阅读说明：本技术 治疗病毒性疾病的rsk抑制剂 (RSK inhibitors for the treatment of viral diseases ) 是由 S·路德维希 O·普兰兹 于 2020-03-19 设计创作，主要内容包括：本发明涉及能够显示一种或多种有益治疗效果的RSK抑制剂。所述RSK抑制剂能够用在预防和/或治疗病毒感染中。RSK抑制剂单独或与其它抗病毒抑制剂化合物组合在病毒性疾病的治疗中能够显示一种或多种有益的治疗效果。(The present invention relates to RSK inhibitors that are capable of exhibiting one or more beneficial therapeutic effects. The RSK inhibitors can be used in the prevention and/or treatment of viral infections. RSK inhibitors can exhibit one or more beneficial therapeutic effects in the treatment of viral diseases, either alone or in combination with other antiviral inhibitor compounds.)

1. An RSK inhibitor for use in a method for the prevention and/or treatment of a viral disease.

2. The RSK inhibitor for use according to claim 1, wherein said RSK inhibitor is selected from the group consisting of BI-D1870, SL0101-1, LJH685, LJI308, BIX 02565, FMK, and a selective RSK1 inhibitor or a derivative, metabolite, or pharmaceutically acceptable salt thereof.

3. The RSK inhibitor for use according to claim 2, wherein the selective RSK1 inhibitor is a selective targeting polypeptide corresponding to a polypeptide having the sequence of SEQ ID NO: 1, or a si-RNA, shRNA or mi-RNA of RSK1 mRNA having the amino acid sequence of SEQ ID NO: 1 of RSK 1.

4. The RSK inhibitor for use according to any preceding claim, wherein the viral disease is an infection caused by a negative strand RNA virus, preferably an influenza virus.

5. The RSK inhibitor for use according to any preceding claim, wherein the viral disease is an infection caused by a positive strand RNA virus, preferably a coronavirus causing a respiratory infection.

6. The RSK inhibitor for use according to claim 4, wherein said influenza virus is resistant to a ceramidase inhibitor selected from oseltamivir, oseltamivir phosphate, zanamivir, peramivir or lanimivir, or a pharmaceutically acceptable salt thereof, or an inhibitor of a viral polymerase complex selected from fapiravir, barlosavir phosphate, Baloxavir Marboxil or pirimodipir, or a pharmaceutically acceptable salt thereof.

7. The RSK inhibitor for use according to claim 4 or 6, wherein said influenza virus is an influenza virus of H1N1, H2N2, H3N2, H5N1, H5N6, H5N8, H6N1, H7N2, H7N7, H7N9, H9N2, H10N7, N10N8 type, or a different influenza A or B virus, such as an influenza virus of Yamagata type or Victoria type.

8. The RSK inhibitor for use according to any of the preceding claims, wherein the RSK inhibitor is administered in combination with a second anti-viral agent.

9. The RSK inhibitor for use according to claim 8, wherein the second antiviral agent is selected from the group consisting of a ceramidase inhibitor, a polymerase complex inhibitor, an endonuclease inhibitor, a hemagglutinin inhibitor, a non-structural protein 1 inhibitor, a nucleoprotein inhibitor and a MEK inhibitor.

10. The RSK inhibitor for use according to claim 9, wherein said neuraminidase inhibitor is selected from oseltamivir, oseltamivir phosphate, zanamivir, peramivir or ranimivir, or a pharmaceutically acceptable salt thereof.

11. The RSK inhibitor for use according to claim 9, wherein the polymerase complex inhibitor is Baloxavir, Baloxavir phosphate, Baloxavir Marboxil, favipiravir or pimozadditionally or a pharmaceutically acceptable salt thereof.

12. The RSK inhibitor for use according to claim 9, wherein the MEK inhibitor is selected from CI-1040, PD-0184264, PLX-4032, AZD6244, AZD8330, AS-703026, GSK-1120212, RDEA-119, RO-5126766, RO-4987655, PD-0325901, GDC-0973, TAK-733, PD98059 and PD184352, or a pharmaceutically acceptable salt thereof.

13. A selective RSK1 inhibitor or a derivative, metabolite or pharmaceutically acceptable salt thereof that binds to a polypeptide having the sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2, and to RSK1 having the nucleotide sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 4, RSK2 having the nucleotide sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 6 and RSK3 having the nucleotide sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: RSK4 of the nucleotide sequence of 8 shows no or low binding affinity.

14. The selective RSK1 inhibitor of claim 13, wherein the inhibitor is si-RNA, shRNA, mi-RNA, an antibody, or a small molecule.

15. A pharmaceutical composition comprising a selective RSK1 inhibitor according to claim 13 or 14, alone or in combination with a second anti-viral agent, for use in the prevention and/or treatment of viral diseases.

16. A method of identifying a specific RSK1 inhibitor, comprising the steps of:

(i) screening for binding to a polypeptide having SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2, a library of potential inhibitors of RSK 1;

(ii) (ii) selecting the inhibitors found in step (i) that bind to RSK1 and screening these inhibitors for binding to a polypeptide having the sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 4, RSK2 having the nucleotide sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 6 and RSK3 having the nucleotide sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 8, RSK4 of the nucleotide sequence of seq id no; and

(iii) selecting from step (ii) an inhibitor that does not bind to RSK2, RSK3 or RSK4 as a specific RSK1 inhibitor.

Technical Field

The present invention relates to RSK inhibitors that are capable of exhibiting one or more beneficial therapeutic effects. The RSK inhibitor can be used in the prevention and/or treatment of viral infections. Combinations of RSK inhibitors with other antiviral compounds can exhibit one or more beneficial therapeutic effects in the treatment of viral diseases.

Background

Viral infections represent a significant threat to human and animal health. For example, influenza virus infection remains a human pandemic and causes a number of deaths each year.

