WO2023170187A1

WO2023170187A1 - INHIBITION OF INTRACELLULAR PATHOGEN UPTAKE BY INHIBITORS OF THE IKK-α/NIK COMPLEX

Info

Publication number: WO2023170187A1
Application number: PCT/EP2023/055949
Authority: WO
Inventors: Andreas PICHLMAIR; Vincent GRASS; Anqi WANG
Original assignee: Technische Universität München
Priority date: 2022-03-09
Filing date: 2023-03-08
Publication date: 2023-09-14

Abstract

The invention refers to an inhibitor of the IκB kinase-α (IKK-α)/ NF-κB-inducing kinase (NIK) complex for use in preventing or treating infection, in particular viral or bacterial infection in a cell and/or subject, in particular for preventing or treating SARS-CoV-2 infection in a subject and/or for use in preventing or treating coronavirus disease 2019 (COVID-19) in a subject. Corresponding pharmaceutical compositions are also contemplated.

Description

Inhibition of intracellular pathogen uptake by inhibitors of the I KK-o/N IK complex

FIELD OF THE INVENTION

The invention refers to an inhibitor of the IKB kinase-a (IKK-a)/ NF-KB-inducing kinase (NIK) complex for use in preventing or treating infection, in particular viral or bacterial infection in a cell and/or subject, in particular for preventing or treating SARS-CoV-2 infection in a subject and/or for use in preventing or treating coronavirus disease 2019 (COVID-19) in a subject. Corresponding pharmaceutical compositions are also contemplated.

Background of the invention

Infectious diseases are a main cause of human illness and death. Infections are caused by pathogens including bacteria, viruses, fungi or parasites. Among existing pathogens, viruses are an ongoing reason of concern, as emerging and re-emerging micro-organisms are arising more and more frequently: SARS-Coronavirus (CoV)-1 (2003), Influenza A (2009) and Zika (2016) epidemics are just few examples. Recently, the 2019 SARS-CoV-2 pandemic has caught the healthcare and economic system worldwide as a surprise and highlighted the necessity to adapt antiviral strategies to treat patients in order to prevent the spread of diseases and infectious pathogenicity. It is clear now that besides intensive vaccination, orthogonal approaches to treat and prevent COVID-19 in patients are still urgently needed. Among symptoms from both infected and recovered patients, multiple pathologies such as fibrosis, edema and coagulation disorders have been observed in lungs and other organs of infected patients. The associated symptoms are a result of uncontrolled virus replication and an associated hyperinflammatory syndrome due to the host’s uncontrolled immune response. This underlines the urgent need to identify drugs for prophylactic and treatment therapies.

On the heels of the SARS-CoV-2 pandemic, an outbreak of the Monkeypox virus (MVP) was confirmed in early 2022. Multiple pathologies linked to chronic inflammation (e.g., cognitive dysfunction, anosmia) can remain long after the virus could not be detected any more also known as long COVID. Thus, there is an urgent need to identify drugs for prophylactic and treatment therapies. The inventors have surprisingly found that chemical inhibition or genetic deletion of IKK-a and NIK kinases prevent infection while other downstream components of the NF-KB signaling cascade have no role in the infection process. Notably, IKK-a is required for SARS-CoV-2 entry and inhibition of the kinase prevents acidification of intracellular vesicles, thereby trapping viral particles in the endolysosomal compartment. Surprisingly, IKK-a was also required for the uptake of dextran beads as well as poxviruses, Rift Valley fever virus and intracellular bacteria such as Salmonella enterica and Shigella flexneri, highlighting a general role of IKKa for endosomal uptake. Thus, the object of the invention is to provide inhibitors of I KK-a and/or the IKK-a/NIK complex for use in treating and preventing infection.

Objectives and Summary of the Invention

The inventors have surprisingly found that inhibitors of the IKB kinase- a (I KK-O)/N F-KB- inducing kinase (NIK) complex prevent the entry of pathogens into host cells. Many pathogens need to enter host cells to survive and spread. Therefore, inhibitors of IKK-a and/or the IKK- a/NIK complex can be used in the prevention or treatment of a wide array of infections.

Hence, a first aspect of the invention relates to an inhibitor of IKK-a or the IKK-a/NIK complex for use in preventing or treating infection. In particular, the invention relates to an inhibitor of IKK-a for use in preventing or treating infection with a pathogen in a cell and/or subject.

In one embodiment, the invention relates to an inhibitor of IKK-a or the IKK-a /NIK complex for use in preventing or treating infection with an intracellular pathogen. The pathogen may be one or more virus, bacterium or fungus. In particular, the one or more bacterium may be an intracellular bacterium. The one or more fungus may be an intracellular fungus. Preferably, the pathogen is an endocytic virus or endocytic bacterium.

In one embodiment, the inhibitor is selected from the group consisting of ACHP, IKK 16 and Amgen 16. In one embodiment, the inhibitor is an IKK-a inhibitor. Preferably, the inhibitor of IKK-a is ACHP or IKK 16. More preferably, the inhibitor of IKK-a is ACHP. In one embodiment, the inhibitor is a NIK inhibitor, preferably Amgen 16.

The intracellular pathogen may enter the host organism’s cell via endocytosis.

Reducing endocytosis in a cell and/or subject reduces the amount of infectious particles, i.e. viruses or bacteria, that enter the cell. Reducing endocytosis is an effective way to treat or prevent infection.

Hence, in another aspect, the invention relates to an inhibitor of IKK-a or the IKK-a/NIK complex for use in reducing endocytosis of an intracellular pathogen in a cell and/or subject. Particularly, the cell may be a mammalian cell. The subject, i.e. the pathogen host, may be a mammal. The inhibitor of IKK-a or the IKK-a/NIK complex may reduce endocytosis compared to an untreated control cell and/or subject. The untreated control cell and/or subject is infected with the same pathogen as the treated cell and/or subject. In one embodiment, endocytosis comprises cell entry via the endolysosomal pathway. Reducing endocytosis means reducing the amount of endosomes, lysosomes, endolysosomes, intracellular vesicles or vacuoles in a cell and/or subject compared to an untreated control cell and/or subject infected with the same pathogen. Hence, in one embodiment, preventing or treating in infection comprises reducing endocytosis of an intracellular pathogen in a cell and/or subject compared to an untreated control cell and/or subject.

Notably, in another aspect, the invention also relates to an inhibitor of IKK-a or the IKK-a/NIK complex for use in reducing the total pathogen load of an intracellular pathogen in a cell and/or subject. The reduction in endocytosis directly results in a reduction of pathogen entry into the cell, which leads to an inability of the pathogen to survive and/or replicate. Hence, the total number of the pathogen is reduced. The inhibitor of IKK-a or the IKK-a/NIK complex may reduce total pathogen load of an intracellular pathogen in a cell and/or subject compared to an untreated control cell and/or subject. In another embodiment, preventing or treating an infection in a cell and/or subject comprises reducing cytoplasmic beta-catenin levels in a cell and/or subject treated with the inhibitor of IKK-a or the IKK-a/NIK compared to untreated control cell and/or subject.

In another aspect, the invention relates to an inhibitor of IKK-a or the IKK-a/NIK complex for use in preventing infection with an intracellular pathogen.

In another aspect, the invention relates to an inhibitor of IKK-a or the IKK-a/NIK complex for use in preventing or treating infection with a pathogen, wherein the pathogen is one or more virus or bacterium.

In another aspect, the invention relates to an inhibitor of IKK-a or the IKK-a/NIK complex for use in preventing or treating a viral infection.

Another aspect of the invention is the provision of an inhibitor of the IKK-a/NIK complex for use in preventing or treating infection with a pathogen in a cell and/or subject, wherein the pathogen belongs to a family selected from the group consisting of Coronaviridae, Poxviridae, Phenuiviridae, Flaviviridae, Picornaviridae, Pneumoviridae, Herpesviridae, Togaviridae, Orthomyxoviridae, Rhabdoviridae, Picornaviridae, Papillomaviridae, Arenaviridae, Enterobacteriaceae, Coxiellaceae, Brucellaceae, Listeriaceae, Rickettsiaceae, Chlamydiaceae, Mycobacteriaceae, Yersiniaceae, Vibrionaceae, Bartonellaceae, Francisellaceae, and Nocardiaceae. In one embodiment, the pathogen belongs to a family selected from the group consisting of Coronaviridae, Poxviridae, Phenuiviridae and Enterobacteriaceae.

In one embodiment, the pathogen is selected from the group consisting of severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome-related coronavirus (MERS- CoV), Vaccinia virus (VACV), Monkeypox virus (MPXV), Rift Valley fever virus (RVFV), Salmonella and Shigella.

In another aspect, the invention provides an inhibitor of IKK-a or the IKK-a/NIK complex for use in preventing or treating an infection with a pathogen in a cell and/or subject, wherein the infection is selected from the group consisting of severe acute respiratory syndrome (SARS), coronavirus disease 2019 (COVID-19), Middle East respiratory syndrome (MERS), Monkeypox, smallpox, Rift Valley fever, hepatitis C, hepatocellular carcinoma, lymphoma, dengue fever, causes foot-and-mouth disease, bronchiolitis, common cold and pneumonia, hemorrhagic fever, Kaposi’s sarcoma, primary effusion lymphoma, HHV8-associated multicentric Castleman’s disease and KSHV inflammatory cytokine syndrome, influenza, vesicular stomatitis, common cold, anal dysplasia, genital cancers such as vulva, vagina, cervix and penis cancer, mouth papilloma, oropharyngeal cancer, aseptic meningitis, encephalitis, meningoencephalitis, pappataci fever, hemorrhagic fever, salmonellosis, typhoid fever, paratyphoid fever, shigellosis, Q fever, brucellosis, listeriosis, gastroenteritis, meningitis, meningoencephalitis, typhus, rickettsial pox, boutonneuse fever, African tick-bite fever, Rocky Mountain spotted fever, Flinders Island spotted fever, Queensland tick typhus, pelvic inflammatory disease, trachoma, gonorrhoea, Legionnaires’ disease, Pontiac fever, tuberculosis, leprosy, Hansen’s disease, lymphadenitis, pulmonary disease, skin disease, plague, yersiniosis, reactive arthritis, pseudoappendicitis, cholera, tularemia, nocardiosis, septicaemia and cat scratch disease.

In one embodiment, the infection is selected from the group consisting of severe acute respiratory syndrome (SARS), coronavirus disease 2019 (COVID-19), Middle East respiratory syndrome (MERS), Monkeypox, Rift Valley fever, salmonellosis, typhoid fever, paratyphoid fever and shigellosis.

The inventors discovered that drugs specifically inhibiting either IKK-a or the NF-KB-inducing kinase (NIK) not only reduced the production of pro-inflammatory cytokines but, surprisingly, also significantly reduced the replication of the severe acute respiratory syndrome (SARS)- associated coronaviruses, SARS-CoV-1 and SARS-CoV-2. This finding is particular surprising since viruses of the phylum Pisuviricota, in particular SARS-CoV-2 does not contain a DNA binding site for NF-KB as their genome consist of single stranded RNA. The inventors found that the antiviral activity is governed by inhibition of IKK-a or the IKK-a/NIK complex, whereas the anti-inflammatory effect is due to the inhibition of the NF-KB pathway.

Hence, another aspect of the invention relates to an inhibitor of IKK-a for use in preventing or treating viral infection in a subject. In particular, the invention relates to an inhibitor of IKK-a for use in preventing or treating infection caused by a virus of the phylum Pisuviricota, more specifically a virus of the family Coronaviridae, in particular SARS-CoV, more specifically SARS-CoV-2. Hence the invention refers to an inhibitor of IKK-a for preventing or treating coronavirus disease 2019 (COVID-19) in a subject and/or for use in preventing or treating SARS-CoV-2 infection in a subject.

The inventors found that an inhibitor of IKB kinase-a (IKK-a) or an inhibitor of NF-KB-inducing kinase (NIK) are highly effective in inhibiting and reducing viral replication, in particular viral replication of SARS-CoV-2. In particular, inhibitors of the IKK-a/NIK complex, such as inhibitors of NIK and IKK-a (e.g. Amgen-16, IKK-16 and ACHP) show a strong antiviral effect. The inventors found that this effect is independent of NF-KB transcription factors.

Thus, in a specific embodiment the inhibitor of IKB kinase (IKK) is ACHP hydrochloride. It could be shown that ACHP is a highly potent antiviral agent inhibiting SARS-CoV-2 replication.

Another aspect of the invention is the provision of a pharmaceutical composition comprising the inhibitor of the invention and a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition further comprises one or more therapeutic agents, preferably anti-inflammatory agents, antibiotic agents or antiviral agents.

Figure Legends

Figure 1 : The canonical NF-KB signaling cascade is not involved in the control of SARS- CoV-2 replication

A. Schematic representation of the canonical (left) and non-canonical (right) NF-KB pathway. B-E. SARS-CoV-2-GFP viral reporter signal normalised to cell confluence (bottom) and confluence of cells as a measure of compounds’ cytotoxicity (top) upon treatment of A549- ACE2 cells with the shown concentrations of the indicated compound, and infection with SARS-CoV-2-GFP virus at MOI of 3. GFP reporter activity was measured by lncucyteS3 live cell fluorescent microscope system as a proxy of virus replication. Data depicts mean of 3 technical replicates, and is representative of 4 independent biological repeats where non- cytotoxic concentrations were used. Figure 2: IKK-a and NIK are the main regulators of SARS-CoV-2 in vitro

A-C. SARS-CoV-2-GFP viral reporter signal normalised to cell confluence (bottom) and confluence of cells as a measure of compounds’ cytotoxicity (top) upon treatment of A549- ACE2 cells with indicated concentrations of each compounds, and infection with SARS-CoV- 2-GFP virus at MOI of 3, as measured by lncucyteS3 live cell fluorescent microscope system. Data depicts mean of 3 technical replicates, and is representative of 4 independent biological repeats. D. Quantification of produced SARS-CoV-2 infectious particles upon inhibitor treatment. VeroE6 cells were pre-treated with the indicated compounds or the vehicle solution at the indicated concentration for 4h, and then infected with WT SARS-CoV-2-MUC1 (MOI 1) for 48h. Supernatant was collected and used to determine the amount of infectious virus particles by plaque assay. Pfu: plaque forming units. Means +/- sd of 3 independent biological experiments. E. Reporter assay quantifying the inhibition of the NF-KB pathway. HEK293T-RI cells expressing a Firefly- NF-KB reporter and EIF1 a-renilla control plasmid were pre-treated with IKK-16, Amgen16, ACHP or the vehicle solution at the indicated concentrations for 3h, and then activated by TNFa (20ng/ml) or I L-1 p (0.1ng/ml) for 6h. Firefly signal was normalized to the Renilla signal. Mean of 3 independent biological experiments.

Figure 3: IKK-a and NIK are essential to SARS coronaviruses replication in a NF-KB- independent manner

A. SARS-CoV-2-GFP viral reporter signal normalised to cell confluence upon treatment of A549-ACE2 cells with indicated concentrations of cytokines promoting the canonical (TNFa ) or the non-canonical (LT ) N FKB pathway, and infection with SARS-CoV-2-GFP virus at MOI of 3, as measured by lncucyteS3 live cell fluorescent microscope system. Data depicts mean of 3 technical replicates, and is representative of 4 independent biological repeats. B. A549- ACE2 cells depleted for NF-KB-related genes by CRISPR/Cas9 and non-targeting control (NTC) KO cell line were infected with SARS-CoV-2-GFP (MOI: 3) and GFP expression measured by lncucyteS3 live cell fluorescent microscopy. Data depicts mean GFP expression normalized to cell density of 4 biological replicates. Left panel: KO cells targeting NF-KB transcription factor (RelA, RelB) were either treated with DMSO or ACHP (2pM) 4h prior infection as indicated. Right panel: IKK-a KO cells were compared to upstream adaptor (NIK) KO cells and previously known interactors (DDX3X KO). C. Quantification of total intracellular RNA levels of SARS-CoV-1 (grey bars) or SARS-CoV-2 (black bars) 24h post-infection of IKK- a KO, NIK KO, RelA KO, RelB KO, DDX3X KO and NTC A549-ACE2 cells with SARS-CoV- FRA1 and SARS-CoV-2-MUC1 , respectively (MOI 1). SARS RNA levels were measured by RT-qPCR and normalized to the house-keeping gene RPLPO. Data depicts SARS levels compared to NTC A549-ACE2 cells set at 100%. Mean +/- sd of 4 independent biological repeats.

Figure 4: The IKK-a inhibitor ACHP can reduce SARS-CoV-2 spread ex vivo by limitating virus entry

A. SARS-CoV-2-GFP viral reporter signal normalised to cell confluence upon treatment of NHBE cells with indicated concentrations of ACHP 4h prior infection, and infection with SARS- CoV-2-GFP virus at MOI of 3, as measured by lncucyteS3 live cell fluorescent microscope system. Data depicts mean of 6 independent healthy donors. B. Quantification of total intracellular WT SARS-CoV-2-MUC1 of NHBE cells 48h post-infection (MOI 1). Cells were treated with indicated concentrations of ACHP 4h prior infection. Mean +/- sd of 6 independent healthy donors. C. Quantification of total intracellular SARS-CoV-2 omicron variant virus of NHBE cells 24h and 48h post-infection (MOI 1). Cells were treated with 2pM ACHP 4h prior infection. Mean +/- sd of 5 independent healthy donors. D. A549-ACE2 cells were pre-treated 4h with either DMSO (vehicle) or ACHP (2pM) before infection by VSV-GFP (MOI: 0.1), SARS- CoV-2 Spike pseudotyped VSV-GFP (MOI 1) and SARS-CoV-2-GFP (MOI 3), respectively. Data depicts mean of viral reporter signal observed at 24h post- infection normalised to cell confluence and compared to DMSO-treated cells from 3 technical replicates measured by lncucyteS3 live cell fluorescent microscope system, and is representative of 3 independent biological repeats.

