 % (v/v) bovine serum albumin (BSA) (35 %) (Sigma-Aldrich) and 1 % (v/v) P/S. Confluent monolayers of VeroE6 cells were infected for 1 h at 37°C with the dilution series and covered with plaque medium (2 % (v/v) FBS, 35 % (v/v) agar (2 %) (Oxoid) and 63 % (v/v) 2x MEM (1 % (v/v) lOOx Penicillin/Streptomycin/L-Glutamine solution (10,000 U/ml Penicillin; 10,000 pg/ml Streptomycin; 29.2 mg/ml L-Glutamine) (Gibco), 1.2 % (v/v) BSA (35 %), 2 % (v/v) HEPES, 3.2 % (v/v) NaHCO₃ (7.5 %) (Gibco), 20 % (v/v) lOx MEM (Gibco)). Plaque forming units were analyzed after 72 h incubation at 37°C.

Cell cytotoxicity assay

The colorimetric MTT assay was used to evaluate the metabolic activity as indicator of the activation, proliferation and cytotoxicity [24]. Calu-3 cells were incubated with the inhibitors in a mono- or double-treatment using the indicated concentrations for 48h. Staurosporine solution (1 pM) (Sigma) served as a positive control for cytotoxic effects. Following 48 h of treatment, MTT 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (1 mg/ml) (Sigma) was added to the cells for 3 h. The supernatant was aspirated, DMSO was added to the cells for 10 min and the OD₅₆₂ was subsequently measured using an Epoch microplate spectrophotometer (BioTek) to evaluate the cell viability according to the manufacturers protocol (Sigma)

Quantification and statistical analysis

Sample size required to detect 90 % reduction in viral titers at powers > 0.8 and a significance level of 0.05 was determined using G*Power 3.1 (RRID:SCR_013726) and a priori power analysis [25 Beck, T.W. The Importance of a Priori Sample Size Estimation in Strength and Conditioning Research. J. Strength Cond. Res. 2013, 27, 2323-2337, doi:10.1519/JSC.0b013e318278eea0.]. Data were analyzed and graphically visualized using GraphPad PRISM Version 8.0 (RRID:SCR_002798). The open-source web-application SynergyFinder Plus [26 Zheng, S.; Wang, W.; Aldahdooh, J.; Malyutina, A.; Shadbahr, T.; Pessia, A.; Tang, J. SynergyFinder Plus: Towards a Better Interpretation and Annotation of Drug Combination Screening Datasets. bioRxiv 2021, 2021.06.01.446564.] was used to evaluate and visualize the drug combinatory screening on the basis of the Bliss independence model (Bliss), Highest single agent model (HSA), Loewe additivity model (Loewe) and the Bliss and Loewe combined Zero Interaction Potency model (ZIP) [27-29 He, L.; Kulesskiy, E.; Saarela, J.; Turunen, L.; Wennerberg, K.; Aittokallio, T.; Tang, J. Methods for High-Throughput Drug Combination Screening and Synergy Scoring. Methods Mol Biol. 2018, 051698, doi:10.1101/051698. - Yadav, B.; Wennerberg, K.; Aittokallio, T.; Tang, J. Searching for Drug Synergy in Complex Dose-Response Landscapes Using an Interaction Potency Model. Comput. Struct. Biotechnol. J. 2015, 13, 504-513, doi:10.1016/j.csbj.2015.09.001. - Malyutina, A.; Majumder, M.M.; Wang, W.; Pessia, A.; Heckman, C.A.; Tang, J. Drug Combination Sensitivity Scoring Facilitates the Discovery of Synergistic and Efficacious Drug Combinations in Cancer. PLoS Comput. Biol. 2019, 15, 1-19, doi:10.1371/journal.pcbi.1006752.]. The reference of the Bliss independence model expects that the two drugs evoke independent effects. The highest single agent model assumes that the reference drug combinational effect equals the maximal effect of a single drug. The Loewe additivity model defines the reference as an effect of a drug combined with itself. The Zero Interaction Potency model compares the changes in the potency of the dose-response curves of single drugs with their combinations, assuming no changes in the response curves if the drugs do not interact [28].

Example 1: Single treatment of ATR-002 or direct acting antivirals (DAA) efficiently inhibits replication of SARS-CoV-2