A problem in the control of RNA viruses in particular is virus adaptation caused by the high error rate of the viral polymerase, which makes the production of suitable vaccines and the development of antiviral substances difficult. Furthermore, it has been found that the use of antiviral drugs directed against viral function leads sooner or later to the selection of mutation-based resistant variants. One example is amantadine and its derivatives, an anti-influenza agent, directed against viral transmembrane proteins. Within a short time after administration, resistant variants of the virus will be produced. Other examples are antiviral drugs for influenza infection that inhibit the influenza virus surface protein neuraminidase, such as Oseltamivir (Oseltamivir), which has also seen widespread emergence of resistant variants in the past.

All viruses are highly dependent on the function of their host cell, due to the very small genome and thus limited coding capacity for the functions necessary for replication. By affecting such cellular functions necessary for viral replication, it is possible to negatively affect viral replication in infected cells. In this case, the virus cannot replace the lacking cell function by adaptation, in particular by mutation, in order to thus escape the selection pressure. This may have been demonstrated in influenza A viruses with relatively non-specific inhibitors of cellular kinases and methyltransferases (Scholtissek and Muller, Arch Virol 119, 111-.

Like any other virus, influenza virus captures infected cells for replication. Viral proteins interact not only with themselves, but also with cellular components. Thus, blocking these components would not only inhibit replication at a broad antiviral level, but would also reduce the occurrence of resistant viral variants (Ludwig, 2003) (Ludwig, 2011) due to the inability of the virus to replace lost cellular functions.

In the influenza life cycle, a newly synthesized ribonucleoprotein complex (vRNP) containing the viral genome must be exported from the nucleus, where replication of the viral genome occurs, into the cytoplasm, for transport to the cell membrane and packaging in progeny virus released from infected cells. RNP is exported out of the nucleus via the Crm 1-mediated nuclear export pathway, as cytoplasmic accumulation is blocked by the Crm1 inhibitor, leptomycin b (Elton, 2001) (Watanabe, 2001). To date, the organization of the vRNP nuclear export complex is not fully understood. One putative model speculates that Nuclear Export Protein (NEP) interaction with the viral polymerase complex creates a supporting binding site for matrix protein 1. The Crm1 interaction is mediated via the NEP N-terminus (bruntotte, 2014). Considering that the output of vRNP does not occur without M1, but that a large drop in the amount of NEP does not affect the process, the exact contribution of M1 and NEP to RNP output remains elusive (Wolstenholme, 1980) (Smith, 1985) (Martin, 1991) (Bui, 2000). RNP export complexes are shown assembled at compact chromatin to enter cellular export machinery. This assembly occurs within the RCC1(Ran nucleotide exchange factor) localization region to ensure that RNPs interact directly with the regenerative Crm 1-RanGTP-complex (Nemergut, 2001) (Chase, 2011).

In addition, the virus needs to activate the Raf/MEK/ERK signaling pathway to ensure successful nuclear export (Pleschka, 2001) (Ludwig, 2004). This signaling cascade is a member of the classical mitogen-activated protein kinase (MAPK) cascade and regulates proliferation, differentiation and cell survival (Lewis, 1998) (Yoon, 2006). Influenza virus infection triggers pathway activation in a biphasic manner both early and late after infection. MEK-specific inhibitors not only inhibit both activation phases but also result in a strong reduction of viral titer caused by nuclear retention of newly synthesized RNPs. These effects are shown in influenza viruses a and B (Pleschka, 2001) (Ludwig, 2004) (Haasbach, 2017). No escape mutants were found after using MEK inhibitors (e.g. U0126) compared to amantadine treatment (Ludwig, 2004). Furthermore, oseltamivir resistant influenza strains can still be inhibited by treatment with MEK inhibitors (Haasbach, 2017). Furthermore, it has recently been disclosed that inhibition of this pathway after respiratory syncytial virus infection reduces viral titers (Preugschas, 2018). These findings suggest that the virus is unable to compensate for the missing cellular functions and have enabled new antiviral strategies. The exact mechanism of how this pathway triggers influenza RNP export is not clear to date.

As described above, influenza viruses utilize the cellular Raf/MEK/ERK signaling pathway to support export of a newly synthesized genome (in the form of an RNA protein complex, called vRNP) from the nucleus of an infected cell. In previous studies, MEK (central kinase of the Raf/MEK/ERK pathway) inhibitors have been shown to block viral replication in vitro and in vivo without side effects, as well as a high barrier to the development of resistance (WO 2014/056894).

However, the direct link between the activated Raf/MEK/ERK pathway and vRNP complex export remains unclear. In general, the role of the Raf/MEK/ERK pathway in cancer has been studied, and ERK and RSK are known to play a role downstream of MEK. However, in the prior art, Rsk inhibition, particularly Rsk2 knock-out, was found to exert viral supporting effects (Kakugawa et al (2009) J Virol.; 83(6): 2510-7).

However, in view of the prior art, there is a clear need for further compounds and compositions that are effective in the treatment of viral diseases, in particular diseases caused by influenza viruses.

Disclosure of Invention

The present invention relates to an RSK inhibitor for use in a method for the prevention and/or treatment of viral diseases. In particular, the RSK inhibitor can be selected from BI-D1870, SL0101-1, LJH685, LJI308, BIX 02565, FMK, and a selective RSK1 inhibitor or a derivative, metabolite, or pharmaceutically acceptable salt thereof. The selective RSK1 inhibitor may be a selective targeting sequence corresponding to a polypeptide having the sequence of SEQ ID NO: 1, or a si-RNA, shRNA or mi-RNA of RSK1 mRNA having the amino acid sequence of SEQ ID NO: 1 of RSK 1.

The viral disease may be an infection caused by a positive or negative strand RNA virus. In a preferred embodiment, the viral disease caused by a negative strand RNA virus is an influenza virus, e.g. an influenza virus of type H1N1, H2N2, H3N2, H5N1, H5N6, H5N8, H6N1, H7N2, H7N7, H7N9, H9N2, H10N7, N10N8 or H5N1, or a different influenza a or b virus, e.g. an influenza virus of type Yamagata or Victoria. In some embodiments, the influenza virus is resistant to a ceramidase inhibitor selected from oseltamivir (oseltamivir), oseltamivir phosphate (oseltamivir phosphate), zanamivir (zanamivir), peramivir (peramivir), or laninamivir (laninamivir), or a pharmaceutically acceptable salt thereof, or an inhibitor of a viral polymerase complex selected from Favipiravir (Favipiravir), baroxavir (Baloxavir), baroxavir phosphate (Baloxavir phosphate), Baloxavir Marboxil, or pirimavir (pimovir), or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the viral disease caused by a positive-stranded RNA virus is a coronavirus causing a respiratory infection, such as SARS, MERS or Covid-19.