Figure 5: Antiviral activity of IKK inhibitors against SARS-CoV-2. (a) A549-ACE2 cells were treated with an IL-1 p or a LT- b agonistic antibody for 4 h and infected with SARS-CoV-2 (strain: IMB-MUC1) (MOI 1) for 24 h. Abundance of SARS-CoV- 2 N mRNA normalized to RPLPO as quantified by qRT-PCR. Shown is the average fold change vs DMSO control of three-four independent experiments, (b) A549-ACE2 cells were pretreated with the indicated NF-KB pathway inhibitors for 4 h and subsequently infected with SARS-CoV-2-GFP reporter virus (MOI 3). GFP signal and cell growth were monitored by fluorescence life cell imaging for 48 h. Heatmap shows GFP positive area normalized to cell confluence of inhibitor treated vs vehicle control conditions at 48 hpi (. Data represents average of three independent biological repeats, (c) Normal human bronchial epithelial cells (NHBE) from five donors were treated with control (DMSO) or inhibitors from (c) for 6 h and infected with SARS-CoV-2 (MOI 1). RNA was isolated at 24 hpi and SARS-CoV-2 N mRNA quantified by RT-qPCR. Graph shows average N abundance normalized to RPLPO of inhibitor treated cells relative to control (DMSO) (n=5). (d) qRT-PCR of SARS-CoV-2 or Omicron infected NHBE cells (MOI 1) that were pre-treated for 6 h with ACHP at 0.2 or 2pM (n=5). Shown is SARS-CoV-2 N RNA expression normalized to RPLPO compared to DMSO-treated cells at 24 hpi. (e-f) A549-ACE2 cells were pre-treated with ACHP for 4 h and infected with SARS-CoV-2 for 24 h. Cells were fixed and immunostained for N. Representative images (e) and quantification of N positive area normalized to cell confluence (f) of three biologically independent experiments. Hpi: hours post infection, FC: fold change. All data are represented as mean +/- sd (Two-sided student t-test, unadjusted p-value). *: p-value < 0.05; **: p-value < 0.01 ; ***: p-value < 10'³.

Figure 6: IKKa has proviral activity that is independent of NF-KB transcription factors, (a) A549-ACE2 cells were knocked-out (KO) for essential components of the NF-KB pathway using CRISPR/Cas9 mediated gene editing. Cells were infected with SARS-CoV-2-GFP (MOI 3) and the GFP signal was quantified at 48 hpi. Graph shows average GFP area normalized to total cell confluence comparing KO and non-targeting control (NTC) cells from four biologically independent experiments, (b) Western blot showing SARS-CoV-2 N and p-actin in SARS-CoV-2 infected (MOI 1) A549-ACE2 cells depleted for IKKa, RelA or RelB and control cells (NTC) at 24 hpi. One representative experiment of three biologically independent experiments is shown, (c) A549-ACE2 IKKa, RelA, RelB, c-Rel KO and NTC cells were infected with SARS-CoV-2 at an MOI of 1 for 24 h and abundance of viral RNA relative to RPLPO mRNA was quantified by qRT-PCR (four biologically independent experiments), (d) A549-ACE2 RelA/RelB double KO (dKO) and NTC cells were pre-treated with ACHP (2pM) for 4 h before infection with SARS-CoV-2 (MOI 1). Viral RNA (relative to RPLPO) was measured at 6 and 24 hpi by RT-qPCR. Data are normalized to DMSO-treated NTC cells (n=3). (e) A549-ACE2 RelA and RelB KO and NTC cells were pre-treated for 4 h with ACHP (2pM) and subsequently infected with SARS-CoV-2-GFP reporter virus (MOI 3). Quantification of GFP signal as in (a) from four biologically independent experiments, (f) A549-ACE2 IKKa, RelA, RelB, c-Rel KO and NTC cells were infected with SARS-CoV-1 (isolate FRA1) at an MOI of 1 for 24 h and analyzed as in (c) in four biologically independent experiments, hpi: hours post-infection; FC: fold change. All data are represented as mean +/- sd (Two-sided student t- test, unadjusted p-value). *: p-value < 0.05; **: p-value < 0.01 ; ***: p-value < 10^-3.

Figure 7: IKK inhibition perturbs endosomal uptake and SARS-CoV-2 entry, (a) A549-ACE2 cells were pre-treated with the indicated concentrations of ACHP for 4 h and infected with SARS-CoV-2 WT. RT-qPCR quantification of viral RNA (SARS-CoV-2 N gene relative to RPLPO) at 1 , 6 and 24 hpi (MOI 1). Data are normalized to DMSO-treated cells (n=3). (b) A549-ACE2 cells were treated for 4 h with the indicated inhibitors (ACHP: 2pM, Celatrol: 1 pM, E64d: 1 pM) and then infected with VSV-GFP (MOI 0.1) or SARS-CoV-2 Spike- pseudotyped VSV-GFP (VSV-Spike) (MOI 1). GFP signal and cell growth were analyzed after 24 hpi by live-cell imaging. The graph shows GFP area normalized to total cell confluence of DMSO- and inhibitor-treated cells (n=4). (c) A549-ACE2 IKKa KO, IKK KO and NTC cells were infected with VSV-GFP or VSV-Spike reporter viruses and were analyzed as in (b)(n=4). (d) A549-ACE2 cells were pre-treated for 4 h with ACHP (2pM) or vehicle control (DMSO) and subsequently treated with virus-like particles (VLPs) bearing SARS-CoV-2 structural proteins and a p-lactamase reporter for 6 h. Images show p-lactamase activity of internalized particles as analyzed by confocal microscopy. Representative images of three biologically independent experiments are shown; scale bar = 20pm. (e-f) A549-ACE2 cells were treated for 4 h with either DMSO, ACHP (2pM), Celastrol (1 M) or E64d (1 M) before addition of pHrodo dextran beads (e) or lysotracker (f). Graphs of fluorescent signals normalized to cell confluence obtained after 24 h (e) or 1 h (f), respectively, by live-cell imaging (three biologically independent experiments), (g-j) A549-ACE2 cells pre-treated with either DMSO or ACHP (2pM) for 4 h and infected with SARS-CoV-2 VLPs (g) or SARS-CoV-2 (i)(MOI 1) for 6 h. Cells were stained with DAPI (nuclei) and antibodies specific for LAMP1 and SARS-CoV-2 S. Colocalization of LAMP1 and S was quantified as double positive LAMP1+/S+ area normalized to the total area of LAMP1 staining for SARS-CoV-2 VLPs treatment (h) or SARS-CoV-2 infection (j) (three biologically independent experiments). Scale bar = 20pm. hpi: hours postinfection; FC: fold change. All data are represented as mean +/- sd (Two-sided student t-test, unadjusted p-value). *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 10'³.

Figure 8: IKKa regulates p-catenin phosphorylation and its transcriptional signature.

(a) Phosphoproteomics analysis of IKKa KO and NTC cells that were infected with SARS-CoV- 2 (MOI 1) for 6 h. (b) The volcano plot shows fold change in host protein phosphosites (x-axis) and corresponding p-value (y-axis) based on a student's t-test (two-sided, permutation-based FDR, 0.05 FDR, n=3). Phosphosites with significant change in phosphorylation are highlighted, (c) Schematic representation of p-catenin (CTNNB1) with known and newly identified phosphosites and the regulating kinases, (d) A549-ACE2 IKKa KO cells, or NTC controls pretreated for 4 h with DMSO or Ipatasertib (10pM), were infected with SARS-CoV-2 (MOI 1) or not for the indicated time points, and and subjected to western blot analysis for the indicated proteins. One representative experiment of three biologically independent experiments, (e) Transcriptome analysis of A549-ACE2 IKKa, RelA or RelB KO and NTC cells infected with SARS-CoV-2 (MOI 1) for 24 h. (f) Figure shows the number of genes that are significantly down- or up-regulated in the indicated KO cell vs NTC control (n=3). (g) KEGG pathway enrichment analysis on genes that were significantly downregulated in IKKa KO vs NTC cells, (h) A549-ACE2 were pre-treated or not (vehicle) with EGF (100ng/ml) or Ipatasertib (10pM) for 6 h and infected with SARS-CoV-2 at (MOI 1). Graph shows RPLP0 normalized SARS- CoV-2 N RNA abundance at 24 hpi in relation to vehicle control (n=3). (i) A549-ACE2 cells were pre-treated with Ipatasertib (10pM) or DMSO and infected with SARS-CoV-2-GFP reporter virus. Graph shows GFP signal normalized to total cell confluence at 48 hpi (three biologically independent experiments), (j) A549-ACE2 cells were pre-treated as in (i) and infected with either VSV-GFP (MOI 0.1) or VSV-S (MOI 1). Graph shows GFP signal normalized to total cell confluence fold change between DMSO- and Ipatasertib-treated cells (three biologically independent experiments), (k-l) A549-ACE2 cells were pre-treated as in (i), and the fluorescence of endocytosed pHrodo dextran beads (k) or lysotracker (I) were analyzed by live-cell imaging (three biologically independent experiments), (m) A549-ACE2 cells were pre-treated for 4 h with either Ipatasertib (10pM), ACHP (2pM) or a combination of both before infection with SARS-CoV-2-GFP (MOI 3). GFP signal normalized to total cell confluence in relation of DMSO- and Ipatasertib- and/or AC HP-treated cells (n=3) is shown, hpi: hours postinfection; FC: fold change. All data are represented as mean +/- sd (Two-sided student t-test, unadjusted p-value). *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 10'³.

Figure 9: IKKa and AKT activity is required for diverse intracellular pathogens, (a-c) A549 cells were pre-treated with either ACHP (2pM), E64d (1 M) or Ipatasertib (10pM) for 4 h and infected with VACV-GFP reporter virus (MOI 0.05)(a), MPXV (MOI 1 )(b) and RVFV- GFP (MOI 0.1)(c). (a, c) GFP signal and cell growth were monitored at 24 hpi by live-cell imaging. Shown is GFP signal normalized to total cell confluence vs vehicle conditions (three biologically independent experiments), (b) At 24 hpi, MPXV DNA and genomic RPLP0 levels were quantified by qPCR. Data of RPLP0 normalized MPXV DNA levels in relation to DMSO control are shown (three biologically independent experiments), (d-e) Immortalized murine bone-marrow derived macrophages (iBMDMs) were pre-treated for 3 h with the indicated concentrations of ACHP. Treated cells were infected with Salmonella enterica serovar Typhimurium SL1344 (d) and Shigella flexneri (e) for 1 h. After cell lysis, internalized bacteria were measured by assessment of colony forming units per ml (CFU/ml) (n=4-6). hpi: hours post-infection; FC: fold change. All data are represented as mean +/- sd (Two-sided student t- test, unadjusted p-value). *: p-value < 0.05; **: p-value < 0.01 ; ***: p-value < 10^-3.

Figure 10: Antiviral activity of drugs targeting IKK and NIK.

(a) Schematic representation of major components of the canonical and non-canonical NF-KB pathway and inhibitors tested in this study, (b) Validation of anti-NF-KB activity of drugs tested in this study. HEK293R1 cells transfected with NF-KB FF-luc and EIF1a-ren reporter plasmids were pretreated with inhibitors at the indicated concentrations for 4 h and treated with I L-1 p (0.1ng/ml) for 4 h. Graphs show Firefly/Renilla signal (n=3). (c) A549-ACE2 were treated with indicated inhibitors at three different concentrations. Cell growth was monitored by live-cell imaging after 48 hours, and total cell confluence was compared to vehicle (DMSO) treated cells (n=3). (d) RT-qPCR quantification of IL-6 mRNA levels (relative to RPLP0) in vehicle (DMSO) or ACHP treated NHBE cells infected with SARS-CoV-2 for 24 h (related to Fig. 1d) (n=5). (e) Representative immunofluorescence images of NHBE cells pre-treated with DMSO or ACHP (0.2 and 2pM) for 4 h and infected with SARS-CoV-2-GFP reporter virus (MOI 3) for 48 h. Scale bar = 200pm. (f) Changes in virus spread from e are displayed as GFP signal normalized to total cell confluence fold change between the treated and vehicle conditions (n=6). FC: fold change. All data are represented as mean +/- sd (Two-sided student t-test, unadjusted p-value). *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 10-3.

Figure 11 : Validation of KO efficiency and identification of IKKa as proviral factor.

(a) The indicated KO cells were infected with SARS-CoV-2 for 24 h (MOI 1) and subjected to western blot analysis for the indicated proteins. One representative experiment of three biologically independent experiments, (b) A549-ACE2 cells genetically depleted for the indicated gene cells were tested for cytotoxic knocked-out genes. Cell growth was monitored by live-cell imaging for 48 hours, and total cell confluence was compared to control (NTC) cells (n=4). hpi: hours post-infection; FC: fold change. All data are represented as mean +/- sd (Two- sided student t-test, unadjusted p-value). *: p-value < 0.05; **: p-value < 0.01 ; ***: p-value < 10-3.

Figure 12: IKKa inhibition perturbs early events of SARS-CoV-2 infection.

(a) NHBE cells were pretreated with the indicated concentration of ACHP for the indicated time and infected with SARS-CoV-2. Graph shows RT-qPCR quantification of IL-6 mRNA levels (relative to RPLP0) after 24 h (related to Fig. 3a) (n=3). (b) Representative image of A549- ACE2 cells pre-treated with E64d (1 M) for 4 h before addition of virus-like particles (VLPs) bearing SARS-CoV-2 structural proteins and a p-lactamase reporter for 6 h. Image shows p- lactamase activity of internalized particles as analyzed by confocal microscopy (related to Fig. 3d); scale bar = 20pm. (c) A549-ACE2 cells were pre-treated with ACHP or E64d for 4 h and infected with Spike decorated VLPs. Co-localization of EEA1 and S were quantified as double positive EEA1+/S+ area normalized to the total area of EEA1 and compared to DMSO- treated cells (n=3 biological independent experiment), (d) A549-ACE2 cells were pre-treated with either DMSO or ACHP (2pM) for 4 h and further infected with SARS-CoV-2 (MOI 1) for 2 h. Cells were stained for nuclei, LAMP1 and SARS-CoV-2 S (n=3). Representative images, scale bar = 20pm. FC: fold change. All data are represented as mean +/- sd (Two-sided student t-test, unadjusted p-value). *: p-value < 0.05; **: p-value < 0.01 ; ***: p-value < 10-3.

Figure 13: p-catenin activity is abrogated in A549-ACE2 IKKa KO cells.

(a) Transcriptome analysis of A549-ACE2 IKKa, RelA, RelB KO and NTC control cells infected with SARS-CoV-2 (MO1 1) for 24 h (n=3). Volcano plots show relative mRNA expression levels in the indicated cell line in comparison to A549-ACE2 NTC cells. Significant hits (p-value < 0.05) are highlighted in black. Diamonds data points that were truncated since the real value lies outside the plotted area, (b) Transcriptom ic analysis of ACHP-treated (2pM) NHBE cells infected with SARS-CoV-2 (MOI 1) for 24 h (n=6). Volcano plot of mRNA expression changes in comparison to infection of DMSO-treated NHBE cells. Significant hits (p-value < 0.05) are highlighted in black. Diamonds indicate that the actual Iog2 fold change or p-value were truncated to fit into the plotted area, (c) Genes, differentially regulated between SARS-CoV-2 infected A549-ACE2 IKKa KO vs NTC control cells (p-adjusted < 0.05) were used for upstream promoter analysis. Top 7 transcription factors, associated with enriched motifs in the up- or down-regulated gene sets, are shown (related to Fig. 4d). The number of genes (ntargets) regulated by individual transcription factors is depicted alongside the enrichment score (NES67). (d) Expression levels of EPHB2 and c-Jun mRNA relative to RPLP0 mRNA in A549- ACE2 IKKa KO and NTC cells infected with SARS-CoV-2 (MOI 1) measured by qRT-PCR 24 hpi. (e) Pathway enrichment on genes that were significantly downregulated in ACHP-treated NHBE cells (related to b). hpi: hours post-infection; FC: fold change. All data are represented as mean +/- sd (Two-sided student t-test, unadjusted p-value). *: p-value < 0.05; **: p-value < 0.01 ; ***: p-value < 10-3.

Figure 14: The EGFR-AKT-IKKa pathway potentiates SARS-CoV-2 entry.