The inventors recently evaluated the potential of MEK1/2 inhibitor zapnometinib, here designated as ATR-002, as a drug candidate against COVID-19, based on its direct inhibitory effect on the SARS-CoV- 2 replication cycle together with a secondary beneficial inhibitory effect on the release of proinflammatory cytokines and chemokines [11 Schreiber, A.; Viemann, D.; Schoning, J.; Schloer, S.; Mecate Zambrano, A.; Brunotte, L.; Faist, A.; Schofbanker, M.; Hrincius, E.; Hoffmann, H.; et al. The MEKl/2-lnhibitor ATR-002 Efficiently Blocks SARS-CoV-2 Propagation and Alleviates pro- inflammatory Cytokine/Chemokine Responses. Cell. Mol. Life Sci. 2022, 79, doi:10.1007/s00018-021- 04085-1.]. While monotherapy with ATR-002 already significantly reduced viral titers, we wanted to assess whether antiviral activity could be potentiated by combinatory treatment of ATR-002 with DAAs including Molnupiravir (MPV), Remdesivir (RDV), Nirmatrelvir (NTV) and Ritonavir (RTV). To define drug concentrations that are suitable for the synergistic evaluation, effective inhibitory concentrations (EC10, EC50, EC90) of all drugs were determined, representing a 10 %, 50 % or 90 % inhibitory effect on the viral replication. Additionally, we calculated the ECI concentrations. As these concentrations do not have an impact on the viral replication propagation, it was used as a defined concentration which does not alter the viral replication, when used in a monotherapy. Calu-3 cells were infected with the D614G SARS-CoV-2 isolate hCoV-19/Germany/FH103201/2020 using a multiplicity of infection (MOI) of 0.01, followed by a treatment of the respective drugs 1 hour postinfection (h.p.i.). for a total incubation time of 48 h. In line with the recently published work of the inventors [11 Schreiber, A.; Viemann, D.; Schoning, J.; Schloer, S.; Mecate Zambrano, A.; Brunotte, L; Faist, A.; Schofbanker, M.; Hrincius, E.; Hoffmann, H.; et al. The MEKl/2-lnhibitor ATR-002 Efficiently Blocks SARS-CoV-2 Propagation and Alleviates pro-inflammatory Cytokine/Chemokine Responses. Cell. Mol. Life Sci. 2022, 79, doi:10.1007/s00018-021-04085-l.], they found a dose-dependent reduction of SARS-CoV-2 titers after ATR-002 treatment, allowing for the calculation of the above- mentioned EC values. Additionally, the inventors could determine EC values for MPV, RDV, NTV and RTV (Figure 1), which are comparable to recently published data [10 Schloer, S.; Brunotte, L.; Mecate-Zambrano, A.; Zheng, S.; Tang, J.; Ludwig, S.; Rescher, U. Drug Synergy of Combinatory Treatment with Remdesivir and the Repurposed Drugs Fluoxetine and Itraconazole Effectively Impairs SARS-CoV-2 Infection in Vitro. Br. J. Pharmacol. 2021, 178, 2339-2350, doi:10.1111/bph.15418., 30-33 Hsu, H.Y.; Yang, C.W.; Lee, Y.Z.; Lin, Y.L.; Chang, S.Y.; Yang, R.B.; Liang, J.J.; Chao, T.L.; Liao, C.C.; Kao, H.C.; et al. Remdesivir and Cyclosporine Synergistically Inhibit the Human Coronaviruses OC43 and SARS-CoV-2. Front. Pharmacol. 2021, 12, 1-11, doi:10.3389/fphar.2021.706901. - Wang, M.; Cao, R.; Zhang, L.; Yang, X.; Liu, J.; Xu, M.; Shi, Z.; Hu, Z.; Zhong, W.; Xiao, G. Remdesivir and Chloroquine Effectively Inhibit the Recently Emerged Novel Coronavirus (2019-NCoV) in Vitro. Cell Res. 2020, 30, 269-271, doi:10.1038/s41422-020-0282-0. - Zarenezhad, E.; Marzi, M. Review on Molnupiravir as a Promising Oral Drug for the Treatment of COVID-19. Med. Chem. Res. 2022, doi:10.1007/s00044-021-02841-3. - Bojkova, D.; Widera, M.; Ciesek, S.; Wass, M.N.; Michaelis, M.; Cinatl, J. Reduced Interferon Antagonism but Similar Drug Sensitivity in Omicron Variant Compared to Delta Variant of SARS-CoV-2 Isolates. Cell Res. 2022, 10- 12, doi:10.1038/s41422-022-00619-9.].

Figure 1 shows the inhibitory effect of ATR-002, MPV, RDV, NTV and RTV on the SARS-CoV-2 replication. According to Figure 1, Calu-3 cells were infected with SARS-CoV-2 (MOI: 0.01). 1 h.p.i. drug treatment was initiated. Untreated (SARS-CoV-2) and DMSO-treated cells served as negative controls. Viral titers were analyzed 48 h.p.i. (a, c, e, g, i) Viral titers in % of DMSO. DMSO was arbitrarily set to 100 %. Shown are data of five independent experiments, each performed in triplicates. Significance was calculated by one-way ANOVA in combination with a Dunnett's multiple comparisons test using DMSO as reference (** p < 0.0021; **** p < 0.0001). (b, d, f, h, j) Calculation of the EC values. Of note, the calculated concentrations required for the antiviral effect are non-cytotoxic for all validated drugs in Calu-3 cells after 48 h treatment (Figure 5 a-e), indicating that the inhibitory effects are not aberrantly caused by impaired cell viability.

Figure 5 shows the determination of the cell viability after single and combinational drug treatment. According to Figure 5, Calu-3 cells were treated with the indicated drug concentrations and combinations. 48 h post-treatment cell viability was evaluated using a MTT-assay. DMSO was used as negative control, Staurosporine (Stauro; 1 pM) as positive control. DMSO and Staurosporine levels are indicated by dashed lines. Shown are means ± SEM of five independent experiments, each performed in triplicates. Significance was calculated using a one-way ANOVA in combination with a Dunnett's multiple comparisons test with DMSO as reference (* p < 0.0332; ** p < 0.0021; *** p < 0.0002; *** p < 0.0001).

Figure 6 shows the drug related inhibitory effect on the SARS-CoV-2 replication. Related to Figure 1. According to Figure 6, SARS-CoV-2 (MOI: 0.01) was used to infect Calu-3 cells. Drug treatment was initiated 1 h.p.i. Untreated (SARS-CoV-2) and DMSO-treated cells served as negative controls. Viral titers were analyzed 48 h.p.i. Shown are viral titers in PFU/ml. Data represents five independent experiments, each performed in triplicates. Significance was calculated by one-way ANOVA in combination with a Dunnett's multiple comparisons test using DMSO as reference (** p < 0.0021; *** p < 0.0002; **** p < 0.0001).

Example 2: Synergistic drug interactions between ATR-002 and DAAs against SARS-CoV-2

The inventors next investigated the synergistic effects of the DAAs MPV, RDV, NTV and RTV in combination with the HTA ATR-002. Calu-3 cells infected with SARS-CoV-2 (MOI 0.01) were either treated with the respective inhibitors as a single treatment (drug + DMSO) or a combinatory treatment (drugl + drug2). Several drug combinations of all tested DAAs (MPV, RDV, NTV, RTV) with ATR-002 showed an overall combinatory effect that by far exceeds the sum of the respective monotherapies (Figure 2; Figure 7-11 a-c).