In some cases, the RSK inhibitor is administered in combination with a second antiviral agent, e.g., a ceramidase inhibitor, a polymerase complex inhibitor, an endonuclease inhibitor, a hemagglutinin inhibitor, a non-structural protein 1 inhibitor, a nucleoprotein inhibitor, or a MEK inhibitor.

The ceramidase inhibitor may be oseltamivir, oseltamivir phosphate, zanamivir, peramivir or ranimivir or a pharmaceutically acceptable salt thereof.

The polymerase complex inhibitor may be Baloxavir, Baloxavir phosphate, Baloxavir Marboxil, favipiravir or pirimovir or a pharmaceutically acceptable salt thereof.

The MEK inhibitor may be CI-1040, PD-0184264, PLX-4032, AZD6244, AZD8330, AS-703026, GSK-1120212, RDEA-119, RO-5126766, RO-4987655, PD-0325901, GDC-0973, TAK-733, PD98059, or PD184352, or a pharmaceutically acceptable salt thereof.

The present invention also relates to a selective RSK1 inhibitor, or a derivative, metabolite or pharmaceutically acceptable salt thereof, that binds to a polypeptide having the sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2, and to RSK1 having the nucleotide sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 4, RSK2 having the nucleotide sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 6 and RSK3 having the nucleotide sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: RSK4 of the nucleotide sequence of 8 shows no or low binding affinity. The selective RSK1 inhibitor can be si-RNA, shRNA, mi-RNA, an antibody or a small molecule. The RSK1 inhibitor may be used alone or in combination with a second antiviral agent as defined above in a pharmaceutical composition for the prevention and/or treatment of viral diseases.

Furthermore, the present invention relates to a method for identifying a specific RSK1 inhibitor comprising the steps of: screening for binding to a polypeptide having SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2, selecting the inhibitors found to bind to RSK1, and screening these inhibitors for binding to a polypeptide having the nucleotide sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 4, RSK2 having the nucleotide sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 6 and RSK3 having the nucleotide sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 8, RSK4 of the nucleotide sequence of seq id no; and selecting from step (ii) an inhibitor that does not bind to RSK2, RSK3 or RSK4 as a specific RSK1 inhibitor.

Definition of

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

As used herein, "consisting of … …" excludes any element, step, or ingredient not specified in the claim element, "consisting essentially of … …" as used herein does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim, in each case herein any of the terms "comprising," "consisting essentially of … …," and "consisting of … …" may be substituted with either of the other two terms.

As used herein, the term "and/or" in connection with a plurality of recited elements should be understood to include both individual and combined options. For example, in the case of two elements connected by "and/or", the first option refers to applying the first element without the second element. The second option refers to applying the second element without the first element. The third option refers to applying the first element and the second element simultaneously. Any of these options is understood to fall within this meaning and thus meet the requirements of the term "and/or" as used herein. Simultaneous application of more than one option is also understood to fall within this meaning and thus meet the requirements of the term "and/or" as used herein.

As used herein, the term "RSK" refers to human RSK (p90 ribosomal S6kinase) which exists in four different isoforms referred to as RSK1(SEQ ID NOS: 1 and 2), RSK2(SEQ ID NOS: 3 and 4), RSK3(SEQ ID NOS: 5 and 6), and RSK4(SEQ ID NOS: 7 and 8). Alignment of the four isoforms as shown in FIG. 1 at the amino acid level and FIG. 2 at the nucleotide level reveals that these four forms are highly conserved, but also have specific regions of diversity.

As used herein, the term "RSK inhibitor" refers to an agent that inhibits RSK. The agent inhibits all four RSK isoforms or at least inhibits RSK 1. The RSK inhibitor is selected from BI-D1870, SL0101-1, LJH685, LJI308, BIX 02565, FMK, and a selective RSK1 inhibitor or a derivative, metabolite, or pharmaceutically acceptable salt thereof. A selective RSK1 inhibitor may be a selective targeting sequence corresponding to a polypeptide having SEQ ID NO: 1 amino acid sequence of RSK1, or a si-, shRNA or mi-RNA of RSK1 mRNA that binds to a polypeptide having the amino acid sequence of SEQ ID NO: 1 amino acid sequence of RSK 1.

As used herein, the term "pharmaceutically acceptable salt, derivative or metabolite" refers to relatively non-toxic, organic or inorganic salts of the active compound, including inorganic or organic acid addition salts of the compound, as well as derivatives and metabolites of the claimed RSK inhibitors that exhibit the same function as the so-called inhibitor RSK inhibitor.

Thus, the terms "treatment" or "treating" include administering an RSK inhibitor, preferably in pharmaceutical form, to an individual suffering from a viral disease, in order to alleviate or ameliorate the symptoms associated with such infection.

Furthermore, the term "prevention" (prophyxiases) as used herein refers to any medical or public health procedure aimed at preventing the medical conditions described herein. As used herein, the term "prevention" (prevention, presence and presence) refers to reducing the risk of acquiring or developing a given condition (i.e., a viral disease as described herein). "preventing" also refers to reducing or inhibiting the recurrence of a viral disease in a patient.