(a-b) A549-ACE2 cells pre-treated for 4 h with 100ng/ml of EGF (a) or with indicated concentrations of Ipatasertib (b) were infected with SARS-CoV-2-GFP reporter virus. GFP signal and cell growth were monitored at 48 hpi by live-cell imaging. Shown is GFP signal normalized to total cell confluence vs vehicle conditions (three biologically independent experiments), (c) A549-ACE2 cells pre-treated for 4 h with the indicated concentrations of Ipatasertib were infected with either VSV-GFP (MOI 0,1) or VSV-S (MOI 1). GFP signal and cell growth were analyzed at 24 hpi by live-cell imaging. The histogram shows GFP area normalized to total cell confluence of DMSO- and inhibitor-treated cells (n=3). (d-f) A549-ACE2 cells were pre-treated for 4 h with 100ng/ml of EGF (d) or with indicated concentrations of Ipatasertib (e-f) and the fluorescence signals of endocytosed pHrodo dextran beads (d-e) or lysotracker (f) were normalized to cell confluence obtained after 24 h or 1 h, respectively. Data shown as relative to vehicle-treated cells (n=3). (g) Analysis of propidium iodide (PI) uptake for 4 h as measured by Fluorescence (ex: 535nm, em. 617nm) of drug-treated cells in biological triplicate (related to Fig. 5d-e). FC: fold change. All data are represented as mean +/- sd (Two- sided student t-test, unadjusted p-value). *: p-value < 0.05; **: p-value < 0.01 ; ***: p-value < 10-3.

Figure 15: ACHP treatment of mice infected with SARS-CoV-2 K18-hACE2 C57BL/6 mice were infected with SARS-CoV-2 alpha variant (2500 pfu, intranasal) and treated at D-1 and DO with ACHP (2 mg/kg, intranasal). Twenty-four hours postinfection, lungs of infected mice were isolated. Ratio of SARS-CoV-2 transcript N and p-Actin was quantified in the lung samples by RT-qPCR as a measure of lung viral load. Mean ± SD of minimum n = 4 animals per condition; statistics were calculated using Student's two-sided t- test (*: p-value <0.05).

DETAILED DESCRIPTION OF THE INVENTION

Before the invention is described in detail with respect to some of its preferred embodiments, the following general definitions are provided.

The present invention as illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

The present invention will be described with respect to particular embodiments and with reference to certain figures but the invention is not limited thereto but only by the claims.

Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of” is considered to be a preferred embodiment of the term “comprising of”. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.

For the purposes of the present invention, the term “obtained” is considered to be a preferred embodiment of the term “obtainable”. If hereinafter e.g. an antibody is defined to be obtainable from a specific source, this is also to be understood to disclose an antibody which is obtained from this source.

Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated. The terms “about” or “approximately” in the context of the present invention denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of ±10%, and preferably of ±5%. Technical terms are used by their common sense. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following in the context of which the terms are used.

The term “inhibitor” includes small molecules, antibodies and binding fragments thereof, nonantibody protein scaffold proteins, aptamers and nucleotide based molecules, such as siRNAs or gRNAs. In preferred embodiments, the inhibitor is a small molecule.

The term “IKK-o/NIK complex” refers to a signaling complex comprising IKB kinase a (IKK-a) and NF-KB inducing kinase (NIK). IKK-a is encoded by the CHUK (component of inhibitor of nuclear factor kappa B kinase complex) gene in humans. IKK-a is also referred to as IKK 1. NIK is encoded by the MAP3K14 (Mitogen-activated protein kinase kinase kinase 14) gene in humans. NIK is a serine/threonine protein kinase. The complex may also comprise I KK-p, also referred to as IKK 2.

The term “inhibitor of IKK-a/NIK complex” refers to inhibitors of IKK-a and NIK. The inhibitors may inhibit both NIK and IKK-a or may inhibit only NIK or only IKK-a. Examples, which are not construed as limiting in any way, include the NIK inhibitor Amgen16 and the IKK-a inhibitors IKK-16 and ACHP. The term includes inhibitors that inhibit at least one molecule of the IKK- a/NIK complex and may further also inhibit other molecules, such as molecules of the NF-KB pathway. For example an inhibitor of IKK-a may also inhibit I KK-p (belonging to the N F-KB pathway). Preferably the inhibitor is an inhibitor of IKK-a. More preferably the inhibitor is ACHP.

In one embodiment, the term “inhibitor of IKK-a/NIK complex” also refers to inhibitors of the IKK complex.

In some embodiments “inhibitor of IKK-a/NIK complex” does not refer to an inhibitor of IKK-a that only acts on the canonical N F-KB pathway. In other words, in some embodiments an inhibitor that only inhibits the IKK-a functioning in combination with I KK-p but does not inhibit IKK-a functioning in combination with NIK, is not encompassed. In some embodiments, the inhibitor does not function through inhibition of N F-KB transcription factors. In some embodiments, the inhibitor does not function through direct inhibition of NF-KB pathway factors downstream of the IKK-a/NIK complex. Preferably, the inhibitor is not an inhibitor of IKK-p.

The phrase “treatment [of a cell and/or subject] with an inhibitor of IKK-a or IKK-a/NIK complex” refers to adding the inhibitor of IKK-a or IKK-a/NIK complex to the cell and/or subject. Adding the inhibitor may involve any route of administration. The skilled person is aware of methods for identification of inhibitors of the I KK-a/N IK complex, in particular of IKK-a and testing of inhibitors of the IKK-a/NIK complex, in particular IKK-a.

ACHP (IIIPAC name: 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4- ylpyridine-3-carbonitrile) also termed compound 4j or IKK-2 inhibitor VIII, is an IKK inhibitor. ACHP is well studied for its aqueous solubility, oral bioavailability, low clearance and great anti-inflammatory activity in vivo (Murata, T. et al. Synthesis and structure-activity relationships of novel IKK-p inhibitors. Part 3: Orally active anti-inflammatory agents. Bioorganic & Medicinal Chemistry Letters 14, 4019-4022 (2004)). ACHP may be in form of the ACHP hydrochloride.

Amgen16 also termed 1-[2-[1-(2-Amino-5-chloro-4-pyrimidinyl)-2,3-dihydro-1 H-indol-6- yl]ethynyl]cyclopentanol and 1-((1-(2-Amino-5-chloropyrimidin-4-yl)indolin-6- yl)ethynyl)cyclopentanol is an NIK inhibitor.

IKK-16 also termed IKK inhibitor VII and is an IKK inhibitor.

The term “endocytosis” means a process by which substances, such as pathogens or pathogenic particles, are brought into the cell. The extracellular substance is enveloped by an area of surrounding cell membrane of the host cell, which then buds off by invagination of the plasma membrane around the extracellular substance to form a vesicle which is mobile within the cytoplasm. The vesicle comprises the extracellular substance on the inside and a plasma membrane, i.e. a lipid bilayer, on the outside. Endocytosis may comprise receptor-mediated endocytosis, caveolae, pinocytosis, macropinocytosis and phagocytosis. The endocytosis pathway comprises endosomes, including early and late endosomes, endolysosome and lysosomes. Hence, the term “endocytosis” refers to the generation of intracellular vesicles comprising endosomes, endolysosomes and lysosomes and vacuoles.

Endocytic viruses and bacteria

All viruses are obligate intracellular pathogens. Many viruses enter the cell via endocytosis. This includes viruses with and without viral envelope. Pathogenic bacteria may also enter the cell via endocytosis.

In the first aspect, the invention provides an inhibitor of the IKK-a/NIK complex for use in preventing or treating infection with a pathogen in a cell and/or subject. In one embodiment, the pathogen is a virus or a bacterium. In one embodiment, the virus or bacterium belongs to a family selected from Coronaviridae, Poxviridae, Phenuiviridae, Flaviviridae, Picornaviridae, Pneumoviridae, Herpesviridae, Togaviridae, Orthomyxoviridae, Rhabdoviridae, Picornaviridae, Papillomaviridae, Arenaviridae, Enterobacteriaceae, Coxiellaceae, Brucellaceae, Listeriaceae, Rickettsiaceae, Chlamydiaceae, Mycobacteriaceae, Yersiniaceae, Vibrionaceae, Bartonellaceae, Francisellaceae, and Nocardiaceae.

In one embodiment, the virus or bacterium belongs to a species selected from the group consisting of the severe acute respiratory syndrome (SARS)-associated coronavirus 1 (SARS- CoV-1), SARS-CoV-2, vaccinia virus (VACV), monkeypox virus (MPXV), rift valley fever virus (RVFV), poliovirus, hepatitis C virus, foot-and-mouth disease virus, Vaccinia virus, respiratory syncytial virus, dengue virus, ebola virus, kaposi sarcoma virus, semliki forest virus, influenza A virus, vesicular stomatitis virus, human rhino 2 virus, mouse polyoma virus, human papilloma 16 virus, lassa virus and LCMV, uukuniemi virus, mimi virus, Salmonella, Shigella, Bartonella, Coxiella, Brucella, Listeria, Rickettsia, Chlamydia, Legionella, Mycobacterium, Yersinia, Vibrio, Francisella, and Nocardia.

In another embodiment, the virus or bacterium belongs to a family selected from Coronaviridae, Poxviridae, Phenuiviridae and Enterobacteriaceae.

In one embodiment, the virus or bacterium belongs to a species selected from the group consisting of severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome-related coronavirus (MERS-CoV), Monkeypox virus, Vaccinia virus and Rift Valley fever virus, Salmonella, and Shigella.

In one aspect of the invention, the inhibitor of the IKK-a/NIK complex is for use in preventing or treating infection, wherein the infection is selected from the group consisting of SARS, COVID-19, MERS, Monkeypox, smallpox, Rift Valley fever, hepatitis C, hepatocellular carcinoma, lymphoma, dengue fever, causes foot-and-mouth disease, bronchiolitis, common cold and pneumonia, hemorrhagic fever, Kaposi’s sarcoma, primary effusion lymphoma, HHV8-associated multicentric Castleman’s disease and KSHV inflammatory cytokine syndrome, influenza, vesicular stomatitis, common cold, anal dysplasia, genital cancers such as vulva, vagina, cervix and penis cancer, mouth papilloma, oropharyngeal cancer, aseptic meningitis, encephalitis, meningoencephalitis, pappataci fever, hemorrhagic fever, salmonellosis, typhoid fever, paratyphoid fever, shigellosis, Q fever, brucellosis, listeriosis, gastroenteritis, meningitis, meningoencephalitis, typhus, rickettsial pox, boutonneuse fever, African tick-bite fever, Rocky Mountain spotted fever, Flinders Island spotted fever, Queensland tick typhus, pelvic inflammatory disease, trachoma, gonorrhoea, Legionnaires’ disease, Pontiac fever, tuberculosis, leprosy, Hansen’s disease, lymphadenitis, pulmonary disease, skin disease, plague, yersiniosis, reactive arthritis, pseudoappendicitis, cholera, tularemia, nocardiosis, septicaemia and cat scratch disease.

In one embodiment, the infection is selected from the group consisting of SARS, COVID-19, MERS, Monkeypox, smallpox, Rift Valley fever, hemorrhagic fever, common cold, pneumonia, encephalitis, salmonellosis, cholera, dysentery, brucellosis, listeriosis, thyphoid fever, paratyphoid fever, shigellosis, legionellosis, tuberculosis, leprosy, tularemia, nocardiosis, pneumonia, chlamydial infection, gonorrhoea, meningitis, septicaemia, typhus, spotted fever, and cat-scratch disease.

In another embodiment, the infection is selected from the group consisting of SARS, COVID- 19, MERS, Monkeypox, hemorrhagic fever, common cold, pneumonia and encephalitis, salmonellosis, typhoid fever, paratyphoid fever and shigellosis.

In another embodiment, the infection is selected from the group consisting of SARS, COVID- 19, MERS, Monkeypox, common cold and pneumonia, salmonellosis, typhoid fever, paratyphoid fever, shigellosis, tularemia, nocardiosis and cholera.

In another embodiment, the infection is selected from the group consisting of SARS, COVID- 19, MERS, Monkeypox, Rift Valley fever, salmonellosis, typhoid fever, paratyphoid fever and shigellosis.

In one embodiment, the invention relates to the use of an inhibitor of the I KK-a/NIK complex in the manufacture for a medicament for the prevention or treatment of infection with a pathogen in a cell and/or subject. Preferably, the pathogen is an endocytic virus or endocytic bacterium.

Many viruses enter the cell via endocytosis. This includes viruses with and without viral envelope. In one embodiment, the pathogen is a virus, preferably a virus that enters the cell via endocytosis. The pathogen may be one or more viruses. The pathogen may be one or more viruses and one or more additional pathogens. The pathogen may be one or more different species of viruses.

When an infection with a virus is referred to herein, this includes infection with one species of virus or with more than one species of virus.

The infection may be with at least one, at least two, at least three, at least four, or at least five species of virus. Viruses that enter the cell via endocytosis comprise severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2), Middle East respiratory syndrome-related coronavirus (MERS-CoV), Monkeypox virus, Rift Valley fever virus, Hepatitis C virus, Foot-and-mouth disease virus, Vaccinia virus, Respiratory syncytial virus, Dengue virus, Ebola virus, Kaposi sarcoma virus, Semliki forest virus, Influenza A virus, Vesicular stomatitis virus, Human rhino 2 virus, Human papilloma 16 virus, Lassa virus, lymphocytic choriomeningitis virus (LCMV), and Uukuniemi virus.

SARS-CoV-1 is a virus of the family Coronavridae and causes severe acute respiratory syndrome (SARS).

SARS-CoV-2 is a virus of the family Coronavridae and causes coronavirus disease 2019 (COVID-19).

MERS-CoV is a virus of the family Coronavridae and causes Middle East respiratory syndrome (MERS).

Monkeypox virus is a virus of the family Poxviridae and causes Monkeypox.

Vaccinia virus is a virus of the family Poxviridae and causes smallpox.

Rift Valley fever virus is a virus of the family Phenuiviridae and causes Rift Valley fever Hepatitis C virus is a virus of the family Flaviviridae and causes hepatitis C, hepatocellular carcinoma and lymphoma.

Dengue virus is a virus of the family Flaviviridae and causes dengue fever.

Foot-and-mouth disease virus is a virus of the family Picornaviridae and causes foot-and- mouth disease.

Respiratory syncytial virus is a virus of the family Pneumoviridae and causes respiratory infections such as bronchiolitis, common cold and pneumonia.

Ebola virus is a virus of the family Filoviridae and causes hemorrhagic fever, i.e. Ebola virus disease.

Kaposi sarcoma-associated herpesvirus or Human herpes virus 8 (HHV 8) is a virus of the family Herpesviridae and causes Kaposi’s sarcoma, primary effusion lymphoma, HHV8- associated multicentric Castleman’s disease and KSHV inflammatory cytokine syndrome.

Semliki forest virus is a virus of the family Togaviridae and causes encephalitis. Influenza A virus is a virus of the family Orthomyxoviridae and causes influenza.

Vesicular stomatitis virus is a virus of the family Rhabdoviridae and causes vesicular stomatitis. Human rhino 2 virus is a virus of the family Picornaviridae and causes the common cold.

Human papilloma 16 virus is a virus of the family Papillomaviridae and causes anal dysplasia, genital cancers such as vulva, vagina, cervix and penis cancer, mouth papilloma and oropharyngeal cancer. Lymphocytic choriomeningitis virus is a virus of the family Arenaviridae and causes aseptic meningitis, encephalitis or meningoencephalitis.

Lassa mammarenavirus is a virus of the family Arenaviridae and causes hemorrhagic fever. Uukuniemi virus is a virus of the family Phenuiviridae and causes pappataci fever, encephalitis and hemorrhagic fever.

The inhibitor may be used for prevention or treatment of viral infection. Accordingly the inhibitor may be administered before viral infection, during viral infection or after viral infection of the subject.

Hence, in one embodiment, the virus belongs to a family selected from Coronaviridae, Poxviridae, Phenuiviridae, Flaviviridae, Picornaviridae, Pneumoviridae, Herpesviridae, Togaviridae, Orthomyxoviridae, Rhabdoviridae, Picornaviridae, Papillomaviridae, and Arenaviridae.

In one embodiment, the virus belongs to a species selected from the group consisting of the severe acute respiratory syndrome (SARS)-associated coronavirus 1 (SARS-CoV-1), SARS- CoV-2, vaccinia virus (VACV), monkeypox virus (MPXV), rift valley fever virus (RVFV), poliovirus, hepatitis C virus, foot-and-mouth disease virus, Vaccinia virus, respiratory syncytial virus, dengue virus, ebola virus, kaposi sarcoma virus, semliki forest virus, influenza A virus, vesicular stomatitis virus, human rhino 2 virus, mouse polyoma virus, human papilloma 16 virus, lassa virus and LCMV, uukuniemi virus, and mimi virus.

In another embodiment, the virus belongs to a family selected from Coronaviridae, Poxviridae and Phenuiviridae.

In one embodiment, the virus belongs to a species selected from the group consisting of severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome-related coronavirus (MERS- CoV), Monkeypox virus, Vaccinia virus and Rift Valley fever virus.

In one aspect of the invention, the inhibitor of the IKK-a/NIK complex is for use in preventing or treating infection, wherein the infection is a virus infection selected from the group consisting of SARS, COVID-19, MERS, Monkeypox, smallpox, Rift Valley fever, hepatitis C, hepatocellular carcinoma, lymphoma, dengue fever, causes foot-and-mouth disease, bronchiolitis, common cold and pneumonia, hemorrhagic fever, Kaposi’s sarcoma, primary effusion lymphoma, HHV8-associated multicentric Castleman’s disease and KSHV inflammatory cytokine syndrome, influenza, vesicular stomatitis, common cold, anal dysplasia, genital cancers such as vulva, vagina, cervix and penis cancer, mouth papilloma, oropharyngeal cancer, aseptic meningitis, encephalitis, meningoencephalitis, pappataci fever, and hemorrhagic fever.