Figure 2 shown the antiviral effect of the combinational treatments in Calu-3 cells. According to Figure 2, combinational drug treatments were initiated 1 h.p.i. Viral titers were analyzed 48 h.p.i. Shown are data of five independent experiments, each performed in triplicates, (a-d) DAAs (MPV, RDV, NTV, RTV) in combination with the HTA ATR-002. (e) Combination of the DAAs NTV with RDV. (a-e) Virus titer in % of DMSO. DMSO (0 pM) was arbitrarily set to 100 % (see also Figure 7-11 a-c).

Figure 7 shows the determination of the ATR-002 + MPV synergism. Related to Figure 3. According to Figure 7, Calu-3 cells were infected with SARS-CoV-2 (MOI: 0.01). 1 h.p.i. drug treatment was initiated. 48 h.p.i. viral titers were deter-mined by plaque titration, (a-c) Viral titers in PFU/ml (a, c) or % of DMSO (b) of the combinational drug treatments. Data represent means ± SEM of five independent experiments, each performed in triplicates, (b) Dashed lines indicate 10 %, 50 % and 90 % titer reduction levels, (d) Dose-response curves of the single treatment in combination with DMSO. (e) 2D dose response matrix of the ATR-002 + MPV drug combination, (f) 3D dose response landscape of the ATR-002 + MPV drug combination, (g) Heat maps of the 2D contour and 3D landscape visualization shown in Figure 3a and Figure 7h. (h) 2D contour visualization of the synergy calculation, (i) Synergy score for the ATR-002 + MPV drug combination calculated with the indicated synergy reference models, (j) Synergy values of the different drug combinations calculated with the indicated synergy reference models (see also Table in Figure 13).

Figure 13 shows the shows a table with the highest synergy score values, calculated for the drug combinations MPV 0.047 pM / ATR-002 12.05 pM, RDV 0.034 pM / ATR-002 12.05 pM, NTV 0.006 pM / ATR-002 12.05 pM, RTV 0.057 pM / ATR-002 12.05 pM and RDV / NTV (Bliss, Loewe, ZIP: RDV 0.034 005 pM + NTV 0.095 pM; Loewe: RDV 0.034 pM + NTV 0.095 pM HSA: RDV 0.199 pM + NTV 0.095 pM). This is related to Figure 3-7 j.

Figure 8 shows the determination of the ATR-002 + RDV synergism. Related to Figure 3. According to Figure 8, Calu-3 cells were infected with SARS-CoV-2 (MOI: 0.01). 1 h.p.i. drug treatment was initiated. 48 h.p.i. viral titers were determined by plaque titration, (a-c) Viral titers in PFU/ml (a, c) or % of DMSO (b) of the combinational drug treatments. Data represent means ± SEM of five independent experiments, each performed in triplicates, (b) Dashed lines indicate 10 %, 50 % and 90 % titer reduction levels, (d) Dose-response curves of the single treatment in combination with DMSO. (e) 2D dose response matrix of the ATR-002 + RDV drug combination, (f) 3D dose response landscape of the ATR-002 + RDV drug combination, (g) Heat maps of the 2D contour and 3D landscape visualization shown in Figure 3b and Figure 8h. (h) 2D contour visualization of the synergy calculation, (i) Synergy score for the ATR-002 + RDV drug combination calculated with the indicated synergy reference models, (j) Synergy values of the different drug combinations calculated with the indicated synergy reference models (see also Table in Figure 13).

Figure 9 shows the determination of the ATR-002 + NTV synergism. Related to Figure 3. According to Figure 9, Calu-3 cells were infected with SARS-CoV-2 (MOI: 0.01). 1 h.p.i. drug treatment was initiated. 48 h.p.i. viral titers were deter-mined by plaque titration, (a-c) Viral titers in PFU/ml (a, c) or % of DMSO (b) of the combinational drug treatments. Data represent means ± SEM of five independent experiments, each performed in triplicates, (b) Dashed lines indicate 10 %, 50 % and 90 % titer reduction levels, (d) Dose-response curves of the single treatment in combination with DMSO. (e) 2D dose response matrix of the ATR-002 + NTV drug combination, (f) 3D dose response landscape of the ATR-002 + NTV drug combination, (g) Heat maps of the 2D contour and 3D landscape visualization shown in Figure 3c and Figure 5h. (h) 2D contour visualization of the synergy calculation. (I) Synergy score for the ATR-002 + NTV drug combination calculated with the indicated synergy reference models, (j) Synergy values of the different drug combinations calculated with the indicated synergy reference models (see also Table in Figure 13).

Figure 10 shows the determination of the ATR-002 + RTV synergism. Related to Figure 3. According to Figure 10, Calu-3 cells were infected with SARS-CoV-2 (MOI: 0.01). 1 h.p.i. drug treatment was initiated. 48 h.p.i. viral titers were deter-mined by plaque titration, (a-c) Viral titers in PFU/ml (a, c) or % of DMSO (b) of the combinational drug treatments. Data rep-resent means ± SEM of five independent experiments, each performed in triplicates, (b) Dashed lines indicate 10 %, 50 % and 90 % titer reduction levels, (d) Dose-response curves of the single treatment in combination with DMSO. (e) 2D dose response matrix of the ATR-002 + RTV drug combination, (f) 3D dose response landscape of the ATR-002 + RTV drug combination, (g) Heat maps of the 2D contour and 3D landscape visualization shown in Figure 3d and Figure 6h. (h) 2D contour visualization of the synergy calculation, (i) Synergy score for the ATR-002 + RTV drug combination calculated with the indicated synergy reference models, (j) Synergy values of the different drug combinations calculated with the indicated synergy reference models (see also Table in Figure 13).