The term "viral disease" as used herein includes diseases caused by viruses, such as diseases caused by positive or negative strand RNA viruses. For example, influenza virus is a negative strand RNA virus; such as influenza a virus and influenza B virus. The influenza virus or influenza virus strain according to the invention may show or have developed resistance to one or more neuraminidase inhibitors (e.g. oseltamivir, oseltamivir phosphate, zanamivir or peramivir), or the influenza virus or influenza virus strain according to the invention shows or does not develop resistance to one or more neuraminidase inhibitors (e.g. oseltamivir, oseltamivir phosphate, zanamivir or peramivir). The influenza virus may be an influenza a virus or an influenza B virus, preferably the influenza a virus is H1N1, H2N2, H3N2, H5N1, H5N6, H5N8, H6N1, H7N2, H7N7, H7N9, H9N2, H10N7, N10N8 or H5N 1. In some embodiments, the influenza virus is resistant to a ceramidase inhibitor selected from oseltamivir, oseltamivir phosphate, zanamivir, peramivir, or laninamivir, or a pharmaceutically acceptable salt thereof, or an inhibitor of a viral polymerase complex selected from favepivir, baroxavir phosphate, Baloxavir Marboxil, or pirimovir, or a pharmaceutically acceptable salt thereof.

The positive-stranded RNA virus can be, for example, a coronavirus, such as SARS-CoV, MERS-CoV or SARS-CoV-2, which causes a respiratory infection, such as SARS, MERS or Covid 19.

The term "second antiviral agent" as used herein refers to a known antiviral agent selected from the group consisting of ceramidase inhibitors, polymerase complex inhibitors, endonuclease inhibitors, hemagglutinin inhibitors, nucleoprotein inhibitors and MEK inhibitors.

A "neuraminidase inhibitor" is an antiviral drug directed against influenza virus that acts by blocking the function of the viral neuraminidase protein, thereby preventing the release of the virus from infected host cells, since the newly produced virus cannot bud from the cells it replicates. Also included are pharmaceutically acceptable salts of neuraminidase inhibitors. Preferred neuraminidase inhibitors are oseltamivir, zanamivir, peramivir, lanimivir, or a pharmaceutically acceptable salt of any of these, such as oseltamivir phosphate, oseltamivir carboxylate, and the like. The ceramidase inhibitor may be oseltamivir, oseltamivir phosphate, zanamivir, peramivir or ranimivir or a pharmaceutically acceptable salt thereof. The most preferred neuraminidase inhibitor is oseltamivir phosphate, zanamivir, oseltamivir or peramivir

Compounds that target polymerase or endonuclease activity by interfering with components PB1, PB2, PA or NP of the viral polymerase complex are for example the NP blocker hydrazinium or the polymerase inhibitor T705 (fabiravir). Preferred "polymerase complex inhibitors" are favipiravir, Baloxavir phosphate, Baloxavir Marboxil or pyridipivir or pharmaceutically acceptable salts thereof.

A "MEK inhibitor" inhibits the mitotic signal kinase cascade Raf/MEK/ERK in a cell or subject by inhibiting MEK (mitogen-activated protein kinase). This signaling cascade is hijacked by many viruses, particularly influenza viruses, to enhance viral replication. Thus, specific blockade of the Raf/MEK/ERK pathway at the MEK bottleneck impairs the growth of viruses, particularly influenza viruses. In addition, MEK inhibitors exhibit low toxicity and low adverse side effects in humans. There was also no tendency to induce virus resistance (Ludwig, 2009). The MEK inhibitor may be CI-1040, PD-0184264, PLX-4032, AZD6244, AZD8330, AS-703026, GSK-1120212, RDEA-119, RO-5126766, RO-4987655, PD-0325901, GDC-0973, TAK-733, PD98059, or PD184352, or a pharmaceutically acceptable salt thereof. Combinations with CI-1040 or PD-0184264 are considered a specific embodiment.

Drawings

FIG. 1 shows an alignment of RSK1-4 at the amino acid level.

FIG. 2 shows an alignment of RSK1-4 at the nucleotide level.

FIG. 3 shows the phosphorylation sites (A, C) of phosphorylated NP at residues S269 and S392 upon Raf/MEK/ERK activation as described in example 1. In addition, (B) shows a comparison of ERK and RSK consensus sequences. FIG. (D) shows the RNA binding site of M1 protein described in example 1 with positively charged histidine, FIG. (F) shows the growth kinetics of the constitutive non-phosphorylated (NP) mutant relative to the wild type (wt), and FIG. (G) shows the growth kinetics of the M1 histidine mutant relative to the wild type (wt).

FIG. 4(A-H) shows the reduction of M1-NP binding by RSK inhibition, nuclear retention of progeny vRNP by RSK inhibition or RSK1 knock-out, as demonstrated in example 2. (I-P) shows that specific RSK inhibitors BI-D1870 and SL0101-1 inhibit viral propagation as described in example 2.

FIG. 5 shows that BI-D1870 treatment as described in example 2 results in chromatin retention and a reduced rate of binding to M1 protein in progeny vRNPs.

FIG. 6(A, B) shows that the pathway inhibition as described in example 3 has a specific effect on the nuclear export of viral proteins, in particular the Raf/MEK/ERK/RSK pathway inhibitors CI-1040 and BI-D1870 have a specific effect on the nuclear export of viral proteins without introducing resistant viral variants. Further, (C, D) shows a comparison of CI-1040, BI-D1870, oseltamivir and balosavir at increasing concentrations of administration.

Figure 7 shows the broad spectrum antiviral effect of RSK inhibition (a-P) and a comparison of the NP region (Q) of different influenza viruses and the crystal structures of influenza viruses a and b (r).

FIG. 8 shows RSK1 nuclear localization during WSN/H1N1 infection. (A) A549 cells were infected or mock-infected with WSN/H1N1(MOI 5). After the indicated time points, the cells were fixed and analyzed by epifluorescence microscopy for cellular localization of vRNP (NP-Alexa-488) and RSK1 (Alexa-561). Nuclei were stained with Dapi. The dashed squares represent the enlargement area. An epifluorescence micrograph of a representative single focal plane is shown. The results of one of three independent experiments are shown. The scale bar represents 50 μm. (B, C) cellular localization of NP and RSK1 of (A) was quantified. 10 epifluorescence micrographs of each sample were analyzed for NP and RSK1 localization using the "Intensity Ratio Nuclear Cytoplasm Tool" of ImageJ. Results are expressed as mean ± SD of three independent experiments. Statistical significance analysis was performed separately for each time point using the Welch-corrected unpaired t-test (ns p > 0.05;. p < 0.01).