In one embodiment, the infection is selected from the group consisting of SARS, COVID-19, MERS, Monkeypox, smallpox, Rift Valley fever, hemorrhagic fever, common cold, pneumonia and encephalitis. In another embodiment, the infection is selected from the group consisting of SARS, COVID-19, MERS, Monkeypox, and Rift Valley fever. In another embodiment, the infection is selected from the group consisting of SARS, COVID-19, MERS, Monkeypox, hemorrhagic fever, common cold, pneumonia and encephalitis. In another embodiment, the infection is selected from the group consisting of SARS, COVID-19, MERS, Monkeypox, common cold and pneumonia.

The virus may be a virus of the phylum Pisuviricota. In one embodiment the virus is of the class Pisoniviricetes. In a more specific embodiment the virus is of the order Nidovirales. In an even more specific embodiment the virus is of the family Coronaviridae. In an even more specific embodiment the virus is of the genus Betacoronavirus, such as the subgenus Sarbecovirus. Preferably, the virus is of the species Severe acute respiratory syndrome-related coronavirus strain. Most preferably, the virus is Severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2).

SARS-CoV-2, also termed 2019-nCoV, refers to severe acute respiratory syndrome coronavirus-2 firstly described by Zhu et al. (Zhu, N. et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019. N. Engl. J. Med. 382, 727-733 (2020)) and variants thereof, e.g. without limitation variant B.1.1.7 (also known as 20I/501Y.V1 , VOC 202012/01), B.1.351 (20H/501Y.V2), P1 (501Y.V3), Delta (B.1.617.2) and Omikron (B1.1.529).

COVID-19 is a contagious disease caused by SARS-CoV-2.

Treating or preventing of COVID-19 may include treating or preventing at least one of lung fibrosis, interstitial pneumonia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), aveolar damage, kidney injury, vasculopathy, cardiac injury, acute myocardial injury, chronic damage to the cardiovascular system, thrombosis and venous thromboembolism, in a patient with COVID-19. In a specific embodiment lung fibrosis, interstitial pneumonia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), kidney injury, such as proteinuria and acute kidney injury, and vasculopathy are triggered by COVID-19. Viral replication is reduced or inhibited means that viral replication is reduced or inhibited compared to a control which is not treated by the inhibitor. Viral replication may be measured by detection of viral nucleic acid levels (e.g. viral RNA levels) by quantitative RT-PCR or by quantifying infectious particles.

In one embodiment, the invention relates to the use of an inhibitor of the I KK-a/NIK complex in the manufacture for a medicament for the prevention or treatment of infection with a virus in a cell and/or subject.

The invention is based on the surprising finding that inhibitors of the I KK-a/NIK complex, in particular of IKK-a inhibit or reduce viral growth. In particular, the invention is based on the surprising finding that inhibitors of the IKK-a/NIK complex show an antiviral effect against a virus of the phylum Pisuviricota, in particular of the family of Coronaviridae, more particular SARS-CoV, even more particular SARS-CoV-2.

Accordingly, another aspect of the invention relates to an inhibitor of IKK-a for preventing or treating viral infection, such as viral infection with a virus of the family Coronaviridae, in particular with SARS-CoV-2. In particular the invention is directed to an inhibitor of IKK-a for preventing or treating infection with a virus of the family Coronaviridae in a subject. In a specific embodiment the invention refers to an inhibitor of IKK-a for preventing or treating SARS-CoV- 2.

The inventors discovered that drugs specifically inhibiting either IKK-a or the NF-KB-inducing kinase (NIK) not only reduced the production of pro-inflammatory cytokines but, surprisingly, also significantly reduced the replication of the severe acute respiratory syndrome (SARS)- associated coronaviruses, SARS-CoV-1 and SARS-CoV-2. This finding is particular surprising since viruses of the phylum Pisuviricota, in particular SARS-CoV-2 does not contain a DNA binding site for NF-KB as their genome consist of single stranded RNA. The inventors found that the antiviral activity is governed by inhibition of IKK-a or the IKK-a/NIK complex.

Hence another embodiment of the invention relates to an inhibitor of IKK-a for use in preventing or treating viral infection in a subject. In particular, the invention relates to an inhibitor of IKK- a for use in preventing or treating infection caused by a virus of the phylum Pisuviricota, more specifically a virus of the family Coronaviridae, in particular SARS-CoV, more specifically SARS-CoV-2. Hence the invention refers to an inhibitor of IKK-a for preventing or treating coronavirus disease 2019 (COVID-19) in a subject and/or for use in preventing or treating SARS-CoV-2 infection in a subject. The inventors found that an inhibitor of IKB kinase-a (IKK-a) or an inhibitor of NF-KB-inducing kinase (NIK) are highly effective in inhibiting and reducing viral replication, in particular viral replication of SARS-CoV-2. In particular, inhibitors of the IKK-a/NIK complex, such as inhibitors of NIK and IKK-a (e.g. Amgen-16, IKK-16 and ACHP) show a strong antiviral effect. The inventors found that this effect is independent of NF-KB transcription factors.

Thus, in a specific embodiment the inhibitor of NF-KB kinase (IKK) is ACHP hydrochloride. It could be shown that ACHP is a highly potent antiviral agent inhibiting SARS-CoV-2 replication. In one embodiment, treatment with an inhibitor of the IKK-a/NIK complex, such as ACHP or a pharmaceutically acceptable salt thereof, leads to a reduction of SARS-CoV-2 viral transcripts and SARS-CoV-2 viral particles compared to untreated control subjects. Accordingly, one embodiment is directed to ACHP or pharmaceutical acceptable salts thereof for use in reducing SARS-CoV-2 viral transcripts in a subject infected with SARS-CoV-2. Another embodiment is directed to ACHP or pharmaceutical acceptable salts for use in reducing SARS-CoV-2 viral particles in a subject infected with SARS-CoV-2.

In one embodiment, treatment with an inhibitor of the IKK-a/NIK complex, such as ACHP or a pharmaceutically acceptable salt thereof, inhibits virus replication in the respiratory system of a subject.

Accordingly, one embodiment is directed to an inhibitor of the IKK-a/NIK complex, such as ACHP or pharmaceutical acceptable salts thereof, for use in inhibiting virus replication in the respiratory system of a subject infected with SARS-CoV-2.

In one embodiment, the respiratory system comprises one or more of nose, nasal cavities, sinuses, pharynx, larynx, trachea, bronchi, bronchiole, alveolar ducts and alveoli. In one embodiment, the respiratory system is lungs.

Another embodiment is directed to an inhibitor of the IKK-a/NIK complex, such as ACHP or pharmaceutical acceptable salts thereof, for use in treating lung fibrosis, interstitial pneumonia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), kidney injury, such as proteinuria and acute kidney injury, and vasculopathy and other extrapulmonary manifestations of COVID-19 (e.g. thrombotic complications, myocardial dysfunction and arrhythmia, acute coronary syndromes, gastrointestinal symptoms, hepatocellular injury, hyperglycemia and ketosis, neurologic illnesses, ocular symptoms, and dermatologic complications). The inhibitor may be used for prevention or treatment of viral infection. Accordingly the inhibitor may be administered before viral infection or after viral infection of the subject.

Pathogenic bacteria may also enter the cell via endocytosis. Hence, in one embodiment, the pathogen is a bacterium, preferably a bacterium that enters the cell via endocytosis. The pathogen may be one or more bacteria. The pathogen may be one or more bacteria and one or more additional pathogens. The pathogen may be one or more different species of bacteria. When an infection with a bacterium or bacteria is referred to herein, this includes infection with one species of bacterium or with more than one species of bacterium.

The infection may be with at least one, at least two, at least three, at least four, or at least five species of bacterium.

Bacteria that can enter the cell via endocytosis include Salmonella, Shigella, Bartonella, Coxiella, Brucella, Listeria, Rickettsia, Chlamydia, Legionella, Mycobacterium and Yersinia.

Salmonella belongs to the family of Enterobacteriaceae and causes salmonellosis, typhoid fever and paratyphoid fever.

Shigella belongs to the family of Enterobacteriaceae and causes shigellosis, i.e. dysentery. Coxiella belongs to the family of Coxiellaceae and causes ,Q fever.

Brucella belongs to the family of Brucellaceae and causes brucellosis, i.e. gastric fever.

Listeria belongs to the family of Listeriaceae and causes listeriosis, gastroenteritis, meningitis and meningoencephalitis.

Rickettsia belongs to the family of Rickettsiaceae and causes typhus, rickettsial pox, boutonneuse fever, African tick-bite fever, Rocky Mountain spotted fever, Flinders Island spotted fever, and Queensland tick typhus.

Chlamydia belongs to the family of Chlamydiaceae and causes genital disease including pelvic inflammatory disease, and trachoma.

Legionella belongs to the family of Legionella and causes Legionnaires’ disease and Pontiac fever.

Mycobacterium belongs to the family of Mycobacteriaceae and causes tuberculosis, leprosy, Hansen’s disease, lymphadenitis, pulmonary disease and skin disease.

Yersinia belongs to the family of Yersiniaceae and plague, yersiniosis, reactive arthritis and pseudoappendicitis.

Vibrio belongs to the family of Vibrionaceae and causes cholera.

Bartonella belongs to the family of Bartonellaceae and causes cat scratch disease. Francisella belongs to the family of Francisellaceae and causes tularemia. Nocardia belongs to the family of Nocardiaceae and causes nocardiosis.

The inhibitor may be used for prevention or treatment of bacterial infection. Accordingly the inhibitor may be administered before bacterial infection, during bacterial infection or after bacterial infection of the subject.

As used herein, “bacterium” and “bacteria” is used interchangeably and refers to one species of bacterium.

In one embodiment, the bacterium belongs to a family selected from Enterobacteriaceae, Coxiellaceae, Brucellaceae, Listeriaceae, Rickettsiaceae, Chlamydiaceae, Mycobacteriaceae, Yersiniaceae, Vibrionaceae, Bartonellaceae, Francisellaceae, and Nocardiaceae.

In one embodiment, one or more bacteria is selected from the group consisting of Salmonella, Shigella, Bartonella, Coxiella, Brucella, Listeria, Rickettsia, Chlamydia, Legionella, Mycobacterium, Yersinia, Vibrio, Francisella, and Nocardia.

In one aspect of the invention, the inhibitor of the IKK-a/NIK complex is for use in preventing or treating infection, wherein the infection is a bacterial infection selected from the group consisting of salmonellosis, typhoid fever, paratyphoid fever, shigellosis, Q fever, brucellosis, listeriosis, gastroenteritis, meningitis, meningoencephalitis, typhus, rickettsial pox, boutonneuse fever, African tick-bite fever, Rocky Mountain spotted fever, Flinders Island spotted fever, Queensland tick typhus, pelvic inflammatory disease, trachoma, gonorrhoea, Legionnaires’ disease, Pontiac fever, tuberculosis, leprosy, Hansen’s disease, lymphadenitis, pulmonary disease, skin disease, plague, yersiniosis, reactive arthritis, pseudoappendicitis, cholera, tularemia, nocardiosis, septicaemia and cat scratch disease.

In one embodiment, the infection is a bacterial infection selected from the group consisting of salmonellosis, cholera, dysentery, brucellosis, listeriosis, thyphoid fever, paratyphoid fever, shigellosis, legionellosis, tuberculosis, leprosy, tularemia, nocardiosis, pneumonia, chlamydial infection, gonorrhoea, meningitis, septicaemia, typhus, spotted fever, and cat-scratch disease.

In another embodiment, the bacterium belongs to the pathogenic enteric bacteria. In one embodiment, the bacterium is of the order Enterobacterales.

In one embodiment, the bacterium is of the family Enterobacteriaceae.

In one embodiment, bacterium is Salmonella or Shigella. In one embodiment, the infection is a bacterial infection selected from the group consisting of salmonellosis, typhoid fever, paratyphoid fever and shigellosis.

Endocytosis of intracellular pathogenic bacteria as used herein does not comprise endocytosis of isolated bacterial proteins or isolated bacterial particles by immune cells for the purpose of antigen presentation.

In one embodiment, the invention relates to the use of an inhibitor of the I KK-a/NIK complex in the manufacture for a medicament for the prevention or treatment of infection with bacteria in a cell and/or subject.

Mechanisms

In one aspect, the invention relates to an inhibitor of IKK-a or the IKK-a/NIK complex for use in reducing endocytosis of an intracellular pathogen in a cell and/or subject.

Particularly, the cell may be a mammalian cell. The subject, i.e. the pathogen host, may be a mammal. The subject may be human. The subject may be a non-human mammal. The subject may be a rodent.

Reducing endocytosis in a cell and/or subject reduces the amount of infectious particles, i.e. viruses or bacteria, which enter the cell. Hence, reducing endocytosis is an effective way to treat or prevent infection. Thus, in one embodiment, preventing or treating infection with a pathogen comprises reducing endocytosis of the pathogen in a cell and/or subject treated with the inhibitor compared to an untreated cell and/or subject.

The inhibitor of IKK-a orthe IKK-a/NIK complex reduces endocytosis compared to an untreated control cell and/or subject. The untreated control cell and/or subject is infected with the same pathogen as the treated cell and/or subject.

In one embodiment, endocytosis comprises cell entry via the endolysosomal pathway. Reducing endocytosis means reducing the amount of endosomes, lysosomes, endolysosomes, intracellular vesicles or vacuoles in a cell and/or subject with an infection compared to an untreated control cell and/or subject infected with the same pathogen.

In one embodiment, treatment with the inhibitor of IKK-a or the IKK-a/NIK complex reduces the amount of endosomes, lysosomes, endolysosomes, intracellular vesicles or vacuoles in a cell and/or subject compared to an untreated control cell and/or subject. In another embodiment, treatment with the inhibitor of IKK-a or the I KK-a/N IK complex reduces the number of an intracellular organelle selected from the group consisting of endosomes, lysosomes, endolysosomes, intracellular vesicles, acidic vesicles and vacuoles in a cell and/or subject compared to an untreated control cell and/or subject.

In another embodiment, treatment with the inhibitor of IKK-a or the I KK-a/N IK complex reduces the number of endosomes, lysosomes or endolysosomes in a cell and/or subject compared to an untreated control cell and/or subject.

In another embodiment, treatment with the inhibitor of IKK-a or the I KK-a/N IK complex reduces the number of endosomes in a cell and/or subject compared to an untreated control cell and/or subject.

In another embodiment, treatment with the inhibitor of IKK-a or the I KK-a/N IK complex reduces the number of lysosomes in a cell and/or subject compared to an untreated control cell and/or subject.

In another embodiment, treatment with the inhibitor of IKK-a or the I KK-a/N IK complex reduces the number of endolysosomes in a cell and/or subject compared to an untreated control cell and/or subject.

In one embodiment treatment with the inhibitor of IKK-a or the IKK-a/NIK complex reduces endocytosis activity as well as the number of acidic vesicles. In one embodiment treatment with the inhibitor of IKK-a or the IKK-a/NIK complex reduces the number of acidic vesicles.

In another embodiment treatment with the inhibitor of IKK-a or the IKK-a/NIK complex reduces endolysosomal maturation. Hence, treatment with the inhibitor of IKK-a or the IKK-a/NIK complex reduces the number of matured endolysosomes in a cell and/or subject compared to an untreated control cell and/or subject.

In one embodiment, treatment with the inhibitor of IKK-a or the IKK-a/NIK complex reduces the amount of endosomes, lysosomes, endolysosomes, intracellular vesicles or vacuoles in a cell and/or subject with a viral infection compared to an untreated control cell and/or subject with the same viral infection, preferably wherein the viral infection is selected from the group consisting of SARS, COVID-19, MERS, Monkeypox, smallpox, Rift Valley fever, hepatitis C, hepatocellular carcinoma, lymphoma, dengue fever, causes foot-and-mouth disease, bronchiolitis, common cold and pneumonia, hemorrhagic fever, Kaposi’s sarcoma, primary effusion lymphoma, HHV8-associated multicentric Castleman’s disease and KSHV inflammatory cytokine syndrome, influenza, vesicular stomatitis, common cold, anal dysplasia, genital cancers such as vulva, vagina, cervix and penis cancer, mouth papilloma, oropharyngeal cancer, aseptic meningitis, encephalitis, meningoencephalitis, pappataci fever, hemorrhagic fever, salmonellosis, typhoid fever, paratyphoid fever, shigellosis, Q fever, brucellosis, listeriosis, gastroenteritis, meningitis, meningoencephalitis, typhus, rickettsial pox, boutonneuse fever, African tick-bite fever, Rocky Mountain spotted fever, Flinders Island spotted fever, Queensland tick typhus, pelvic inflammatory disease, trachoma, gonorrhoea, Legionnaires’ disease, Pontiac fever, tuberculosis, leprosy, Hansen’s disease, lymphadenitis, pulmonary disease, skin disease, plague, yersiniosis, reactive arthritis, pseudoappendicitis, cholera, tularemia, nocardiosis, septicaemia and cat scratch disease.