Figure 11 shows the determination of the RDV + NTV synergism. Related to Figure 3. According to Figure 11, Calu-3 cells were infected with SARS-CoV-2 (MOI: 0.01). 1 h.p.i. drug treatment was initiated. 48 h.p.i. viral titers were deter-mined by plaque titration, (a-c) Viral titers in PFU/ml (a, c) or % of DMSO (b) of the combinational drug treatments. Data rep-resent means ± SEM of five independent experiments, each performed in triplicates, (b) Dashed lines indicate 10 %, 50 % and 90 % titer reduction levels, (d) Dose-response curves of the single treatment in combination with DMSO. (e) 2D dose response matrix of the RDV + NTV drug combination, (f) 3D dose response landscape of the RDV + NTV drug combination, (g) Heat maps of the 2D contour and 3D landscape visualization shown in Figure 3e and Figure 7h. (h) 2D contour visualization of the synergy calculation, (i) Synergy score for the RDV + NTV drug combination calculated with the indicated synergy reference models, (j) Synergy values of the different drug combinations calculated with the indicated synergy reference models (see also Table in Figure 13).

Of note, the strong antiviral effects are caused by the inhibitory actions of the drugs and are not the result of enhanced cell cytotoxicity (Figure 5f-j). To analyze drug synergy of the drug pairs, the inventors used the open-source web application SynergyFinder Plus. Four different reference models (Bliss, HSA, Loewe, ZIP) were used to quantify the degree of interactions (synergy score) and the overall treatment efficacy (combination sensitivity score; CSS). Examination of the drug interactions and landscape visualizations revealed strong synergistic effects for ATR-002 combined with all tested DAAs (Figure 3; Figure 7-10; Figure 13, 14 with respective Tables). Figure 3 shows the synergistic evaluation of ATR-002 and with DAAs in Calu-3 cells. Figure 3 shows Landscape visualization of the synergy evaluation shown in Fig. 2. Bliss independence (Bliss), Loewe additivity (Loewe), highest single agent (HSA) and Zero Interaction Potency (ZIP) reference models were used to calculate and visualize synergistic areas. Surface is color coded. Red indicates synergistic interactions and green indicates antagonistic interactions (see also Figure 7-11).

For all reference models, highest synergy values were found for the combinations MPV 0.047 pM, RDV 0.034 pM, NTV 0.006 pM or RTV 0.057 pM when administered with 12.05 pM ATR-002 indicating the robustness of the data (Figure 7-10; Figure 13, 14 with respective Tables). All tested reference models showed positive overall synergy scores for the different drug combinations, confirming average synergistic mode of actions (Figure 7-10 i). To compare the synergistic potency of ATR-002 with a treatment strategy that only targets the virus (DAA1 + DAA2), we further tested the combination of the viral RdRp-inhibitor RDV with the 3C-like protease inhibitor NTV. Interaction determination revealed synergisms if NTV was used in higher concentrations (NTV: 0.096 pM, 2.162 pM) combined with all tested RDV concentrations. Whereas lower NTV concentrations (NTV: 0.0002 pM, 0.006 pM) led to an antagonistic effect (Figure 2e, 3e; Figure 11). In line with this results, the overall synergy scores, as well as the CSS, were lower compared to the HTA + DAA treatments (Figure Hi; Figure 13 with respective Table).

Example 3: Strong synergistic effects of ATR-002 combinations with DAAs against different SARS- CoV-2 variants

Because of the strong synergy of the drug pairs, the inventors further evaluated the effect of the drug combinations on viral replication for other SARS-CoV-2 variants. For this purpose, the drug combinations with the highest synergy values detected for the D614G isolate (MPV: 0.047 pM / ATR- 002: 12.05 pM; RDV: 0.034 pM /ATR-002: 12.05 pM; NTV: 0.006 pM / ATR-002: 12.05 pM; RTV: 0.057 pM /ATR-002: 12.05 pM) (Figure 4; Figure 12) were chosen and tested against Wuhan-like virus (Munchen-01), a-Bl.1.7, 0-B1.351 and 6-B1.617.2.

Figure 4 shows the synergistic effects of MPV, RDV, NTV and RTV in combination with ATR-002 for different SARS-CoV-2 VOCs in Calu-3 cells. According to Figure 4, drug treatments were initiated 1 h.p.i. MPV (0.047 pM), RDV (0.034 pM), NTV (0.006 pM) or RTV (0.057 pM) were combined with ATR- 002 (12.05 pM). Untreated (SARS-CoV-2), DMSO treated and single treated (drug + DMSO) cells served as controls. Viral titers were analyzed 48 h.p.i. Shown are virus titers in % of DMSO. DMSO was arbitrarily set to 100 %. Data represents five independent experiments, each performed in triplicates. Significance was calculated using an unpaired t-test for each inhibitor individually, comparing the single treated (MPV, RDV, NTV, RTV) vs. the double treated results (**** p < 0.0001) (see also Figure 12).

Figure 12 shows the synergistic effects of different drug combinations on the replication of SARS- CoV-2 VOCs. Related to Figure 4. According to Figure 12, Calu-3 cells were infected with SARS-CoV-2 (MOI: 0.01). 1 h.p.i. drug treatment was initiated. MPV (0.047 pM), RDV (0.034 pM), NTV (0.006 pM) or RTV (0.057 pM) were combined with ATR-002 (12.05 pM). Untreated (SARS-CoV-2), DMSO treated and single treated (drug + DMSO) cells served as controls. 48 h.p.i. viral titers were determined by plaque titration. Data represents five independent experiments, each performed in triplicates. Significance was calculated using an unpaired t-test for each inhibitor individually, comparing the single treated (MPV, RDV, NTV, RTV) vs. the double treated results (* p < 0.0332; ** p < 0.0021).

Overall reduction of viral titers after single drug treatments was comparable between the different VOCs a-Bl.1.7, 0-B1.351 and 6-B1.617.2 and stronger as for the Wuhan-like virus which is in line with recently published data [34]. All drug combinations reduced viral titers of the different variants far beyond an additive effect of the single treatments (Figure 4; Figure 12). The most pronounced differences between the investigated combinational inhibitory effect and a calculated additive effect of the titer inhibition was found for ATR-002 + MPV (0-B1.351, 6-B1.617.2), ATR-002 + RDV (a-Bl.1.7) or ATR-002 + NTV (Munchen-01), indicating that these combinations might exhibit the highest synergy.