FIG. 9 shows no change in RSK2 cell localization during WSN/H1N1 infection. (A) A549 cells were infected or mock-infected with WSN/H1N1(MOI 5). After the indicated time points, the cells were fixed and analyzed by epifluorescence microscopy for cellular localization of vRNP (NP-Alexa-488) and RSK2 (Alexa-561). Nuclei were stained with Dapi. The dashed squares represent the enlargement area. An epifluorescence micrograph of a representative single focal plane is shown. The results of one of three independent experiments are shown. The scale bar represents 50 μm. (B, C) cellular localization of NP and RSK2 of (A) was quantified. 10 epifluorescence micrographs of each sample were analyzed for NP and RSK2 localization using the "Intensity Ratio Nuclear Cytoplasm Tool" of ImageJ. Results are expressed as mean ± SD of three independent experiments. Statistical significance analysis was performed separately for each time point using the Welch-corrected unpaired t-test (ns p > 0.05;. p < 0.05).

FIG. 10 shows no change in ERK1/2 cell localization during WSN/H1N1 infection. (A) A549 cells were infected or mock-infected with WSN/H1N1(MOI 5). After the indicated time points, the cells were fixed and analyzed by epifluorescence microscopy for cellular localization of vRNP (NP-Alexa-488) and ERK1/2 (Alexa-561). Nuclei were stained with Dapi. The dashed squares represent the enlargement area. An epifluorescence micrograph of a representative single focal plane is shown. The results of one of three independent experiments are shown. The scale bar represents 50 μm. (B, C) cellular localization of NP and ERK1/2 of (A) was quantified. 10 epifluorescence micrographs of each sample were analyzed for NP and ERK1/2 localization using the "Intensity Ratio Nuclear Cytoplasm Tool" of ImageJ. Results are expressed as mean ± SD of three independent experiments. Statistical significance analysis was performed separately for each time point using the Welch-corrected unpaired t-test (ns p > 0.05).

FIG. 11 shows nuclear localization of RSK2 and ERK1/2 caused by TPA stimulation. (A, C) A549 cells were stimulated with TPA (200 nM). The solvent DMSO was used as a negative control. After 1 hour, the cells were fixed and analyzed by epifluorescence microscopy for cell localization of RSK2 or ERK1/2 (Alexa-561). Nuclei were stained with Dapi. The dashed squares represent the enlargement area. An epifluorescence micrograph of a representative single focal plane is shown. The results of one of three independent experiments are shown. The scale bar represents 50 μm. (B, D) quantitated the cellular localization of RSK2 or ERK1/2 of (A, C). The 15 epifluorescence micrographs of each sample were analyzed for location according to RSK2 using the "Intensity Ratio Nuclear Cytoplasm Tool" of ImageJ. Results are expressed as mean ± SD of three independent experiments. Statistical significance analysis was performed separately for each time point using Welch-corrected unpaired t-test (p < 0.01).

FIG. 12 shows that chronic treatment with either MEK or RSK inhibitors CI-1040 or BI-D1870 does not introduce resistance in WSN/H1N 1. (A) A549 cells were infected with WSN/H1N1(MOI 0.01) and treated with MEK inhibitor CI-1040, RSK-inhibitor BI-D1870, viral NA-inhibitor oseltamivir acid, or viral cap-dependent endonuclease inhibitor of the polymerase subunit PA Baloxavir Marboxil. Progeny virus titers were determined by standard plaque titration 24 hours after infection. In the next few rounds, fresh a549 cells were infected with collected supernatant (MOI0.01) and treated with different inhibitors at increasing concentrations. The concentration remains constant as marked by the arrow. The solvent DMSO (1%) served as a negative control. Relative viral titers are shown. The titer of the DMSO control was arbitrarily set at 100% (marked with dashed line). Data represent mean ± SD of two independent experiments. Each experiment was performed in triplicate. Statistical significance (. times.p < 0.001) was calculated by two-way analysis of variance (two-way ANOVA), followed by Bonferroni post-test. (B) Summary of inhibitor concentrations used for each round. The concentration remained constant at round 5 (CI-1040, BI-D1870) or round 6 (Oseltamivir, Balosavir).

Examples

Example 1: Raf/MEK/ERK pathway dependent phosphorylation of specific motifs within nucleoproteins