In one embodiment, treatment with the inhibitor of IKK-a or the IKK-a/NIK complex reduces the amount of matured endolysosomes in a cell and/or subject with a viral infection compared to an untreated control cell and/or subject with the same viral infection, preferably wherein the viral infection is selected from the group consisting of SARS, COVID-19, MERS, Monkeypox, smallpox, Rift Valley fever, hepatitis C, hepatocellular carcinoma, lymphoma, dengue fever, causes foot-and-mouth disease, bronchiolitis, common cold and pneumonia, hemorrhagic fever, Kaposi’s sarcoma, primary effusion lymphoma, HHV8-associated multicentric Castleman’s disease and KSHV inflammatory cytokine syndrome, influenza, vesicular stomatitis, common cold, anal dysplasia, genital cancers such as vulva, vagina, cervix and penis cancer, mouth papilloma, oropharyngeal cancer, aseptic meningitis, encephalitis, meningoencephalitis, pappataci fever, hemorrhagic fever, salmonellosis, typhoid fever, paratyphoid fever, shigellosis, Q fever, brucellosis, listeriosis, gastroenteritis, meningitis, meningoencephalitis, typhus, rickettsial pox, boutonneuse fever, African tick-bite fever, Rocky Mountain spotted fever, Flinders Island spotted fever, Queensland tick typhus, pelvic inflammatory disease, trachoma, gonorrhoea, Legionnaires’ disease, Pontiac fever, tuberculosis, leprosy, Hansen’s disease, lymphadenitis, pulmonary disease, skin disease, plague, yersiniosis, reactive arthritis, pseudoappendicitis, cholera, tularemia, nocardiosis, septicaemia and cat scratch disease.

In one embodiment, treatment with the inhibitor of IKK-a or the IKK-a/NIK complex reduces the amount of matured endolysosomes in a cell and/or subject with a viral infection compared to an untreated control cell and/or subject with the same viral infection, preferably wherein the viral infection is selected from the group consisting of SARS, COVID-19, MERS, Monkeypox, smallpox, Rift Valley fever, salmonellosis, typhoid fever, paratyphoid fever, and shigellosis.

In one embodiment, the invention provides use of an inhibitor of IKK-a or the IKK-a/NIK complex in the manufacture of a medicament for reducing the amount of matured endolysosomes in a cell and/or subject with a viral infection compared to an untreated control cell and/or subject with the same viral infection. In another aspect, the invention also relates to an inhibitor of IKK-a or the IKK-a/NIK complex for use in reducing the total pathogen load of an intracellular pathogen in a cell and/or subject. Methods of measuring total pathogen load in a cell and/or subject are known in the art. Methods include, for example, quantitative PCR, next-generation sequencing, enzyme-linked immunosorbent assay (ELISA), reporter assays (e.g. luciferase assay), quantification of infectious particles (e.g. plaque assay), and imaging (e.g. cell viability).

For example, for viral infections, reducing the total pathogen load comprises reducing accumulation of viral genomes, reducing virus replication or reducing virus spread. For bacterial infections, reducing the total pathogen load comprises reduction of Colony Forming Units (CFU) of internalized bacteria.

Hence, in one embodiment, treatment with the inhibitor of IKK-a or the IKK-a/NIK complex reduces accumulation of viral genomes, virus replication, virus spread or Colony Forming Units of internalized bacteria.

Another aspect of the invention relates to an inhibitor of IKK-a or the IKK-a/NIK complex for use in preventing or treating an infection with a pathogen, wherein preventing or treating infection with a pathogen comprises reducing activity of cytoplasmic p-catenin levels in a cell and/or subject treated with the inhibitor compared to untreated control cell and/or subject. In one embodiment, treatment with an inhibitor of IKK-a or the IKK-a/NIK complex of the invention reduces p-catenin Ser⁵⁵² phosphorylation.

The compounds of the present invention may be administered in the form of pharmaceutically acceptable salts.

The term "pharmaceutically acceptable salt" refers to a salt which possesses the effectiveness of the parent compound and which is not biologically or otherwise undesirable (e.g., is neither toxic nor otherwise deleterious to the recipient thereof). Suitable salts include acid addition salts which may, for example, be formed by mixing a solution of the compound of the present invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, acetic acid, trifluoroacetic acid, or benzoic acid. When the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof can include alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., calcium or magnesium salts), and salts formed with suitable organic ligands such as quaternary ammonium salts. Also, in the case of an acid (-COOH) or alcohol group being present, pharmaceutically acceptable esters can be employed to modify the solubility or hydrolysis characteristics of the compound. The pharmaceutically acceptable salt of ACHP may be for example the hydrochloride salt of ACHP.

In some embodiments, the inhibitor is administered in combination with a further therapeutic ingredient.

Another aspect of the invention refers to a pharmaceutical composition comprising the inhibitor according to invention together with a pharmaceutically acceptable carrier and an optionally further therapeutic ingredient for use in preventing or treating infection with a pathogen in a subject.

In particular, the invention refers to a pharmaceutical composition comprising the inhibitor according to any one of the preceding claims together with a pharmaceutically acceptable carrier and an optionally further therapeutic ingredient for use in preventing or treating COVID- 19 in a subject, or for use in preventing or treating SARS-CoV-2 infection in a subject.

Another aspect of the invention refers to a pharmaceutical composition comprising the inhibitor according to invention together with a pharmaceutically acceptable carrier and optionally a further therapeutic ingredient for use in preventing or treating infection with a pathogen in a subject, wherein the further therapeutic ingredient is an anti-inflammatory agent, antibiotic agent or antiviral agent.

In one embodiment, the invention refers to the use of a pharmaceutical composition comprising the inhibitor according to invention together with a pharmaceutically acceptable carrier and optionally a further therapeutic ingredient for the manufacture of a medicament for preventing or treating infection with a pathogen in a subject, wherein the further therapeutic ingredient is an anti-inflammatory agent, antibiotic agent or antiviral agent. The further therapeutic ingredient may be selected from the group consisting of protease inhibitors, nucleotide analogues, inhibitors of autophagy, AKT kinase inhibitor, penicillins, cephalosporins, monobactams, quinolones, aminoglycosides, tetracyclines, glycopeptiddes, macrolides, anitmetabolites, nitroimidazoles, nonsteroidal anti-inflammatory drugs, antileukotrienes, corticosteroids or interferons. In another embodiment, the further therapeutic ingredient may be selected from the group consisting of protease inhibitors, nucleotide analogues, inhibitors of autophagy, AKT kinase inhibitor, corticosteroids or interferons. In one embodiment, the further therapeutic ingredient is selected from the group consisting of Pegylated interferon, Nirmatrelvir with Ritonavir (Paxlovid), Remdesivir (Veklury), Bebtelovimab, Molnupiravir (Lagevrio), Ribavirin, Ciprofloxacin, Ceftriaxone, Azithromycin, Trimethoprim/sulfamethoxazole (Bactrim), Fluoroquinolone, Ampicillin, Cotrimoxazole, Penicillin, Chloramphenicol, Erythromycin, Isoniazid, Rifampicin, Pyrazinamide, Ethambutol, Dapsone, Rifampin, Streptomycin, Tetracyclin, Doxycyclin, Gentamycin, meclofenamate sodium, zileuton, montelukast, zafirlukast, aspirin, ibuprofen, naproxen, paracetamol, hydrocortisone, dexamethasone, fludrocortisone, and prednisone.

Another aspect of the invention refers to a pharmaceutical composition comprising the inhibitor according to invention together with a pharmaceutically acceptable carrier and an optionally further therapeutic ingredient for use in preventing or treating infection with a pathogen in a subject, wherein the further therapeutic ingredient is an antiviral.

In one embodiment, the antiviral is selected from the group consisting of Pegylated interferon, Nirmatrelvir with Ritonavir (Paxlovid), Remdesivir (Veklury), Bebtelovimab, Molnupiravir (Lagevrio), Ribavirin and nucleoside analogues.

Another aspect of the invention refers to a pharmaceutical composition comprising the inhibitor according to invention together with a pharmaceutically acceptable carrier and an optionally further therapeutic ingredient for use in preventing or treating infection with a pathogen in a subject, wherein the further therapeutic ingredient is an antibiotic.

In one embodiment, the antibiotic is selected from the group consisting of Ciprofloxacin, Ceftriaxone, Azithromycin, Trimethoprim/sulfamethoxazole (Bactrim), Fluoroquinolone, Ampicillin, Cotrimoxazole, Penicillin, Chloramphenicol, Erythromycin, Isoniazid, Rifampicin, Pyrazinamide, Ethambutol, Dapsone, Rifampin, Streptomycin, Tetracyclin, Doxycyclin, and Gentamycin.

In one embodiment, the pharmaceutical composition comprising the inhibitor according to invention together with a pharmaceutically acceptable carrier further comprises one, two, three, four or five further therapeutic ingredients. In one embodiment, the pharmaceutical composition comprising the inhibitor according to invention together with a pharmaceutically acceptable carrier further comprises more than five further therapeutic ingredients.

Another aspect of the invention refers to a pharmaceutical composition comprising the inhibitor according to the invention together with a pharmaceutically acceptable carrier and an optionally further therapeutic ingredient for use in preventing or treating COVID-19 in a subject, or for use in preventing or treating SARS-CoV-2 infection in a subject. Another aspect of the invention refers to a method of preventing or treating infection in a human or non-human animal in need thereof, comprising administering an inhibitor of the IKK-a/NIK complex as described herein to said human or non-human animal.

In another aspect, the present invention relates to the use of an inhibitor of the IKK-a/NIK complex as described herein for the manufacture of a medicament for use in preventing or treating viral infection and/or for use in preventing or treating coronavirus disease 2019 (COVID-19) in a subject.

Examples

Materials and Methods

Cell lines and reagents

HEK293R1 , A549, A549-ACE2, and Vero E6 cells were cultured as previously described (Stukalov, A. et al. Nature 594, 246-252 (2021)). Primary normal human bronchial epithelial cells (NHBE, Lonza) and culturing conditions were performed as described previously (Jakwerth, C. A. et al. J Mol Med 100, 613-627 (2022)). iBMDMs cells were seeded in Greiner tissue culture treated 96-well plates the day prior to the experiment in their growth media (DMEM, Glutamax, 10% FCS, 10% MCSF). All cells were tested to be mycoplasma-free.

Generation of knockout A549-ACE2 cells was performed by cloning multiplexed gRNA sequences into pLentiCRISPRv2 plasmid (Addgene plasmid #52961). Lentivirus production, transduction of cells, and antibiotic selection were performed as described (Bergant, V. et al. EMBO J e111608 (2022) doi: 10.15252/embj.2022111608). In short, lentiviruses encoding puromycin resistance, Cas9 and gRNAs were added to A549-ACE2, followed by a 7 days puromycin selection (2pg/ml). All cells were validated for their respective knockout by Sanger sequencing of PCR products containing Cas9 cleavage site and western blot. gRNA sequences and sequencing primers can be provided upon request.

For stimulation or inhibition of cells, following compounds were used: LT- agonistic antibody (kindly gifted by Prof. Mathias Heikenwalder), IL-ip (Peprotech, 200-01 B), recombinant human EGF was a kind gift from Kirti Sharma, TPCA-1 (Tocris, 2559), Amlexanox (Tocris, 4857), Celastrol (Tocris, 3203), ML 130 (Tocris, 4354), CID 2858522 (Tocris, 4246), IKK 16 (Tocris, 2539), ACHP (Sigma, 401487), Ro 106-9920 (Tocris, 1778), JSH-23 (Sigma, J4455), SC75741 (Sigma, SML2382), Amgen16 (Sigma, SML2457), and Ipatasertib (Cayman Chemical, 18412).

Protein abundance measurement by western blotting was done with following antibodies: I KBO (Cell Signaling, 4814, 1 :1000 dilution), phospho-lKBa (Ser32/36) (Cell Signaling, 9246, 1 :1000 dilution), N F-KB p65 (Cell Signaling, 8242, 1 :1000 dilution), phospho-NF-KB p65 (Ser536) (Cell Signaling, 3033, 1 :1000 dilution), NF-KB2 p100/p52 (Cell Signaling, 4882, 1 : 1000 dilution), phospho-NF-KB2 p100/p52 (Ser866/870) (Cell Signaling, 4810, 1 : 1000 dilution), IKKy (Cell Signaling, 2695, 1 :2000 dilution), phospho-IKKy (Ser376) (Cell Signaling, 2689, 1: 1000 dilution), IKKa (Cell Signaling, 11930, 1 :1000 dilution), IKKp (Cell Signaling, 8943), phospho- IKKa/p (Ser176/180) (Cell Signaling, 2697, 1 : 1000 dilution), SARS-CoV-1/SARS-CoV-2 N protein (Sino Biological, 40143- MM 05, 1 :1000 dilution), p-Catenin (Sigma, C7207, 1 :1000 dilution), phospho-p-Catenin (Ser552) (Cell Signaling, 9566, 1 :1000 dilution) and ACTB-HRP (Santa Cruz, sc-47778, 1 :5000 dilution). Secondary antibodies detecting mouse (Cell Signaling, 7076, 1:5000 dilution), and rabbit IgG (Dako, P0448, 1 :5000 dilution) were coupled to HRP.

Virus strains and stock preparation

SARS-CoV-Frankfurt-1 , SARS-CoV-2-MUC-IMB-1 and SARS-CoV-2-GFP strains were produced by infecting Vero E6 cells (2 days, MOI 0.01), as described previously. VSV-GFP was produced as previously described (Pichlmair, A. et al. Nat Immunol 12, 624-630 (2011)), and SARS-CoV-2 Spike-pseudotyped VSV-GFP (VSV-S) as previously described (Jocher, G. et al. EMBO Rep 23, e54305 (2022)). VACV-V300-GFP was a kind gift from Joachim Bugert, and was produced as described (McGuigan, C. et al. J. Med. Chem. 56, 1311-1322 (2013). MPXV was produced by infection of Vero E6 cells (MOI 0.01) for 2 days. Infectious particles were harvested by three freeze-thaw cycles of infected cells and supernatant was spun twice (10min, 1000g) before being aliquoted. RVFV-GFP was a kind gift from Friedemann Weber, and virus stocks were prepared as described (Kainulainen, M. et al. J Virol 88, 3464-3473 (2014)). qRT-PCR analysis

RNA isolation of either uninfected or infected cells was performed using NucleoSpin RNA Plus kit (Macherey-Nagel) according to the manufacturer's protocol. Total RNA was used for reverse transcription with PrimeScript RT with gDNA eraser (Takara). In the case of MPXV infected cells, total DNA isolation was done using DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's protocol. For relative transcript quantification Powerllp SYBR Green (Applied Biosystems) was used. Primer sequences can be provided upon request.

Viral inhibitor screening assay

A549-ACE2 cells were seeded into 96-well plates the day before infection. Cells were pretreated for 4 hours before infection with SARS-CoV-2-GFP reporter virus (MOI 3). Infected and uninfected plates were placed into an IncuCyte S3 Live-Cell Analysis System (Essen Bioscience), where real-time images of mock (phase channel) and infected (GFP and phase channel) cells were captured every 4 h for 48 hpi. Cell growth (mock and infected) and virus signal (infected) were assessed as the cell confluence per well (phase area) and GFP area normalized by cell confluence per well (GFP area/phase area) respectively using IncuCyte S3 Software (Essen Bioscience; version 2020C rev1). Similar treatments, infection and analysis were performed for NHBE cells.

Endocytic activity and acidic vesicle measurement

A549-ACE2 cells were seeded in 96-well plates the day before treatment. Cells were treated with indicated inhibitors 4 hours before addition of either pHrodo Green Dextran 10,000 MW (Thermo Fisher, P35368) for endocytosis tracking (final concentration: 20pg/ml) or Lysotracker Red (Thermo Fisher, L7528) for acidic vesicle labeling (final concentration: 50nM). Plates were placed inside an IncuCyte S3 Live-Cell Analysis System (Essen Bioscience), where real-time images of mock (phase channel), pHrodo- treated (GFP and phase channel) and Lysotracker- treated cells (RFP and phase channel) were captured every 2 hours for 24 hours. Labeled organelles were assessed as the fluorescent-positive area normalized by cell confluence per well (GFP area/phase area or RFP area/phase area) respectively using IncuCyte S3 Software (Essen Bioscience; version 2020C rev1).

Confocal microscopy with virus-like particles

Engineered SARS-CoV-2 VLPs were generated by transient transfection of HEK293T cells with plasmids, encoding S, M, N, E and CD63~BlaM, and were harvested from conditioned medium 72 h post-transfection as described earlier (Roessler, J. et al. PNAS Nexus 1 , pgac045 (2022)). A549-ACE2 cells were pre-treated with ACHP (2pM), E64d (1 M) or DMSO prior to inoculation for 6 h at 37°C. Thereafter, cells were stained overnight with CCF4-AM (Thermo Fisher Scientific, K1095), a fluorescent BlaM substrate, which accumulates within cells and undergoes a shift in fluorescence emission upon BlaM mediated cleavage. Cells were analyzed for BlaM cleaved CCF4 by confocal fluorescence microscopy.

Intracellular Spike protein in ACHP, E64d or DMSO treated A549-ACE2 cells loaded with VLPs or infected with SARS-CoV-2 (MOI 1) was stained with the fluorescently labeled monoclonal anti-Spike full-length antibody 43A11_AlexaFluor488 or 43A11_AlexaFluor647 and visualized by confocal fluorescence microscopy. The 43A11 antibody is published (Roessler, J. et al. PNAS Nexus 1 , pgac045 (2022)).. Data were analyzed with FIJI (Imaged; version 1.53q).