REFERENCES

1. Chams, N.; Chams, S.; Badran, R.; Shams, A.; Araji, A.; Raad, M.; Mukhopadhyay, S.; Stroberg, E.; Duval, E.J.; Barton, L.M.; et al. COVID-19: A Multidisciplinary Review. Front. Public Heal. 2020, 8, 1-20, doi:10.3389/fpubh.2020.00383.

2. Stasi, C.; Fallani, S.; Voller, F.; Silvestri, C. Treatment for COVID-19: An Overview. Ear. J. Pharmacol. 2020.

3. Chen, Y.T. The Effect of Vaccination Rates on the Infection of Covid-19 under the Vaccination Rate below the Herd Immunity Threshold. Int. J. Environ. Res. Public Health 2021, 18, doi:10.3390/ijerphl8147491.

4. Wall, E.C.; Wu, M.; Harvey, R.; Kelly, G.; Warchai, S.; Sawyer, C.; Daniels, R.; Hobson, P.; Hatipoglu, E.; Ngai, Y.; et al. Neutralising Antibody Activity against SARS-CoV-2 VOCs B.1.617.2 and B.1.351 by BNT162b2 Vaccination. Lancet 2021, 397, 2331-2333, doi:10.1016/S0140-6736(21)01290- 3.

5. Kustin, T.; Harel, N.; Finkel, U.; Perchik, S.; Harari, S.; Tahor, M.; Caspi, I.; Levy, R.; Leshchinsky, M.; Ken Dror, S.; et al. Evidence for Increased Breakthrough Rates of SARS-CoV-2 Variants of Concern in BNT162b2-MRNA-Vaccinated Individuals. Nat. Med. 2021, 27, 1379-1384, doi:10.1038/s41591-021-01413-7.

6. Fierabracci, A.; Arena, A.; Rossi, P. COVID-19: A Review on Diagnosis, Treatment, and Prophylaxis. Int. J. Mol. Sci. 2020, 21, 1-16, doi:10.3390/ijms21145145.

7. Ludwig, S.; Wolff, T.; Ehrhardt, C.; Wurzer, W.J.; Reinhardt, J.; Planz, O.; Pleschka, S. MEK Inhibition Impairs Influenza B Virus Propagation without Emergence of Resistant Variants. FEBS Lett. 2004, 561, 37-43, doi:10.1016/S0014-5793(04)00108-5.

8. Zumla, A.; Hui, D.S.; Azhar, E.L; Memish, Z.A.; Maeurer, M. Reducing Mortality from 2019- NCoV: Host-Directed Therapies Should Be an Option. Lancet 2020, 395, e35-e36, doi:10.1016/S0140- 6736(20)30305-6.

9. Brunotte, L.; Zheng, S.; Mecate-Zambrano, A.; Tang, J.; Ludwig, S.; Rescher, U.; Schloer, S. Combination Therapy with Fluoxetine and the Nucleoside Analog Gs-441524 Exerts Synergistic Antiviral Effects against Different Sars-Cov-2 Variants in Vitro. Pharmaceutics 2021, 13, doi:10.3390/pharmaceuticsl3091400.

10. Schloer, S.; Brunotte, L.; Mecate-Zambrano, A.; Zheng, S.; Tang, J.; Ludwig, S.; Rescher, U. Drug Synergy of Combinatory Treatment with Remdesivir and the Repurposed Drugs Fluoxetine and Itraconazole Effectively Impairs SARS-CoV-2 Infection in Vitro. Br. J. Pharmacol. 2021, 178, 2339- 2350, doi:10.1111/bph.15418. 11. Schreiber, A.; Viemann, D.; Schoning, J.; Schloer, S.; Mecate Zambrano, A.; Brunotte, L; Faist,

A.; Schofbanker, M.; Hrincius, E.; Hoffmann, H.; et al. The MEKl/2-lnhibitor ATR-002 Efficiently Blocks SARS-CoV-2 Propagation and Alleviates pro-inflammatory Cytokine/Chemokine Responses. Cell. Mol. Life Sci. 2022, 79, doi:10.1007/s00018-021-04085-l.

12. Preugschas, H.F.; Hrincius, E.R.; Mewis, C.; Tran, G.V.Q.; Ludwig, S.; Ehrhardt, C. Late Activation of the Raf/MEK/ERK Pathway Is Required for Translocation of the Respiratory Syncytial Virus F Protein to the Plasma Membrane and Efficient Viral Replication. Cell. Microbiol. 2019, 21, 1- 14, doi:10.1111/cmi.12955.

13. Albarnaz, J.D.; De Oliveira, L.C.; Torres, A.A.; Palhares, R.M.; Casteluber, M.C.; Rodrigues, C.M.; Cardozo, P.L.; De Souza, A.M.R.; Pacca, C.C.; Ferreira, P.C.P.; et al. MEK/ERK Activation Plays a Decisive Role in Yellow Fever Virus Replication: Implication as an Antiviral Therapeutic Target. Antiviral Res. 2014, 111, 82-92, doi: 10.1016/j. antiviral.2014.09.004.

14. Menzel, N.; Fischl, W.; Hueging, K.; Bankwitz, D.; Frentzen, A.; Haid, S.; Gentzsch, J.; Kaderali,

L.; Bartenschlager, R.; Pietschmann, T. MAP-Kinase Regulated Cytosolic Phospholipase A2 Activity Is Essential for Production of Infectious Hepatitis C Virus Particles. PLoS Pathog. 2012, 8, 21, doi: 10.1371/journal.ppat.1002829.

15. Cai, Y.; Liu, Y.; Zhang, X. Suppression of Coronavirus Replication by Inhibition of the MEK Signaling Pathway. J. Virol. 2007, 81, 446-456, doi:10.1128/jvi.01705-06.