Under physiological conditions, the Raf/MEK/ERK kinase cascade transmits extracellular signals within cells to promote cellular processes such as proliferation and differentiation. This signal is transmitted by sequential phosphorylation of protein kinases (Yoon, 2006). To analyze putative phosphorylation sites within the viral NP protein, HEK293T cells were infected with recombinant WSN/H1N1 virus expressing Strep-tagged PB2 protein (MOI5) and treated with DMSO (1%) or CI-1040(10 μ M) 3H after infection. vRNP was purified from total protein lysates 7h after infection. The phosphorylation pattern of the DMSO control and the CI-1040 samples were analyzed by mass spectrometry. It was found that the phosphorylation status of both serine residues decreased after inhibition of the pathway with the MEK inhibitor CI-1040. The crystal structure of vRNP binding to RNA indicates that the two serine residues 269 and 392 are in close proximity to each other near the RNA binding groove of Nuclear Export Signal (NES) and NP. There is a vRNA loop (vRNA represented by spheres) around both amino acids. Furthermore, S269 is located within NES2 of nucleoprotein and S392 is located near NES2 and NES3 of nucleoprotein (fig. 3A). The loop points to the interior of the helical vRNP complex (fig. 3). Frozen electronic reconstitution of the helical portion of the ribonucleoprotein of influenza virus a/Wilson-smith (wsn)/1933(H1N1) shown in figure 3C was obtained from Protein Database (PDB) ID 4BBL (aranz et al, 2012). NP protein (LILRGS)²⁶⁹V，AIRTRS³⁹²G) The recognition region within shows no similarity to the consensus target sequence of serine/threonine kinase ERK (Pro-Xaa-Ser/Thr-Pro), as shown in fig. 3B, which provides a comparison of ERK (Gonzalez et al, 1991) and RSK (Romeo et al, 2012) consensus sequences with the recognized phosphorylation motifs. Thus, recognized serine residues are unlikely to be encoded by kinase ERK is directly phosphorylated. The consensus sequence of the downstream kinase 90kDa Ribosomal S6Kinase (RSK) (Arg/Lys-Xaa _ Arg-Xaa-Xaa-Ser/Thr; Arg-Arg-Xaa-Ser/Thr) shows a higher homology to the identified NP region (Romeo, 2012). WSN mutants with non-phosphorylated amino acids (aa) were generated at positions 269 and 392 (S269A, S392A, S269A/S392A) to investigate the importance of these residues in the viral life cycle. It should be considered that phosphomimetic mutants could not be rescued, indicating that the permanent negative charges at these positions were not tolerated by the virus. As can be seen from fig. 3D, the RNA binding site of the M1 protein shows a comparable shape to the recognized vRNP loop. The positively charged histidine (H110) that can interact with phosphorylated serine residues is shown in the bottom line of fig. 3E. The crystal structure of the N-terminal domain of the M1 protein of influenza A/Puerto Rico/8/1934(H1N1) was obtained from the Protein Database (PDB) ID 1EA3(Arzt et al, 2001). Structural comparison of the vRNA loop with the RNA binding site of the M1 protein shows that it has a shape comparable to a positively charged histidine. We hypothesize that this histidine may interact with the negative charge of the phosphorylated serine residue. WSN mutants were generated that mimic neutral (H110A) and negative (H110D) charges, and the results are shown in fig. 3F and 3G, which give the growth kinetics of constitutive non-phosphorylated WSN-NP (S269A, S392A, S269A/S392A) and WSN-M1(H110D, H110A) mutants. Here, a549 cells were infected with different WSN virus mutants or wild type viruses (MOI 0.01). Progeny virus titers were determined by standard plaque assays at 8h, 24h, and 32h post infection. Data represent the average of three independent experiments. Each experiment was performed in triplicate. Statistical significance was calculated by two-way analysis of variance followed by a bonofurany post-hoc test.

Example 2: RSK1 as a linker between the Raf/MEK/ERK path and the vrNP output

To elucidate whether RSK is a linker between Raf/MEK/ERK signaling pathway activation and nuclear export of newly synthesized vRNP, its activation in the viral life cycle was analyzed. This activation could be blocked by incubation with the MEK inhibitor CI-1040, suggesting that virus-induced activation of RSK is mediated by the Raf/MEK/ERK pathway. Specifically, A549 cells were infected with WSN/H1N1(MOI 5). At 7h and 9h post infection, cells were lysed and cell lysates were used for immunoblot (western blot) analysis of phosphorylation states of ERK1/2, RSK1 and GSK-3 β. Viral replication was determined by protein expression of PB1, NP, and M1. Tubulin was detected as an internal reference. The results of one of the two independent experiments are shown in fig. 4A. In the late stages of infection, not only ERK but also RSK and its downstream target GSK-3 β are phosphorylated and activated (fig. 4A). In further experiments, cells were treated with DMSO (1%) or BI-D1870 at the indicated concentrations 3h post infection. 7h after infection, cells were lysed. Cell lysates were used for immunoblot analysis of phosphorylation states of ERK1/2 and GSK-3 β. Viral replication was determined by protein expression of PB1, NP, and M1. Tubulin was detected as an internal reference. The results of one of the three independent experiments are shown in fig. 4B.

In further experiments, 3h post-infection, cells were treated with DMSO (1%), CI-1040 (10. mu.M) or BI-D1870 (15. mu.M) and the results are shown in FIGS. 4C, 4D and 4E. 9h post infection, cells were fixed and analyzed by epifluorescence microscopy for localization of vRNP (NP-Alexa488) and (C) PA (Alexa-561), (D) M1(Alexa-561) or (E) NEP (Alexa-561). Nuclei were stained with Dapi. Epifluorescence micrographs of a single focal plane are representative images of (C, D) three or (E) two independent experiments, with a scale bar representing 20 μ M.

In further experiments to rule out off-target effects of RSK inhibitors, RSK1 and RSK2 were targeted by siRNA-mediated knockdown in a549 cells and analyzed for effects on viral life cycle, as shown in fig. 4F. Specifically, a549 cells were transfected with siRNA against RSK1 or RSK2 at the indicated concentrations. Untransfected and control transfected cells served as negative controls. Total protein amounts of RSK1, RSK2, and ERK1/2 were determined by immunoblot analysis 48 hours after transfection. Tubulin was detected as an internal reference. As shown in FIG. 4G, A549 cells on coverslips were transfected with siRNA at a concentration of 100 nM. 48H after transfection, cells were infected with WSN/H1N1(MOI 5). 9h post infection, cells were fixed and analyzed for the localization of vRNP (NP-Alexa488) and M1(Alexa-561) by epifluorescence microscopy. Nuclei were stained with Dapi. Epifluorescence micrographs of a single focal plane are representative images of three independent experiments using a scale bar representing 20 μm. The data indicate that only RSK1 knockdown, but not RSK2 knockdown, resulted in retention of vRNP in the nucleus of infected cells.

In addition, as shown in fig. 4H, RSK1 or RSK2 knockouts were introduced into a549 cells using siRNA at a concentration of 100 nM. 48H after transfection, cells were infected with WSN/H1N1(MOI 0.01). Progeny virus titers were determined by standard plaque assays 24h post-infection. The control siRNA titers were set at 100%. In addition, the absolute viral titer is expressed as PFU/ml. Mean ± SD of three independent experiments are shown. Each experiment was performed in triplicate. Statistical significance was analyzed by paired two-tailed t-test (p.ltoreq.0.05;. p.ltoreq.0.01, p.ltoreq.0.001). Consistent with the effect of the knockdown on nuclear export, it was found that the RSK1 knockdown had an antiviral effect in the multiplex replication assay, as evidenced by a reduction in viral titer. However, the RSK2 knockout appears to have a slight proviral effect. In 2009 Kakugawa et al have described a supporting role for the RSK2 knockout on viral replication. This suggests that the two RSK subtypes RSK1 and RSK2 have different roles in the influenza virus life cycle.