Mass spectrometric sample processing of SARS-CoV-2 infected cell lines For the full proteome and phosphoproteome analysis A549-ACE2 knockout (IKKa) and control (NTC) were infected with SARS-CoV-2 (MOI 1). The samples were harvested 6 h postinfection, lysed with 200pl lysis buffer (100 mM Tris HCI pH 8.5; 4% SDC), heat inactivated (95°C, 5min) and sonicated (5 min, 4 °C, 30 s on, 30 s off, low settings; Bioruptor, Diagenode). The protein concentration of the lysates was determined with the Pierce 660nm protein assay (Pierce 660, ThermoFisher, #22660). The samples were further processed as published with 200pg for each replicate (Humphrey, S. J. et al. Nat Protoc 13, 1897-1916 (2018)).

For phosphoproteome measurement peptides were loaded on a 50 cm reverse-phase analytical column (75 pm diameter, 60 °C; ReproSil-Pur C18-AQ 1.9 pm resin; Dr. Maisch) and separated using an EASY-nLC 1200 system (Thermo Fisher Scientific). For chromatographic separation, a 100 min gradient with a flow rate of 350 nl/min and a binary buffer system consisting of buffer A (0.1 % formic acid in H2O) and buffer B (80 % acetonitrile, 0.1 % formic acid in H2O) was used: 3-19 % buffer B (60 min), 19-41 % buffer B (30 min), 41-90% buffer B (5 min), and kept at 90% buffer B (5 min). Eluting peptides were directly analyzed on a Q- Exactive HF mass spectrometer (Thermo Fisher Scientific) equipped with a nano-electrospray source (Thermo Fisher Scientific). All measurements were performed in positive ion mode, the spray voltage was set to 2.4 kV, funnel RF level at 60, and heated capillary at 250 °C. Data- dependent acquisition included repeating cycles of one MS1 full scan (300-1650 m/z, R = 60000 at 200 m/z) at an ion target of 3 * 10⁶ with an injection time of 120 ms, followed by 10 MS2 scans of the highest abundant isolated and higher-energy collisional dissociation (HCD) fragmented peptide precursors (R = 15000 at 200 m/z). For MS2 scans, collection of isolated peptide precursors was limited by an ion target of 1 x 10⁵ and a maximum injection time of 100 ms. Isolation and fragmentation of the same peptide precursor was eliminated by dynamic exclusion for 30 s. The isolation window of the quadrupole was set to 1.6 m/z and HCD was set to a normalized collision energy of 27%.

For full proteome measurement purified peptides were loaded on a 50 cm reverse-phase analytical column (75 pm diameter, 60 °C; ReproSil-Pur C18-AQ 1.9 pm resin; Dr. Maisch) and separated using an EASY-nLC 1200 system (Thermo Fisher Scientific). For peptide separation, a 120 min gradient with a flow rate of 300 nl/min and a binary buffer system consisting of buffer A (0.1 % formic acid in H2O) and buffer B (80 % acetonitrile, 0.1 % formic acid in H2O) was used: 5-30 % buffer B (95 min), 30-95 % buffer B (10 min), wash out at 95 % buffer B (5 min), decreased to 5% buffer B (5 min), and kept at 5% buffer B (5 min). Eluting peptides were directly analyzed on a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific) equipped with a nano-electrospray source (Thermo Fisher Scientific). All measurements were performed in positive ion mode, the spray voltage was set to 2.4 kV, funnel RF level at 60, and heated capillary at 250 °C. Data-dependent acquisition included repeating cycles of one MS1 full scan (300-1650 m/z, R = 60000 at 200 m/z) at an ion target of 3 x io⁶ with an injection time of 20 ms, followed by 15 MS2 scans of the highest abundant isolated and higher-energy collisional dissociation (HCD) fragmented peptide precursors (R = 15000 at 200 m/z). For MS2 scans, collection of isolated peptide precursors was limited by an ion target of 1 x 10⁵ and a maximum injection time of 25 ms. Isolation and fragmentation of the same peptide precursor was eliminated by dynamic exclusion for 20 s. The isolation window of the quadrupole was set to 1.4 m/z and HCD was set to a normalized collision energy of 27 %.

Mass spectrometric data analysis

Raw MS data files of the phosphoproteomics experiments were processed with MaxQuant (Version 2.0.3) using the default settings for DDA measurement. Additionally, the Phospho (STY) was enabled as a variable modification. The spectra were searched against the reviewed human proteome including isoforms (Uniprot, UP000005640) and SARS-CoV-2 (Uniprot, UP000464024) by the built-in Andromeda search engine.

The phosphorylated peptide sites (Phospho(STY)sites.txt) were further analyzed with Persues (Version: 1.6.15.0). The columns were filtered by reverse and contaminants default settings and the Iog2 transformed intensities were normalized to the row- and column median, leading to 4.887 identified phosphosites. The proteins were further filtered for a localization probability (>0.75) and minimum 2 valid values in at least one grouping. The missing values were imputed (downshift 1.8, width 0.3, total matrix) and a two-sample t-test (two-sided, permutation-based FDR, 0.05 FDR, 250 randomizations) was performed, leading to 105 significantly regulated sites.

Raw MS data files of the full proteome experiments were processed with MaxQuant (version: 2.0.1.0) using the default settings for DDA measurement, intensity Based Absolute Quantification (iBAQ) and label-free quantification (LFQ) enabled (LFQ min ratio count 2, normalization type classic). Spectra were searched against the reviewed human proteome including isoforms (Uniprot, UP000005640) and SARS-CoV-2 (Uniprot, UP000464024) by the built-in Andromeda search engine.

The protein groups were further analyzed using Perseus (Version: 1.6.15.0). The LFQ values were imported from the proteinGroups.txt and the columns were filtered for the default settings of reverse, only identified by site and contaminants, leading to 3.839 protein groups. The LFQ values were Iog2 transformed and the protein groups were filtered for at least 2 valid values per grouping. The missing values were replaced by the normal distribution (1.8 downshift, 0.3 width, separately for each column). The two conditions were analyzed with a two-sample t-test (two-sided, permutation-based FDR, 0.05 FDR, 250 randomizations).

Assessment of cytotoxicity by uptake of propidium iodide To assess putative cytotoxic effects of ACHP in iBMDMs cells, cells were treated by adding increasing concentrations of ACHP (ranging from OpM to 10pM) in OptiMEM (Gibco) containing 12.5 pg/ml propidium iodide (Thermo Fisher). Fluorescence was measured on a BioTek Cytation 5 in intervals of 10 minutes and cell death was quantified as the difference to the untreated control, divided by the difference between the 100% lysis control and the untreated control.

Bacterial infection of iBMDMs and quantification of bacterial entry iBMDMs cells were seeded the day prior to infection as described above. Overnight cultures of Salmonella enterica serovar Typhimurium SL1344 and Shigella flexneri were grown in LB and used for infection without subculturing. Cells were treated for three hours with a range of concentrations of ACHP and subsequently infected with either Salmonella or Shigella at an MOI of 10 after washing the bacterial cells once in PBS to remove residual growth media. Cells were centrifuged at 300g for 5 minutes and incubated at 37°C for 25 minutes. Infected cells were washed in pre-warmed PBS and subsequently treated with gentamicin (Gibco) containing media (100 pg/ml) for 30 minutes to kill remaining extracellular bacteria.

For quantification of bacterial entry, infected and gentamicin-treated cells were washed twice in pre-warmed PBS and fully lysed by adding 0.1 % Triton-X100 in PBS. A series of 5-fold dilutions was performed in PBS and 5pL bacterial suspension of several dilutions were plated onto LB-agar and grown overnight. Colony numbers were counted and normalized to the untreated control to compare replicates more easily.

Western blotting

Cells were lysed in SDS buffer (62.5 mM Tris-HCI pH 6.8, 50 mM Dithiothreitol, 10% Glycerol, 2% Sodium dodecyl sulfate and 0.01 % Bromophenol blue). Proteins were separated on 10% polyacrylamide gels and then transferred to a nitrocellulose membrane. Membranes were incubated with primary antibodies (1 : 1,000) at 4 °C overnight and HRP-conjugated secondary antibodies at room temperature for 1 h. Immunoblots were developed and imaged with BioRad ChemiDoc Imaging System.

Luciferase reporter assay

HEK293R1 cells were seeded into 24-well plates the day before transfection. The NF-KB luciferase reporter vector was cotransfected with the EF1a-F?en/7/a reporter vector. Twenty-four hours after transfection, cells were pretreated with different dose drugs for 4 hours and then treated with 0.1ng/ml I L-1 p for an additional 4 hours. The luciferase activity in these cells was measured using the Dual-Luciferase Reporter Assay System (Promega, E1960). The relative luciferase activity was determined by firefly luciferase activity normalizing to Renilla luciferase activity, as published (Stukalov, A. et al. Nature 594, 246-252 (2021)).

Transcriptomics - sample preparation and measurements

A549-ACE2 knockout cells and pre-treated NHBE cells used for transcriptional profiling of SARS-CoV-2 infection were cultured and infected as described above. RNA isolation was performed using the NucleoSpin RNA Plus kit (Macherey-Nagel) according to the manufacturer's protocol. Library preparation for bulk 3’-sequencing of poly(A)-RNA was done. Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs) and an adapter. 5’ ends of the cDNAs were extended by a template switch oligo (TSO) and after pooling of all samples full-length cDNA was amplified with primers binding to the TSO-site and the adapter. cDNA was fragmented and TruSeq-Adapters ligated with the NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® (NEB) and 3’-end-fragments were finally amplified using primers with Illumina P5 and P7 overhangs. The P5 and P7 sites were exchanged to allow sequencing of the cDNA in readl and barcodes and UMIs in read2 to achieve better cluster recognition. The library was sequenced on a NextSeq 500 (Illumina) with 75 cycles for the cDNA in readl and 16 cycles for the barcodes and UMIs in read2.

Transcriptomics - data analysis

For RNA-seq data analysis Gencode gene annotations v35 and the human reference genome GRCh38 were derived from the Gencode homepage (EMBL-EBI). Dropseq tools v1.12 was used for mapping raw sequencing data to the reference genome. The resulting UMI filtered count matrix was imported into R v3.4.4. Lowly expressed genes were filtered prior to differential expression analysis with DESeq2 v1.18.1. Dispersion of the data was estimated with the genotype or treatment as explanatory variable during model fitting with DESeq2. The Wald test was used for determining differentially regulated genes between all conditions of interest. Shrunken Iog2 fold changes were calculated afterwards. A gene was considered to be significantly differentially expressed at an FDR level of 0.05.

For Gene Ontology (GO) term enrichment on DAVID, downregulated genes in IKKa knockout A549-ACE2 compared to control cells with adjusted p-value <0.05 were defined as significant hits and were compared to all detected genes. The top ten KEGG pathway terms were then used.

For upstream promoter analysis (UPA), genes with adjusted p-value < 0.05 were considered significantly differentially expressed and were further separated into downregulated and upregulated sets. These gene sets were individually imported into Cytoscape (version 3.8.1), wherein iRegulon plugin was used to perform UPA using default settings. Data visualization was performed in R (version 4.0.2).

Data and Code availability

The files of the proteomic datasets and Maxquant output have been deposited to the ProteomeXchange Consortium) via the PRIDE partner repository. This includes the phosphoproteomic datasets PXD036627. The raw sequencing data for this study have been deposited with the ENA at EMBL-EBI under accession numbers PRJEB56503 and PRJEB56504.

Example 1

Adaptors of the pro-inflammatory NF-KB pathway were targeted. More specifically TBK1 , P105, RelA, NFKB1 (involved in the canonical NF-KB pathway) (Fig. 1A) were inhibited. In order to understand the role of cytokine production upon SARS-CoV-2 infection, the etiologic agent responsible of COVID-19 in patients, human lung-derived A549 cells, complemented with the SARS coronaviruses entry receptor Ace2 (A549-ACE2), were used. By employing a recombinant SARS-CoV-2-GFP reporter virus (Stukalov, A., et al. Nature 594, 246-252; 2021) and time-resolved live imaging, cell viability and virus spread was followed over time in order to establish the function of the tested inhibitors (Fig. 1 B-E). These experiments showed lack of cytotoxicity by the used compounds, even at the highest concentration, evidenced by normal cell growth rates in the absence of virus infections. The tested inhibitors exerted no detected effect on SARS-CoV-2-GFP (Fig. 1 B-E). As pro-inflammatory cytokines are a result of activated innate immune responses, it was expected that the used inhibitors have no impact or even favor replication of SARS-CoV-2. Surprisingly, a strong inhibition of the virus was observed when the IKK complex (involved in both the canonical and non-canonical N F-KB pathway) and NIK (mostly involved in the non-canonical NF-KB pathway) were inhibited (Fig. 2A-C). Focusing on expression of GFP as proxy for virus replication, the antiviral effect of IKK (IKK-16 and ACHP) and NIK (Amgen16) inhibitors were observed, with IKK-16 and Amgen16 having a strong antiviral effect when used at 1 pM (Fig. 2A-B), and ACHP completely abolishing virus replication when used at 2pM concentration (Fig. 2C). ACHP showed a consistent doseresponse when used at lower concentrations. To validate this first observation, Vero cells were treated with IKK and NIK inhibitors prior to infection with SARS-CoV-2, and generation of infectious particles were quantified 48h post- infection. Surprisingly, a strong inhibition of virus production up to 100-fold was observed in case of IKK-16 treatment at a concentration of 10pM, and a 10-fold reduction of SARS-CoV-2 when used at 1 pM (Fig. 2D). The ability of IKK inhibitors (IKK-16 and ACHP) and NIK inhibitor (Amgen16) to reduce the TNFa and IL-1p induced NF-KB pathway activation was then tested. Treatment of HEK293T-RI reporter cells by NIK inhibitor Amgen16 led to a dose-dependent inhibition of the NF-KB pathway, as tested by NF-KB dependent reporter gene activation (Fig. 2E). However, both IKK inhibitors, IKK-16 and ACHP exerted a strong and dose-dependent inhibition of the response (Fig. 2E), supporting the known activities of the used compounds. Notably, all compounds significantly reduced expression of the NF-KB in response of IL-ip treatment in the nanomolar range indicating that treatment with NF-KB inhibitory drugs efficiently reduces inflammatory conditions. Collectively, these findings are showing that IKK and NIK inhibitors are not leading to cytopathic effects in vitro and that IKK and NIK inhibitors are inhibiting virus replication.

As inhibitors can have broad-range effect, especially IKK inhibitors targeting both IKK-a (involved in both the canonical and non-canonical NF-KB pathway) and IKK-p (involved in the canonical NF-KB pathway) (Fig. 1A), A549-ACE2 cells were treated with agonists of the canonical (TNFa) and non-canonical (LTP) NF-KB pathway prior to infection with SARS-CoV- 2-GFP. This experiment showed an enhanced and dose-dependent viral replication of SARS- CoV-2-GFP upon LTp treatment, but not when TNFa was used. To validate the necessity of the non-canonical NF-KB pathway, RelA (involved in the canonical NF-KB pathway) and RelB (involved in the non-canonical NF-KB pathway) (Fig. 1A) were genetically depleted (KO) in A549-ACE2 cells using CRISPR/Cas9 targeting. Compared to the non-targeting control (NTC) cell line, all KO cells led to almost no reduction of SARS-CoV-2 growth in vitro (Fig. 3B, left panel). However, treatment of the NTC and KO cells with the IKK inhibitor (ACHP) led to a comparable and clear reduction of the GFP signal (Fig. 3B, left panel). IKK-a, NIK and a known interactor of IKK-a (DDX3X) were genetically depleted (KO) in A549-ACE2 cells using CRISPR/Cas9 targeting, in order to reproduce the involvement of the targeted genes in SARS- CoV-2 infection. Compared to the non-targeting control (NTC) cell line, IKK-a and NIK KO cells led to a clear reduction of SARS-CoV-2 growth in vitro, with IKK-a KO cells exerting the most prominent effect (Fig. 3B, right panel). Surprisingly, DDX3X KO cells exerted a strong gain of SARS-CoV-2 growth, thus abolishing a similar role of IKK-a and DDX3X as upon HCV infection. To corroborate these findings growth of clinical isolates of SARS-CoV-1 or SARS- CoV-2 was tested and virus growth was monitored using quantitative RT-PCR in order to accurately quantify the number of intracellular viral RNA genomes being replicated. As expected, IKK-a and NIK KO cell lines led to a significant decrease of SARS-CoV-2 replication (black bars), with IKK-a KO cells showing more than 70% reduction of SARS-CoV-2 RNA levels compared to NTC cells. Surprisingly, accumulation of SARS-CoV-1 RNA mostly required IKK-a, but NIK to a lesser extent (grey bars) (Fig. 3C). While RelA and RelB KO cells had only a minor effect on both SARS-CoV-1 and SARS-CoV-2 replication, DDX3X KO cells allowed an enhanced viral replication for both viruses (Fig. 3C). Collectively, these findings are showing that IKK-a/NIK complex inhibitors are inhibiting virus replication in a NF-KB and DDX3X-independent manner. Finally, to corroborate these findings in disease relevant cell types from healthy donors, Normal Human Bronchial Epithelial (NHBE) cells were used, a well described ex vivo model to study respiratory infectious diseases. NHBE cells were treated with the IKK inhibitor ACHP and the cells were infected with either SARS-CoV-2-GFP (Fig. 4A) or clinical isolates of SARS-CoV-2, including the more rapidly spreading omicron virus variant (Fig. 4B-C). In line with observations in cell lines, it could be observed that ACHP has strong and dose-dependent antiviral activity, as it was able to reduce accumulation of all SARS-CoV-2 RNA strains tested by 80% (Fig. 4B- C), underlining that inhibition of the IKK-a/NIK complex can restrict virus growth in diseaserelevant cell types. In order to understand which step of the viral replication cycle could be impacted by the use of IKK inhibitor, a non-related RNA virus, Vesicular Stomatitis Virus (VSV) was genetically modified to express the glycoprotein Spike of SARS-CoV-2 instead of its own. By this mean, the newly produce virus have an altered host tropism and entry mechanism comparable to SARS-CoV-2. A549-ACE2 cells were treated with ACHP prior infection with VSV-GFP, VSV-GFP-Spike or a clinical isolate of SARS-CoV-2. Surprisingly, IKK inhibition led to a comparable decrease of VSV-Spike or SARS-CoV-2, while having no significant effect on VSV (Fig. 4D). Thus, it seems that inhibition of the IKK-a/NIK complex can restrict SARS-CoV- 2 entry.