16. Planz, O.; Pleschka, S.; Ludwig, S. MEK-Specific Inhibitor U0126 Blocks Spread of Borna Disease Virus in Cultured Cells. J. Virol. 2001, 75, 4871-4877, doi:10.1128/jvi.75.10.4871-4877.2001.

17. Pleschka, S.; Wolff, T.; Ehrhardt, C.; Hobom, G.; Planz, O.; Rapp, U.R.; Ludwig, S. Influenza Virus Propagation Is Impaired by Inhibition of the Raf/MEK/ERK Signalling Cascade. Nat. Cell Biol. 2001, 3, 301-305, doi:10.1038/35060098.

18. Laure, M.; Hamza, H.; Koch-Heier, J.; Quernheim, M.; Muller, C.; Schreiber, A.; Muller, G.; Pleschka, S.; Ludwig, S.; Planz, O. Antiviral Efficacy against Influenza Virus and Pharmacokinetic Analysis of a Novel MEK-lnhibitor, ATR-002, in Cell Culture and in the Mouse Model. Antiviral Res. 2020, 178, 104806, doi:10.1016/j. antiviral.2020.104806.

19. Lee, C.; Hsieh, C. Molnupiravir — A Novel Oral Anti-SARS-CoV-2 Agent. 2021, 10, 1-8.

20. Warren, T.K.; Jordan, R.; Lo, M.K.; Ray, A.S.; Mackman, R.L.; Soloveva, V.; Siegel, D.; Perron,

M.; Bannister, R.; Hui, H.C.; et al. Therapeutic Efficacy of the Small Molecule GS-5734 against Ebola Virus in Rhesus Monkeys. Nature 2016, 531, 381-385, doi:10.1038/naturel7180.

21. Owen, D.R.; Allerton, C.M.N.; Anderson, A.S.; Aschenbrenner, L.; Avery, M.; Berritt, S.; Boras,

B.; Cardin, R.D.; Carlo, A.; Coffman, K.J.; et al. An Oral SARS-CoV-2 Mproinhibitor Clinical Candidate for the Treatment of COVID-19. 2021, 3, 1586-1593. 22. Nukoolkarn, V.; Lee, V.S.; Malaisree, M.; Aruksakulwong, O.; Hannongbua, S. Molecular

Dynamic Simulations Analysis of Ritronavir and Lopinavir as SARS-CoV 3CLpro Inhibitors. J. Theor.

Biol. 2008, 254, 861-867, doi:10.1016/j.jtbi.2008.07.030.

23. Nutho, B.; Mahalapbutr, P.; Hengphasatporn, K.; Pattaranggoon, N.C.; Simanon, N.; Shigeta, Y.; Hannongbua, S.; Rungrotmongkol, T. Why Are Lopinavir and Ritonavir Effective against the Newly Emerged Coronavirus 2019? Atomistic Insights into the Inhibitory Mechanisms. Biochemistry 2020, 59, 1769-1779, doi:10.1021/acs.biochem.0c00160.

24. Mosmann, T. Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. J. Immunol. Methods 1983, 65, 55-63, doi:10.1016/0022- 1759(83)90303-4.

25. Beck, T.W. The Importance of a Priori Sample Size Estimation in Strength and Conditioning Research. J. Strength Cond. Res. 2013, 27, 2323-2337, doi:10.1519/JSC.0b013e318278eea0.

26. Zheng, S.; Wang, W.; Aldahdooh, J.; Malyutina, A.; Shadbahr, T.; Pessia, A.; Tang, J. SynergyFinder Plus: Towards a Better Interpretation and Annotation of Drug Combination Screening Datasets. bioRxiv 2021, 2021.06.01.446564.

27. He, L.; Kulesskiy, E.; Saarela, J.; Turunen, L.; Wennerberg, K.; Aittokallio, T.; Tang, J. Methods for High-Throughput Drug Combination Screening and Synergy Scoring. Methods Mol Biol. 2018, 051698, doi:10.1101/051698.

28. Yadav, B.; Wennerberg, K.; Aittokallio, T.; Tang, J. Searching for Drug Synergy in Complex Dose-Response Landscapes Using an Interaction Potency Model. Comput. Struct. Biotechnol. J. 2015, 13, 504-513, doi:10.1016/j.csbj.2015.09.001.

29. Malyutina, A.; Majumder, M.M.; Wang, W.; Pessia, A.; Heckman, C.A.; Tang, J. Drug Combination Sensitivity Scoring Facilitates the Discovery of Synergistic and Efficacious Drug Combinations in Cancer. PLoS Comput. Biol. 2019, 15, 1-19, doi:10.1371/journal.pcbi.1006752.

30. Hsu, H.Y.; Yang, C.W.; Lee, Y.Z.; Lin, Y.L.; Chang, S.Y.; Yang, R.B.; Liang, J.J.; Chao, T.L.; Liao, C.C.; Kao, H.C.; et al. Remdesivir and Cyclosporine Synergistically Inhibit the Human Coronaviruses OC43 and SARS-CoV-2. Front. Pharmacol. 2021, 12, 1-11, doi:10.3389/fphar.2021.706901.

31. Wang, M.; Cao, R.; Zhang, L.; Yang, X.; Liu, J.; Xu, M.; Shi, Z.; Hu, Z.; Zhong, W.; Xiao, G. Remdesivir and Chloroquine Effectively Inhibit the Recently Emerged Novel Coronavirus (2019-NCoV) in Vitro. Cell Res. 2020, 30, 269-271, doi:10.1038/s41422-020-0282-0.

32. Zarenezhad, E.; Marzi, M. Review on Molnupiravir as a Promising Oral Drug for the Treatment of COVID-19. Med. Chem. Res. 2022, doi:10.1007/s00044-021-02841-3.