In further experiments, A549 cells were infected with WSN/H1N1(MOI 5). 3h after infection, cells were treated with DMSO (1%) or CI-1040 (10. mu.M). Untreated cells served as negative controls. At 7h and 9h post infection, cells were lysed and cell lysates used for immunoblot analysis of the phosphorylation status of ERK1/2 and RSK 1. Viral replication was determined by protein expression of PB1, NP, and M1. Tubulin was detected as an internal reference. The results of one of the three independent experiments are shown in fig. 4I. Furthermore, it was found that ERK activation increased after inhibition of RSK, which could be explained by inhibition of the negative feedback loop under normal conditions would prevent over-activation of the pathway (fig. 4I).

In another experiment, A549 cells were infected with WSN/H1N1(MOI 0.01), as shown in FIG. 4J. Following infection, cells were treated with BI-D1870 (10. mu.M), CI-1040 (10. mu.M), a combination of two inhibitors (10. mu.M each), or solvent control DMSO (0.2%). Progeny virus titers were determined by standard plaque methods 24h post-infection. Data represent the average of four independent experiments. Each experiment was performed twice. Statistical significance (ns p > 0.05;. p ≦ 0.01;. p ≦ 0.001) was assessed by two-way analysis of variance (two-way ANOVA), followed by Bonferroni post-test. The results showed no significant difference in progeny viral titers between CI-1040 treatment and BI-D1870 and CI-1040 combination treatment, further indicating that MEK and RSK are sequentially activated in the same pathway (i.e., the virus-induced Raf/MEK/ERK pathway) (FIG. 4J).

After viral infection, RSK inhibition by specific inhibitor BI-D1870 results in a concentration-dependent decrease in GSK-3 β phosphorylation, confirming its inhibitory effect on RSK activation during the viral life cycle. An increase in inhibitor BI-D1870 concentration negatively affects vRNP nuclear output production. To analyze the antiviral effect of RSK inhibitors, we used BI-D1870 and SL0101-1 and measured the progeny virus titer 24 hours after treatment at increasing concentrations from 1.56. mu.M to 100. mu.M (FIG. 4K, N). Both inhibitors reduced viral titers, but BI-D1870(EC50 ═ 2.808 μ M) showed higher efficacy against influenza infection than SL0101-1(EC50 ═ 10.54 μ M) (fig. 4L, O). Specifically, A549 cells were infected with WSN/H1N1(MOI 0.01). After infection, cells were treated with BI-D1870(I), SL0101-1(L), or DMSO (0.1%) at the concentrations described. Progeny virus titers were analyzed by standard plaque assay 24h post infection. The titer of DMSO-treated cells was set as 100%. Data represent the average of three independent experiments. Each experiment was performed in triplicate. Statistical significance (. p. < 0.001) was assessed by two-way analysis of variance (two-way ANOVA), followed by Dunnett's multiple comparison test. The results are shown in FIGS. 4K and 4N). Furthermore, in FIGS. 4L and 4O, the progeny viral titers from (I, L) were used to calculate EC₅₀The value is obtained. Finally, in FIGS. 4M and 4P, A549 cells were treated at BI-D1870 at the concentrations (L) described. Cell viability and cell membrane integrity were measured 24h post infection with LDH cytotoxicity assay. Use of the data of union (E) for the calculation of CC₅₀The value is obtained.

Chromatin fractionation analysis following Strep-PB2-WSN virus infection and RSK inhibition was performed to reveal the effect on vRNP-M1 interaction. A549 cells were infected with Strep-PB2-WSN/H1N1(MOI 5). 2.5h post infection, cells were incubated with DMSO (1%) or BI-D1870 (10. mu.M). 7h after infection, cells were isolated and vRNP-complexes were purified from the isolated lysates by PB 2-Strep-Tag. The protein amounts of Strep-PB2, PB1, PA, NP, and M1 were verified by immunoblot analysis. The results of one of three independent experiments are shown. BI-D1870 treatment produced results comparable to the CI-1040 inhibitor, as shown in FIG. 5A. In fig. 5B, the total protein amount of the cell lysate isolated in fig. 5A was analyzed by immunoblotting. In the RSK-inhibited sample, a reduced amount of M1 was co-purified with Strep-PB2-vRNP, with a higher amount of protein in the ch500 dense chromatin fraction (FIG. 5B). In addition, the protein amount of the total cell lysate in fig. 5A was analyzed by immunoblotting. The phosphorylation status of ERK was analyzed by using an ERK-specific antibody. The phosphorylation status of GSK-3 β was analyzed using phospho-GSK and GSK specific antibodies. Fibricin (Lamin A/C) was detected as an internal reference. The phospho-ERK blot in fig. 5C shows that ERK1/2 phosphorylation status is induced due to the suppressed negative feedback loop (fig. 5C).

Example 3: blocking specific export of vRNP by MEK inhibitors and RSK inhibitors

Leptomycin B broadly blocks the active nuclear export pathway by alkylating and inhibiting the major cellular export factor Crm 1. This process is irreversible, although the inhibited cells die. As a control for cellular Crm1 export pathways, we used the Ran binding protein RanBP 1. The 23-kDa RanBP1 is small enough to diffuse through the nuclear pore in the nucleus. The results indicate that high nuclear concentrations of RanBP1 are toxic to cells. Therefore, it must be permanently exported back into the cytoplasm through the Crm1 pathway. Leptomycin B causes nuclear accumulation of RanBP1 and can be used as a control for general blockade of the Crm1 export pathway (Plafker, 2000). We used MEK inhibitors CI-1040 and ATR-002 and RSK inhibitors BI-D1870 and SL0101-1 to examine their effects on the Crm1 pathway. Cells were infected with influenza A/WSN (H1N1) and treated with inhibitors. Immunofluorescent staining was performed 9h after infection. Specifically, A549 cells were infected with WSN/H1N1(MOI 5). 3h post infection, cells were treated with MeOH (0.1%), leptin B (LMB) (5nM), DMSO (1%), CI-1040 (10. mu.M), or BI-D1870 (15. mu.M). At 9h post-infection, cells were fixed and analyzed by epifluorescence microscopy for cellular localization of vRNP (NP-Alexa488) and RanBP1(Alexa561), as shown in fig. 6A, where nuclei were stained with Dapi, dashed squares representing magnified regions, and scale bars representing 20 μ M. Quantitative analysis showed nuclear retention of viral NP protein for all inhibitors tested. Approximately 1000 cells of each of the substances in FIG. 6A were analyzed using ImageJ's "Intensity Ratio Nuclear Cytoplasm Tool" (Intensity Ratio Nuclear Cytoplasm Tool) and scored for protein localization. The results are depicted in fig. 6B as mean ± SD of three independent experiments. As shown in fig. 6B, the highest retention of both leptomycin B and the two MEK inhibitors was found. As can be seen from fig. 6B, only the leptomycin B treated sample detected nuclear retention of RanBP 1. This indicates that inhibition of the Raf/MEK/ERK/RSK pathway has no general effect on the Crm1 export pathway, but is specific for vRNP export.