These pre-clinical data show that IKK-a/NIK complex inhibitors, and more precisely IKK inhibitors such as ACHP, have strong antiviral efficacy against SARS-CoV-2 in vitro and ex vivo. Thus, the inventors of the present invention propose that ACHP and other inhibitors of the IKK-a/NIK complex can be considered as a preventive treatment by blunting virus growth as well as pro-inflammatory activities. The use of such inhibitors may prevent viral spread, damage of the respiratory tract as well as other organs besides the expected effects of N F-KB inhibition on hyperinflammation.

Example 2

Pro-inflammatory signaling through IKB kinases promotes SARS-CoV-2 replication

SARS-CoV-2 infection induces hyperinflammation in patients and production of pro- inflammatory cytokines in infected cells in vitro and ex vivo. To evaluate if such a hyperinflammatory milieu affects SARS-CoV-2 replication per se, we monitored the virus replication in A549 cells overexpressing ACE2 (A549-ACE2) treated with IL-i p or a LT-p agonistic antibodies to mimic activation of the canonical and non-canonical NF-KB signaling pathways, respectively (Fig. 10a). Surprisingly, treatment with IL-ip and LT-p agonistic antibody led to a significantly higher accumulation of SARS-CoV-2 RNA levels at 24 h post infection (Fig. 5a). The increased abundance of SARS-CoV-2 mRNA in IL-i p and LT-p agonistic antibody treated cells led us to test whether inhibition of NF-KB signaling would negatively affect SARS-CoV-2 replication. To this aim, we used inhibitors targeting different steps of the NF-KB signaling cascade (Fig. 10a). As expected, IL-ip treatment induced prominent NF-KB activation in non-infected conditions, confirming activation of the canonical NF-KB pathway, and most tested compounds suppressed IL-i p-mediated activation of a NF- KB luciferase reporter construct (Fig. 10b) and did not affect cell growth rates at 1 pM concentration (Fig. 10c). We next used time-lapse microscopy to monitor whether the NF-KB inhibitors influenced replication of a GFP-expressing SARS-CoV-2 strain. The majority of tested inhibitors did not show a pro- or antiviral effect. Surprisingly, inhibitors targeting the IKK complex (IKK 16, ACHP) or NIK (Amgen16), a kinase activating IKKa, led to a significant dosedependent reduction of the GFP signal (Fig. 5b). This prompted us to test the antiviral activities of all selected drugs against SARS-CoV-2 replication in ex vivo primary normal human bronchial epithelial cells (NHBEs). Notably, while most inhibitors did not significantly affect SARS-CoV-2 accumulation, ACHP, an inhibitor for IKKa and IKKp, exerted a prominent antiviral effect (Fig. 5c). ACHP caused a reduction in expression of IL-6 mRNA in response to SARS-CoV-2 infection (Fig. 10d), as expected and confirming its activity in primary cells. Employing NHBEs from different donors further confirmed that nanomolar concentrations of ACHP strongly inhibited accumulation of viral RNA in cells infected with SARS-CoV-2. Moreover, ACHP inhibited accumulation of viral RNA in NHBEs infected with the variant of concern SARS-CoV-2 BA.1 (Omicron) (Fig. 5d), showing that the inhibitory effect of the IKK complex inhibitor is conserved between SARS-CoV-2 isolates. In addition, ACHP treatment significantly reduced the spread of SARS-CoV-2-GFP in NHBE cells (Fig. 10e-f) as well as syncytia formation (Fig. 10e). We confirmed the antiviral activity of ACHP at nanomolar concentrations by staining for the nucleoprotein (N) in SARS-CoV-2 infected A549-ACE2 cells (Fig. 5e-f).

Overall, our data show that activation of the NF-KB pathway has a proviral effect. Surprisingly, however, only IKKa/p and NIK inhibitors curbed SARS-CoV-2 replication while other inhibitors did not or only mildly affect the virus. Collectively, these data point towards a central role of IKKs as proviral host factors for SARS-CoV-2 infection both in cell lines and in primary human lung cells.

Example 3

IKKa proviral activity is independent of NF-KB transcription factors

ACHP has been described as an anti-inflammatory molecule targeting IKKa (IC50=250 nM) and IKKp (IC50=40 nM), which contribute to the canonical and non-canonical N F-KB pathway (Fig. 10a). To validate the drug screening approach and identify if any of the two IKK kinases are responsible for the antiviral phenotype induced by ACHP, we genetically deleted components of the canonical and non-canonical NF-KB signaling pathways using CRISPR/Cas9. Successful targeting of the individual NF-KB components was verified by western blotting (Fig. 11a). Cell growth was not affected in any of the knockout cell lines (Fig. 11 b). In accordance to the chemical compound inhibitor data, depletion of RelA or RelB, the main transcription factors involved in the canonical and non-canonical pathways, respectively, only led to a minor reduction of virus replication and did not significantly affect SARS-CoV-2- GFP infection (Fig. 6a). In contrast, knocking out c-Rel, which is involved in canonical N F-KB signaling, increased virus replication, indicating that NF-KB signaling contributes to antiviral activities. Interestingly, IKKa knockout as well as depletion of its upstream kinase NIK showed significant reduction of the SARS-CoV-2 GFP signal (Fig. 6a). IKKp knockout did not result in a prominent reduction of virus replication. Similar results were achieved when repeating this knockout screen with SARS-CoV-2 virus and testing for viral protein and RNA accumulation. We observed less SARS-CoV-2 N expressed in IKKa KO cells while RelA KO and RelB KO cells allowed similar accumulation of N as compared to control cells (NTC) (Fig. 6b, Fig. 11a). In addition, IKKa depletion significantly affected SARS-CoV-2 RNA accumulation while deletion of RelA, RelB or c-Rel did not lead to significant differences (Fig. 6c). Moreover, RelA/RelB double knockout (dKO) A549-ACE2 cells allowed similar accumulation of SARS- CoV-2 RNA as compared to NTC cells, further confirming that NF-KB transcription factors are not required for SARS-CoV-2 replication (Fig. 6d). However, ACHP had a similar antiviral effect on both RelA, RelB KO and control cells (Fig. 6e) further underlining a transcription factor independent activity of the IKKa inhibitor. To understand whether IKKa’s requirement is specific for SARS-CoV-2 infection or whether it is more generally required for Betacoronaviruses, we tested infection of cells depleted for N F-KB components with SARS- CoV-1 (strain: FRA1), which was responsible for the 2003 SARS epidemic. Notably, while NF- KB transcription factors did not significantly affect SARS-CoV-1 replication, depletion of IKKa prominently reduced SARS-CoV-1 RNA accumulation (Fig. 6f).

Altogether, these observations led us to conclude that the proviral effect of IKKa was independent of canonical and non-canonical NF-KB pathway transcription factors and was relevant for SARS-CoV-2 and SARS-CoV-1.

Example 4

IKKa is a key factor of SARS-CoV-2 entry into host cells

We next investigated which steps of the virus life cycle were influenced by IKKa. We performed a time- and dose-response analysis of SARS-CoV-2 sensitivity towards ACHP treatment in A549-ACE2 cells. We pre-treated A549-ACE2 cells with ACHP for 4 h and infected cells for 1 , 6 and 24 h, which are representative time-points of virus attachment, early steps of infection and virus spread, respectively. At 1 hour post infection (hpi), ACHP did not affect SARS-CoV- 2 RNA levels, indicating that viral attachment was unaffected (Fig. 7a). Interestingly, at this time point the IL-6 mRNA expression of SARS-CoV-2 infected cells was already reduced in ACHP treated cells (Fig. 8a), further indicating that the transcriptional inhibition of pro- inflammatory cytokines by ACHP did not correlate with the antiviral effect of the inhibitor. However, we were able to observe a prominent and dose-dependent decrease of viral RNA as early as 6 hpi (Fig. 7a). The degree of ACHP’s antiviral effect was comparable at 6 hpi and 24 hpi suggesting that the inhibitor affected early events of the viral life cycle, such as entry or early replication. Since the entry process is governed by the viral S protein we tested whether S-driven viral entry indeed requires IKKa. For this we employed a S-pseudotyped vesicular stomatitis virus (VSV-S) expressing GFP, and compared its infection dynamics with VSV-GFP expressing its native G glycoprotein. E64d treatment (a well described cathepsin inhibitor) led to selective inhibition of VSV-S while it did not affect VSV, which is in line with expected results since VSV does not rely on cathepsins for endosomal egress (Fig. 7b). Notably, VSV-S but not VSV was inhibited by ACHP treatment, underlining a role of IKKa in the S-dependent entry pathway (Fig. 7b). Celastrol (a canonical NF-KB inhibitor) treatment had no significant effects on VSV-S and VSV, showing that NF-KB transcriptional responses per se are not required for VSV replication (Fig. 7b). Accordingly to our inhibitor data, IKKa- but not IKKp-depleted A549- ACE2 cells showed significantly decreased VSV-S infection, while the parental VSV was not affected (Fig. 7c). Overall, the kinetic of SARS-CoV-2 infection and the use of VSV-S showed that IKKa controls Spike protein mediated entry of SARS-CoV-2.

Example 5

IKKa is required for endosomal uptake and maturation

To further identify which step of the viral entry process is affected by IKKa inhibition, we used purified virus-like particles (VLPs) consisting of the SARS-CoV-2 proteins Spike, M, N and E and engineered to carry CD63~BlaM, a protein fusion of human CD63 with p-lactamase. The BlaM enzyme, which resides intraluminally in the SARS-CoV-2 based VLPs allows their tracking upon cellular uptake or membrane fusion with target cells. We pretreated A549-ACE2 cells with either ACHP, the cathepsin L inhibitor E64d or their vehicle (DMSO), incubated the cells with the VLPs carrying BlaM, loaded the cells with its CCF4 substrate and visualized the turnover of the substrate by confocal microscopy at 6 hpi. As expected, vehicle-treated cells showed a diffuse signal, which is in line with the successful delivery of BlaM into the cells' cytoplasmic compartment (Fig. 7d). In contrast, E64d blocked the VLPs resulting in concentrated, dot-like signals indicative of VLPs trapped in intracellular vesicles and incapable of escaping from endosomes. Notably, in ACHP-treated cells, the BlaM signal was also punctate and reminiscent of E64d treatment suggesting that IKKa inhibition prevented VLP fusion with endosomal membranes and thus cytoplasmic entry (Fig. 7d). This surprising result prompted us to test the effect of ACHP on general endocytic activity. To this aim we employed a dextran bead (coupled with pHrodo-green) uptake assay, which allows to monitor general uptake of beads as well as the proportion of endosomes that mature into acidified vesicles. Compared to DMSO- or E64d-treated cells, ACHP-treated A549-ACE2 cells showed a dramatically reduced uptake of dye-coupled dextran beads into matured endosomes (Fig. 7e). To evaluate whether ACHP affects endosomal trafficking or endolysosomal maturation more specifically, we monitored organelle acidification in live cells using a low pH-sensitive dye (Lysotracker). ACHP but not DMSO or E64d treatment led to a significant decrease in vesicle acidification (Fig. 7f), pointing towards a severe defect in endosomal maturation in IKKa inhibited cells. We next stained VLP-inoculated A549-ACE2 cells for SARS-CoV-2 Spike (S) and the early endosomal marker EEA1 or lysosomal marker LAMP1 , to identify whether incoming VLPs are trapped in specific intracellular compartments of ACHP treated cells. As expected, control treated cells showed little S signal, in line with Spike degradation after successful infection (Fig. 7g-h). In contrast, the S signal accumulated in dot-like structures upon ACHP and E64d treatment, similar to the pattern observed in the enzymatic VLPs uptake assay (Fig. 7g-h). Notably, in both ACHP and E64d treatment the Spike signal colocalized with LAMP1 (Fig. 7h) but not with EEA1 (Fig. 12c) indicating that IKKa inhibition leads to accumulation of VLPs in lysosome-derived vesicles. To further confirm this in the context of virus infection we infected A549-ACE2 cells for different times and stained them for SARS- CoV-2 S and LAMP1. At early times after SARS-CoV-2 infection (e.g. 2 hpi), the S intensity was similarly low in DMSO and ACHP treated conditions (Fig. 12d). In general, S and LAMP1 colocalization was rare regardless of the treatment, in accordance with our previous results that attachment and early entry steps of the viral particles do not require IKKa activity (Fig. 7a). However, at later stages of the infection process (6 hpi) a higher colocalization score of S and LAMP1 was obtained, particularly when cells were treated with ACHP (Fig. 7i-j). In addition, Spike-positive dot-like structures were also detectable in ACHP and E64d conditions while DMSO-treated cells revealed a diffuse Spike-positive signal (Fig. 7i), likely due to de novo Spike synthesis. We concluded that IKKa inhibition affects SARS-CoV-2 entry by perturbing lysosomal acidification, thus preventing Spike activation and membrane fusion, which are required for viral egress from late endosomes. Consequently, IKKa inhibition trapped the incoming particles in endolysosomal vesicles.

Example 6

IKKa mediates unconventional phosphorylation of p-catenin

IKKa is best described as a kinase contributing in the signaling cascade of the canonical and non-canonical NF-KB pathway culminating in transcriptional responses governed by RelA and RelB, respectively. However, since inhibition or genetic depletion of these transcription factors did not phenocopy the effect of IKKa depletion (Fig. 2), and since IKKa was required for a very early step in the infection cycle of SARS-CoV-2, we hypothesized that IKKa kinase activity may directly influence proteins involved in endosomal maturation. To identify potential targets of IKKa, we analyzed the phosphorylation profile of IKKa KO and control cells (NTC) infected with SARS-CoV-2 for 6 h (Fig. 8a). Overall, this phosphoproteomic analysis identified 1 ,314 phosphorylated peptides. Direct comparison of IKKa KO and NTC cells revealed 98 peptides that were significantly less phosphorylated in IKKa deficient cells. Among the significantly decreased phosphorylated phosphosites we identified six that originated from proteins that were reported to physically interact with IKKa (Fig. 8b). Surprisingly, this analysis identified p- catenin (CTNNB1), a key component of the Wnt signaling pathway that has already been reported to contribute to endosomal maturation. Interestingly, IKKa was shown to coprecipitate and regulate the phosphorylation status of p-catenin. Most regulatory phosphorylation sites of CTNNB1 are located in the N-terminus of the protein (Ser³³, Ser³⁷, Thr⁴¹ and Ser⁴⁵) (Fig. 8c). However, phosphorylation at position Ser⁵⁵² has been shown to be impaired in AKT kinase inhibited cells. Notably, AKT is a well described regulator of IKKa and has been shown to be hyperactive in SARS-CoV-2 and SARS-CoV-1 infected cells. Differential phosphorylation of Ser⁵⁵² has been linked to a specific p-catenin transcriptional signature involved in angiogenic and metastatic gene expression in colorectal cancer. We confirmed that Ser⁵⁵² phosphorylation was slightly reduced in A549-ACE2 treated with Ipatasertib, a highly selective pan-AKT inhibitor (IC50=5 nM/18 nM/8 nM against AKT1/2/3, respectively) and infected with SARS-CoV-2 for 24h. However, in comparison to Ipatasertib treatment and to NTC controls, IKKa-depleted A549-ACE2 cells showed much more profound reduction of p- catenin Ser⁵⁵² phosphorylation levels (Fig. 8d). Given the effects on p-catenin phosphorylation we expected that IKKa depletion would result in an altered p-catenin transcriptional signature. To validate this hypothesis, we performed transcriptome analysis of 24 h SARS-CoV-2 infected IKKa, RelA and RelB KO A549-ACE2 cells and its corresponding NTC cell line (Fig. 8e, Fig. 13a). Comparing infected IKKa deficient to NTC cells revealed 491 genes that were differentially regulated in an IKKa dependent manner (Fig 4f). Given the involvement of IKKa in NF-KB signaling, a subset of genes with reduced expression in IKKa knockout cells were involved in the inflammatory response, such as the IL-17 and TNF signaling pathways (Fig. 8g). Notably, IKKa depletion led to a prominent subset of regulated genes that were not dysregulated in infected RelA or RelB KO cells (Fig. 8f), indicating that IKKa depletion led to a distinct transcriptional profile as compared to depletion of NF-KB transcription factors. Gene enrichment analysis revealed that the majority of downregulated genes were linked to processes that are transcriptionally regulated by p-catenin, i.e. genes involved in gap junction, EGFR and ErbB signaling (Fig. 8g). Additionally, unbiased upstream promoter analysis linked IKKa KO transcriptional regulations in A549-ACE2 cells to transcriptional signatures involved in the p-catenin dependent differentiation (TCF12, BCL3, EP300) and the decreased detection of factors involved in proliferative p-catenin activity (TEAD4, FOSL2) (Fig. 13c). We further validated these results in A549-ACE2 IKKa KO cells and observed significantly decreased mRNA expression of p-catenin regulated genes such as c-Jun and EPHB2, the latter being known as a receptor for paramyxovirus entry and involved in endocytosis (Fig. 13d). To gain insights in IKKa-dependent regulation of p-catenin in primary cells and to exclude a bias by using a cell line, we performed transcriptome analysis in ACHP and control (DMSO) pretreated and SARS-CoV-2 infected primary NHBE cells. As seen in A549-ACE2 cells, pathways downregulated by ACHP treatment could be linked to p-catenin activity and contained regulation of carcinogenic processes, atherosclerosis and transcriptional dysregulation in cancer (Fig. 13b, e,). Collectively, these data point to a role of IKKa in phosphorylating p- catenin at Ser⁵⁵² resulting in differential activity of p-catenin upon virus entry and corresponding transcriptional regulation at a later time of infection.