33. Bojkova, D.; Widera, M.; Ciesek, S.; Wass, M.N.; Michaelis, M.; Cinatl, J. Reduced Interferon Antagonism but Similar Drug Sensitivity in Omicron Variant Compared to Delta Variant of SARS-CoV-2 Isolates. Cell Res. 2022, 10-12, doi:10.1038/s41422-022-00619-9. 34. Vangeel, L; Chiu, W.; De Jonghe, S.; Maes, P.; Slechten, B.; Raymenants, J.; Andr, E.; Leyssen, P.; Neyts, J.; Jochmans, D. Remdesivir , Molnupiravir and Nirmatrelvir Remain Active against SARS- CoV-2 Omicron and Other Variants of Concern. Antiviral Res. 2022, 198, 10-12, doi:10.1016/j. antiviral.2022.105252.

35. Yin, N.; Ma, W.; Pei, J.; Ouyang, Q.; Tang, C.; Lai, L. Synergistic and Antagonistic Drug Combinations Depend on Network Topology. PLoS One 2014, 9, doi:10.1371/journal. pone.0093960.

36. Lehar, J.; Krueger, A.; Avery, W.; Heilbut, A.; Johansen, L. Synergistic Drug Combinations Improve Therapeutic Selectivity. Nat Biotechnol 2009, 27, 659-66, doi:10.1038/nbt.l549. Synergistic.

37. Dancey, J.E.; Chen, H.X. Strategies for Optimizing Combinations of Molecularly Targeted Anticancer Agents. Nat. Rev. Drug Discov. 2006, 5, 649-659, doi:10.1038/nrd2089.

38. Schloer, S.; Goretzko, J.; Kuhnl, A.; Brunotte, L.; Ludwig, S.; Rescher, U. The Clinically Licensed Antifungal Drug Itraconazole Inhibits Influenza Virus in Vitro and in Vivo. Emerg. Microbes Infect. 2019, 8, 80-93, doi:10.1080/22221751.2018.1559709.

39. Schloer, S.; Goretzko, J.; Pleschka, S.; Ludwig, S.; Rescher, U. Combinatory Treatment with Oseltamivir and Itraconazole Targeting Both Virus and Host Factors in Influenza A Virus Infection. Viruses 2020, 12, doi:10.3390/vl2070703.

40. Lin, B.; He, S.; Yim, H.J.; Liang, T.J.; Hu, Z. Evaluation of Antiviral Drug Synergy in an Infectious HCV System. Antivir. Ther. 2016, 21, 595-603, doi:10.3851/IMP3044.

41. Murga, J.D.; Franti, M.; Pevear, D.C.; Maddon, P.J.; Olson, W.C. Potent Antiviral Synergy between Monoclonal Antibody and Small-Molecule CCR5 Inhibitors of Human Immunodeficiency Virus Type 1. Antimicrob. Agents Chemother. 2006, 50, 3289-3296, doi:10.1128/AAC.00699-06.

42. Muratov, E.N.; Varlamova, E. V.; Artemenko, A.G.; Polishchuk, P.G.; Nikolaeva-Glomb, L.; Galabov, A.S.; Kuz'Min, V.E. QSAR Analysis of Poliovirus Inhibition by Dual Combinations of Antivirals. Struct. Chem. 2013, 24, 1665-1679, doi:10.1007/sll224-012-0195-8.

43. Sun, W.; He, S.; Martinez-Romero, C.; Kouznetsova, J.; Tawa, G.; Xu, M.; Shinn, P.; Fisher, E.; Long, Y.; Motabar, O.; et al. Synergistic Drug Combination Effectively Blocks Ebola Virus Infection. Physiol. Behav. 2016, 176, 139-148, doi: 10.1016/j. antiviral.2016.11.017. Synergistic.

44. Bafna, K.; White, K.; Harish, B.; Rosales, R.; Ramelot, T.A.; Acton, T.B.; Moreno, E.; Kehrer, T.; Miorin, L.; Royer, C.A.; et al. Hepatitis C Virus Drugs That Inhibit SARS-CoV-2 Papain-like Protease Synergize with Remdesivir to Suppress Viral Replication in Cell Culture. Cell Rep. 2021, 35, doi:10.1016/j.celrep.2021.109133.

45. Tan, Y.L.; Tan, K.S.W.; Chu, J.J.H.; Chow, V.T. Combination Treatment With Remdesivir and Ivermectin Exerts Highly Synergistic and Potent Antiviral Activity Against Murine Coronavirus Infection. Front. Cell. Infect. Microbiol. 2021, 11, 1-10, doi:10.3389/fcimb.2021.700502. 46. Drugs. Com / Drug Interaction Checker. Available online: https://www.drugs.com/ (accessed on 24 January 2022).

Claims

CLAIMS A MEK inhibitor or a pharmaceutically acceptable salt, a metabolite or derivate thereof for use in a method for the treatment and/or prophylaxis of a coronavirus associated disease in a subject, wherein the MEK inhibitor is administered in combination with one or more direct acting antiviral (DAA), wherein the DAA is selected from the group consisting of a nucleoside analogue, nucleoside reverse transcriptase inhibitor, RNA polymerase inhibitor, and a protease inhibitor. The MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof for the use of claim 1, wherein the MEK inhibitor is selected from the group consisting of PD-0184264, CI-1040, GSK-1120212, GDC-0973, Binimetinib, Selumetinib, PLX-4032, AZD6244, AZD8330, AS- 703026, RDEA-119, RO-5126766, RO-4987655, PD-0325901, TAK-733, AS703026, PD98059 and PD184352. The MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof for the use of claim 1 or 2, wherein the MEK inhibitor is PD-0184264. The MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof for the use of any one of claims 1 to 3, wherein the nucleoside analogue is selected from the group consisting of Molnupiravir and its prodrugs, Ribavirin, GS-441524 monophosphate, GS-441524 triphosphate, beta-D-N4-hydroxycytidine (NHC, EIDD-1931, EID 2801 prodrug) and Sofosbuvir; wherein the nucleoside reverse transcriptase inhibitor is selected from the group consisting of Azvudine (RO-0622), Emtricitabine, and Tenofovir; wherein the RNA polymerase inhibitor is selected from Remdesivir (GS-5734), Favipiravir, and Galidesivir (BCX4430); and wherein the protease inhibitor is selected from the group consisting of Nirmatrelvir, Ritonavir, the combination of Nirmatrelvir and Ritonavir, Darunavir, Danoprevir, Lopinavir, Nafamostat, and TMC-310911 (ASC-09). The MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof for the use of any one of claims 1 to 4, wherein the MEK inhibitor is PD-0184264 and the nucleoside analog is Molnupiravir; or wherein the MEK inhibitor is PD-0184264 and the RNA polymerase inhibitor is Remdesivir (GS-5734); or wherein the MEK inhibitor is PD-0184264 and the protease inhibitor is Nirmatrelvir; or wherein the MEK inhibitor is PD-0184264 and the protease inhibitor is Ritonavir. The MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof for the use of any one of claims 1 to 5, wherein the coronavirus associated disease is a disease comprising respiratory insufficiency, fever or chills, cough, shortness of breath or difficulty breathing, Fatigue, muscle or body aches, headache, new loss of taste or smell, sore throat, Congestion or runny nose, nausea or vomiting, diarrhea, cytokine storm, hyperinflammatory processes, or the coronavirus disease 2019 (COVID-19), wherein COVID-19 is selected from the group consisting of Stage I COVID-19, Stage II COVID-19, Stage III COVID-19, and a COVID-19 cytokine storm, such as a hyperinflammatory cytokine storm associated with COVID-19. The MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof for the use of any one of claims 1 to 6, wherein the COVID-19 is caused by a SARS-CoV-2 variant, preferably selected from the group consisting of D614G, B.1.1.7, B.1.351, Pl, P2, B.1.617, B.1.427, B.1.429, B.1.525, B.1.526, and Bl.617.2. The MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof for the use of any one of claims 1 to 7, wherein the MEK inhibitor or a pharmaceutically acceptable salt thereof is administered contemporaneously, previously or subsequently to the DAA. A pharmaceutical composition comprising a MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof and one or more DAA selected from the group consisting of a nucleoside analogue, nucleoside reverse transcriptase inhibitor, RNA polymerase inhibitor, and a protease inhibitor. A pharmaceutical composition comprising a MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof and one or more DAA selected from the group consisting of a nucleoside analogue, nucleoside reverse transcriptase inhibitor, RNA polymerase inhibitor, and a protease inhibitor for use as a medicament. The pharmaceutical composition of claim 9 or 10, wherein the MEK inhibitor is selected from the group consisting of PD-0184264, CI-1040, GSK-1120212, GDC-0973, Binimetinib, Selumetinib, PLX-4032, AZD6244, AZD8330, AS-703026, RDEA-119, RO-5126766, RO-4987655, PD-0325901, TAK-733, AS703026, PD98059 and PD184352, or a metabolite or derivate thereof. The pharmaceutical composition of any one of claims 9 to 11, wherein the nucleoside analogue is selected from the group consisting of Molnupiravir and its prodrugs, Ribavirin, GS- 441524 monophosphate, GS-441524 triphosphate, beta-D-N4-hydroxycytidine (NHC, EIDD- 1931, EID 2801 prodrug) and Sofosbuvir; wherein the nucleoside reverse transcriptase inhibitor is selected from the group consisting of Azvudine (RO-0622), Emtricitabine, and Tenofovir; wherein the RNA polymerase inhibitor is selected from Remdesivir (GS-5734), Favipiravir, and Galidesivir (BCX4430); and wherein the protease inhibitor is selected from the group consisting of Nirmatrelvir, Ritonavir, the combination of Nirmatrelvir and Ritonavir, Darunavir, Danoprevir, Lopinavir, Nafamostat, and TMC-310911 (ASC-09). The pharmaceutical composition of any one of claims 9 to 12 for use in the prophylaxis and/or treatment of a coronavirus associated disease. The pharmaceutical composition of claim 13, wherein the coronavirus associated disease is a disease comprising respiratory insufficiency, fever or chills, cough, shortness of breath or difficulty breathing, Fatigue, muscle or body aches, headache, new loss of taste or smell, sore throat, Congestion or runny nose, nausea or vomiting, diarrhea, cytokine storm, hyperinflammatory processes, or the coronavirus disease 2019 (COVID-19), wherein COVID-19 is selected from the group consisting of Stage I COVID-19, Stage II COVID-19, Stage III COVID- 19, and a COVID-19 cytokine storm, such as an hyperinflammatory cytokine storm associated with COVID-19. A kit-of-parts of a MEK inhibitor or a pharmaceutical acceptable salt, a metabolite or derivate thereof with one or more DAA, wherein the MEK inhibitor is selected from the group consisting of PD-0184264, CI-1040, GSK- 1120212, GDC-0973, Binimetinib, Selumetinib, PLX-4032, AZD6244, AZD8330, AS-703026, RDEA-119, RO-5126766, RO-4987655, PD-0325901, TAK-733, AS703026, PD98059 and PD184352, and wherein the DAA is selected from the group consisting of a nucleoside analogue, nucleoside reverse transcriptase inhibitor, RNA polymerase inhibitor, and a protease inhibitor, wherein the nucleoside analogue is selected from the group consisting of Molnupiravir and its prodrugs, Ribavirin, GS-441524 monophosphate, GS-441524 triphosphate, beta-D-N4- hydroxycytidine (NHC, EIDD-1931, EID 2801 prodrug) and Sofosbuvir; wherein the nucleoside reverse transcriptase inhibitor is selected from the group consisting of Azvudine (RO-0622), Emtricitabine, and Tenofovir; wherein the RNA polymerase inhibitor is selected from Remdesivir (GS-5734), Favipiravir, and Galidesivir (BCX4430); and wherein the protease inhibitor is selected from the group consisting of Nirmatrelvir, Ritonavir, the combination of Nirmatrelvir and Ritonavir, Darunavir, Danoprevir, Lopinavir, Nafamostat, and TMC-310911 (ASC-09).