IKKa inhibition or genetic depletion controlled SARS-CoV-2 at early steps of infection. We hypothesized that a comparable phenotype may be seen when inhibiting AKT, being an IKKa activating kinase and that was reported to be involved in regulation of the same p-catenin phosphorylation site. To confirm this we first studied the effect of AKT activation (e.g. by EGF treatment) or inhibition (e.g. Ipatasertib treatment) on SARS-CoV-2 replication. Indeed, in line with this notion, EGF treatment of A549-ACE2 cells led to increased SARS-CoV-2 and SARS- CoV-2-GFP spread (Fig. 8h, Fig. 14a). Inversely, Ipatasertib inhibited SARS-CoV-2 and SARS- CoV-2-GFP in a dose-dependent manner (Fig. 8h-i, Fig. 14b). Consistent with a role of AKT in the infection process, Ipatasertib selectively inhibited infection with S-pseudotyped VSV (VSV- Spike) but not VSV (Fig. 8j, Fig. 14c).

Previous studies have linked the Wnt pathway, and thus p-catenin activity, to the induction of lysosomal acidification. Since we observed that IKKa inhibition reduced endocytosis activity as well as the number of acidic vesicles, we tested whether EGF treatment, which is known to activate p-catenin, similarly affects endolysosomal maturation. Indeed, EGF treatment led to increased endosomal maturation as tested by uptake of pHrodo dextran beads (Fig. 14d). Conversely, as seen for ACHP treatment, inhibition of AKT by Ipatasertib led to a significant, dose-dependent reduction of pHrodo dextran bead uptake into matured endolysosomes (Fig. 8k and Fig. 14e). Moreover, Ipatasertib treatment significantly reduced the accumulation of acidified vesicles as tested by staining with lysotracker (Fig. 8I and Fig. 14f). Furthermore, as compared to ACHP treatment alone, co-treatment of ACHP and Ipatasertib did not show significant additive effects on SARS-CoV-2-GFP spread (Fig. 8m), indicating that both IKKa and AKT operate in the same pathway supporting SARS-CoV-2 infection.

Example 7 IKKa is generally required for pathogens infecting through late endosomes/endolysosomes

Given that other pathogens including a subset of viruses and intracellular bacteria rely on the lysosomal pathway for entry and productive infection, we set out to test the effect of ACHP treatment on additional pathogens with the hypothesis that these should be similarly affected as SARS-CoV-2. We first tested non-related DNA viruses belonging to the Orthopoxvirus genus, which require late vacuolar conditions for entry. A549 WT cells treated with DMSO and E64d showed similar accumulation of GFP when infected with GFP-expressing Vaccinia virus (VACV-GFP) (strain: VACV-V300-GFP). Notably, ACHP treatment significantly reduced infection with VACV-GFP, in line with the necessity of lysosomal acidification for poxvirus infection (Fig. 9a). Similarly, A549 cells infected with Monkeypox virus (MPXV), an Orthopoxvirus responsible for a recent worldwide epidemic, showed more than 75% reduction of genomic DNA when treated with the ACHP (Fig. 9b). As for VACV, E64d treatment did not significantly affect MPXV replication (Fig. 9b). For both poxviruses tested, we could also observe a significant inhibition of virus growth when cells were pre-treated with Ipatasertib, emphasizing that viruses relying on endolysosomal entry require IKKa and AKT. We furthermore evaluated the effect of IKKa and AKT inhibition on Rift Valley fever virus (RVFV) expressing GFP (strain: RVFV-GFP), a negative single-strand RNA virus from the Phlebovirus genus, which also enters cells by endocytosis and lysosomal endocytosis. Both ACHP and Ipatasertib significantly reduced RVFV-GFP accumulation, while E64d did not affect GFP levels (Fig. 9c). Besides viruses, intracellular bacteria often infect cells through the endolysosomal pathway. We thus tested whether IKKa inhibition by ACHP would also affect the entry of Salmonella enterica and Shigella flexneri in immortalized bone marrow-derived macrophages (iBMDMs) by analyzing Colony Forming Unit (CFUs) of internalized bacteria. Indeed, 1 h after infection, we observed a reduction in CFUs of both pathogens in a ACHP concentration-dependent manner (Fig. 9d-e). ACHP did not show cytotoxic effects in iBMDMs, excluding that the observed phenotypes are mediated by drug toxicity (Fig. 9g).

In sum, our data highlight that IKKa is required for successful infection by a broad range of microbial pathogens and that inhibition of IKKa impairs both viruses and pathogenic bacteria that usurp the endolysosomal entry route for their infection process.

Example 8

ACHP treatment reduces SARS-CoV-2 transcripts in vivo

K18-hACE2 C57BL/6J mice (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J) were obtained from The Jackson Laboratory. Age matched male and female mice, were randomly grouped, fed standard chow diets and were housed in a pathogen free facility. Animals were administered 2.5x10³ p.f.u. SARS-CoV-2 (a clinical isolate of B.1.1.7, alpha strain) under anaesthesia (75pg/g Ketamin, and 1 pg/g Medetomidin) the effect of anaesthesia was reversed by using Antisedan (1 pg/g). The mice received DMSO or ACHP (2mg/kg) via intranasal administration at -1day and -1 hr before SARS-CoV-2 infection under isoflurane anaesthesia.

Lungs harvested at 24 h.p.i were homogenized using steel beads (Qiagen) in a Tissuelyser (II) (Qiagen) in PBS and immediately used for RNA isolation. RNA were isolated using the High Pure RNA Isolation Kit (Roche) and an equal amount of RNA was used for standard One-Step RT-PCR (Applied Biosystems TaqMan RNA to CT One Step Kit). SARS-CoV-2 N gene was identified using specific qPCR primers and probes. For p-Actin primers and probe (Mm00607939_s1) from ThermoFisher were used. RNA levels of SARS-CoV-2 N gene were normalized to the mouse housekeeping gene p-Actin using the formula 2^A(Ct (P-Actin)- Ct(SARS-CoV-2 RNA)). Treatment with ACHP significantly reduced SARS-CoV-2 transcripts in the lungs of mice infected with SARS-CoV-2 24h earlier (Fig. 15). This demonstrates that treatment with an inhibitor of the IKK-a/NIK complex has antiviral effects in vivo. Since many of the severe symptoms of SARS-CoV-2 infection in patients results from lung infection, it is particularly relevant that the treatment was able to reduce SARS-CoV-2 transcripts in lungs.

Further, the Invention includes the following embodiments:

Embodiment 1 : Inhibitor of IKK-a/NIK complex for use in preventing or treating viral infection in a subject.

Embodiment2: Inhibitorfor use according to embodiment 1 , wherein viral replication is reduced or inhibited.

Embodiment 3: Inhibitor of for use in embodiment 1 or embodiment 2, wherein the viral infection is caused by a virus of the phylum Pisuviricota.

Embodiment 4: Inhibitor for use according to embodiment 3, wherein the viral infection is caused by a virus of the family Coronaviridae.

Embodiment 5: Inhibitor for use according to embodiment 4, wherein the viral infection is SARS-CoV infection.

Embodiment 6: Inhibitor for use according to embodiment 5, wherein SARS-CoV is selected from the group consisting of SARS-CoV-1 and SARS-CoV-2.

Embodiment 7: Inhibitor for use according to embodiment 6, wherein SARS-CoV is SARS- CoV-2. Embodiment 8: Inhibitor for use of according to any one of embodiments 1 to 6, wherein the inhibitor is selected from the group consisting of inhibitor of IKK-a and inhibitor of NIK.

Embodiment 9: Inhibitor for use according to embodiment 8, wherein the inhibitor does not function through inhibition of NF-KB transcription factors.

Embodiment 10: Inhibitor for use according to embodiment 9, wherein the inhibitor is an inhibitor of IKK-a.

Embodiment 11 : Inhibitor for use according to any one of embodiment 10, wherein the inhibitor of IKK-a is IKK-16 or ACHP hydrochloride.

Embodiment 12: Inhibitor for use according to embodiment 11 , wherein the inhibitor is ACHP hydrochloride.

Embodiment 13: Inhibitor for use according to embodiment 8, wherein the NIK inhibitor is Amgen16.

Embodiment 14: Inhibitor for use according to any one of the preceding embodiments, wherein the inhibitor is combined with interferon therapy or antiviral therapy.

Embodiment 15: A pharmaceutical composition comprising the inhibitor according to any one of the preceding embodiments together with a pharmaceutically acceptable carrier.

Embodiment 16: Inhibitor of IKK-a/NIK complex for use in preventing or treating viral infection in a subject and/or for use in preventing or treating coronavirus disease 2019 (COVID-19) in a subject.

Embodiment 17: Inhibitor for use according to embodiment 16, wherein viral replication is reduced or inhibited.

Embodiment 18: Inhibitor for use according to embodiment 16 or 17, wherein the virus is not HCV, Herpesviruses and/or HIV.

Embodiment 19: Inhibitor for use according to any one of embodiments 16 to 18, wherein the viral infection is caused by a virus of the phylum Pisuviricota. Embodiment 20: Inhibitor for use according to embodiment 19, wherein the viral infection is caused by a virus of the family Coronaviridae.

Embodiment 21 : Inhibitor for use according to embodiment 20, wherein the viral infection is SARS-CoV infection.

Embodiment 22: Inhibitor for use according to embodiment 21 , wherein SARS-

CoV is selected from the group consisting of SARS-CoV-1 and SARS-CoV-2, preferably wherein SARS-CoV is SARS-CoV-2.

Embodiment 23: Inhibitor for use of according to any one of embodiments 16 to

22, wherein the inhibitor is selected from the group consisting of inhibitor of the IKK-a and inhibitor of NF-KB-inducing kinase (NIK).

Embodiment 24: Inhibitor for use according to embodiment 23, wherein the inhibitor of the IKK-a/NIK complex is an inhibitor of NIK.

Embodiment 25: Inhibitor for use according to embodiment 24, wherein the inhibitor of the IKK-a/NIK complex is an inhibitor of IKK-a.

Embodiment 26: Inhibitor for use of according to any one of embodiments, wherein the inhibitor does not function through inhibition of NF-KB transcription factors.

Embodiment 27: Inhibitor for use according to any one of embodiment 26, wherein the inhibitor of IKK-a is IKK-16 or ACHP hydrochloride.

Embodiment 28: Inhibitor for use according to embodiment 27, wherein the inhibitor is

ACHP hydrochloride.

Embodiment 29: Inhibitor for use according to embodiment 28, wherein the NIK inhibitor is Amgen16.

Embodiment 30: Inhibitor for use according to any one of the preceding embodiments, wherein the inhibitor is combined with interferon therapy or antiviral therapy.

Embodiment 31 : Inhibitor for use according to any one of the preceding embodiments, wherein the inhibitor is administered before viral infection. Embodiment 32: Inhibitor for use according to any one of the preceding embodiments, wherein the inhibitor is administered after viral infection.

Embodiment 33: A pharmaceutical composition comprising the inhibitor as defined in any one of the preceding embodiments together with a pharmaceutically acceptable carrier.

Embodiment 34: A method of preventing or treating viral infection and/or for use in preventing or treating coronavirus disease 2019 (COVID-19) in a human or non-human animal in need thereof, comprising administering an inhibitor of the IKK-a/NIK complex as defined in any one of embodiments 1 to 17 or the pharmaceutical composition according to embodiment 18 to said human or non-human animal.

Embodiment 35: A method of preventing or treating viral infection in a human or non- human animal in need thereof, comprising administering an inhibitor of the IKK-a/NIK complex to said human or non-human animal.

Embodiment 36: Use of an inhibitor of the IKK-a/NIK complex for the manufacture of a medicament for use in preventing or treating viral infection and/or for use in preventing or treating coronavirus disease 2019 (COVID-19) in a subject.

Claims

1. Inhibitor of the IKK-a/NIK complex for use in preventing or treating infection with a pathogen in a cell and/or subject.

2. Inhibitor for use according to claim 1 , wherein the inhibitor is an IKK-a inhibitor.

3. Inhibitor for use according to any one of claims 1 or 2, wherein preventing or treating infection with a pathogen comprises reducing endocytosis of the pathogen in a cell and/or subject treated with the inhibitor compared to an untreated cell and/or subject.

4. Inhibitor for use according to any one of claims 1 to 3, wherein preventing or treating infection with a pathogen comprises reducing the total pathogen load of the pathogen in a cell and/or subject treated with the inhibitor compared to an untreated cell and/or subject.

5. Inhibitor for use according to any one of claims 1 to 4, wherein preventing or treating infection with a pathogen comprises reducing activity of cytoplasmic p-catenin levels in a cell and/or subject treated with the inhibitor compared to untreated control cell and/or subject.

6. Inhibitor for use according to any one of claims 1 to 5, wherein the inhibitor is ACHP or IKK 16, or a pharmaceutically acceptable salt thereof.

7. Inhibitor for use according to any one of claims 1 to 6, wherein the pathogen is one or more virus or bacterium.

8. Inhibitor for use according to any one of claims 1 to 7, wherein the pathogen is an intracellular pathogen.

9. Inhibitor for use according to any one of claims 1 to 8, wherein the pathogen belongs to a family selected from the group consisting of Coronaviridae, Poxviridae, Phenuiviridae, Flaviviridae, Picornaviridae, Pneumoviridae, Herpesviridae, Togaviridae, Orthomyxoviridae, Rhabdoviridae, Picornaviridae, Papillomaviridae, Arenaviridae, Enterobacteriaceae, Coxiellaceae, Brucellaceae, Listeriaceae, Rickettsiaceae, Chlamydiaceae, Mycobacteriaceae, Yersiniaceae, Vibrionaceae, Bartonellaceae, Francisellaceae, and Nocardiaceae.

10. Inhibitor for use according to any one of claims 1 to 9, wherein the pathogen belongs to a family selected from the group consisting of Coronaviridae, Poxviridae, Phenuiviridae and Enterobacteriaceae. Inhibitor for use according to any one of claims 1 to 10, wherein the pathogen is selected from the group consisting of severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome-related coronavirus (MERS-CoV), Vaccinia virus (VACV), Monkeypox virus (MPXV), Rift Valley fever virus (RVFV), Salmonella and Shigella. Inhibitor for use according to any one of claims 1 to 10, wherein the infection is selected from the group consisting of severe acute respiratory syndrome (SARS), coronavirus disease 2019 (COVID-19), Middle East respiratory syndrome (MERS), Monkeypox, smallpox, Rift Valley fever, hepatitis C, hepatocellular carcinoma, lymphoma, dengue fever, causes foot-and-mouth disease, bronchiolitis, common cold and pneumonia, hemorrhagic fever, Kaposi’s sarcoma, primary effusion lymphoma, HHV8-associated multicentric Castleman’s disease and KSHV inflammatory cytokine syndrome, influenza, vesicular stomatitis, common cold, anal dysplasia, genital cancers such as vulva, vagina, cervix and penis cancer, mouth papilloma, oropharyngeal cancer, aseptic meningitis, encephalitis, meningoencephalitis, pappataci fever, hemorrhagic fever, salmonellosis, typhoid fever, paratyphoid fever, shigellosis, Q fever, brucellosis, listeriosis, gastroenteritis, meningitis, meningoencephalitis, typhus, rickettsial pox, boutonneuse fever, African tick-bite fever, Rocky Mountain spotted fever, Flinders Island spotted fever, Queensland tick typhus, pelvic inflammatory disease, trachoma, gonorrhoea, Legionnaires’ disease, Pontiac fever, tuberculosis, leprosy, Hansen’s disease, lymphadenitis, pulmonary disease, skin disease, plague, yersiniosis, reactive arthritis, pseudoappendicitis, cholera, tularemia, nocardiosis, septicaemia and cat scratch disease. Inhibitor for use according to any one of claims 1 to 12, wherein the infection is selected from the group consisting of severe acute respiratory syndrome (SARS), coronavirus disease 2019 (COVID-19), Middle East respiratory syndrome (MERS), Monkeypox, Rift Valley fever, salmonellosis, typhoid fever, paratyphoid fever and shigellosis. Pharmaceutical composition comprising the inhibitor of any one of the claims 1 to 13 and a pharmaceutically acceptable carrier. Pharmaceutical composition according to claim 14, further comprising one or more therapeutic agents, preferably anti-inflammatory agents, antibiotic agents or antiviral agents.