To test the development of resistance, multiple passage experiments were performed. As shown in FIGS. 6C and 6D, A549 cells were infected with WSN/H1N1(MOI 0.01) and treated with CI-1040, BI-D1870, oseltamivir or baroxavir at increasing concentrations as shown in FIG. 6D. At 24h post-infection, supernatants were collected and progeny virus titers were determined by standard plaque method. In the next few rounds, fresh a549 cells were infected with the supernatant (MOI0.01) and incubated with increasing amounts of the substance. Data represent mean ± SD of two independent experiments. The titer of the DMSO control was set at 100%. Each experiment was performed in triplicate. Statistical significance was analyzed by comparing each material to DMSO by two-way analysis of variance (two-way ANOVA), followed by bonveroni post-test (Bonferroni post-test) (. x.p < 0.001). As shown in FIG. 6C, the viral titer of CI-1040 and BI-D1870 treated infected cells decreased with increasing inhibitor concentration at passage and the titer remained at a lower level at subsequent passages, indicating a high barrier to resistance development. This is in sharp contrast to oseltamivir, where the virus titer increases dramatically from passage 3 and returns to control levels from passage 5, indicating complete resistance of the virus population. Although balosavir did not show similar behavior to oseltamivir, it must be noted here that the titer also increased with increasing concentration up to passage 6, contrary to the trend of CI-1040 or BI-D1870, which indicates a slightly reduced sensitivity to the drug. In summary, CI-1040 and BI-D1870 present a high barrier to the development of resistance, in sharp contrast to oseltamivir, which can rapidly induce resistant viral variants

Furthermore, in FIGS. 6E and 6F, A549 cells were infected with WSN/H1N1(MOI 5). 3h after infection, cells were treated with DMSO (1%), ATR-002 (150. mu.M) or SL0101-1 (100. mu.M). 9h post infection, cells were fixed and analyzed for cellular localization of vRNP (NP-Alexa488) and RanBP1(Alexa561) by epifluorescence microscopy. Nuclei were stained with Dapi. The dashed squares represent the enlargement area. Approximately 1000 cells of each of the substances in FIG. 6A were analyzed using ImageJ's "Intensity Ratio Nuclear Cytoplasm Tool" (Intensity Ratio Nuclear Cytoplasm Tool) and scored for protein localization. Results are described as mean ± SD of three independent experiments. Scale bar: 20 μ M. Quantitative analysis showed nuclear retention of viral NP protein of ATR-002(MEK inhibitor PD-0184264) and SL 0101. ATR-002 and SL0101 did not detect a nuclear retention of RanBP 1. This suggests a specific inhibition of nuclear export of vRNP, rather than the general Crm1 mediated export, which can be reproduced with other inhibitors pf MEK and RSK, and is not limited to inhibitors CI-1040 or BI-D1870.

Example 4: broad spectrum anti-influenza activity of BI-D1870

Since the Raf/MEK/ERK/RSK pathway is critical for influenza virus replication, we used different influenza a subtypes, including porcine H1N1 pandemic virus, seasonal H3N2 virus, different highly pathogenic avian influenza viruses and influenza B virus to determine broad-spectrum antiviral activity (fig. 7). Specifically, A549 cells (FIGS. 7A-O) or MDCKII cells (FIG. 7P) were infected with human IAV (A-H) (WSN/H1N1, PR8M/H1N1, pdm09Hamburg/H1N1, Panama/H3N2), avian IAV (I-L) (KAN-1/H5N1, Anhui/H7N9), IAV/SC35M/H7N7(M-N), or IBV (O-P) (B/Lee) (MOI5 for immunofluorescence analysis (A, C, E, G, I, K, M, O), MOI0.01 for multiple replication cycle analysis (B, D, F, H, J, L, N, P)). 3h after infection, cells were treated with BI-D1870 (15. mu.M) or DMSO (0.1%). Cells were fixed 9h (a, C, E, G, I, K, M) or 12h (o) post infection and analyzed for vRNP (NP-Alexa488) localization by epifluorescence microscopy. Nuclei were stained with Dapi. A fluorescence microscope photograph of a single focal plane is shown (B, D, F, H, J, L, N, P). Cells were treated with BI-D1870 (15. mu.M) or DMSO (0.1%) immediately after infection. Progeny virus titers were analyzed by standard plaque assay 24h post infection. Data represent the average of three independent experiments. The titer of the DMSO control was set at 100%. In addition, the absolute viral titer is expressed as PFU/ml. Mean + SD of three independent experiments are shown. Each experiment was performed in triplicate. Statistical significance was analyzed by paired two-tailed t-test (p.ltoreq.0.05;. p.ltoreq.0.01, p.ltoreq.0.001). Scale bar: 20 μm. In fig. 7Q, a sequence comparison of the NP regions containing S269 and S392 for different viruses is shown, indicating that the phosphorylation sites in NP are conserved in all of these viruses. Sequences were obtained from influenza study databases and aligned using Jalview. In FIG. 7R, the crystal structures of the influenza A/WSN (H1N1) and influenza B/Lee NP regions reveal similar structures. The crystal structure was obtained from the protein database (Influenza virus A/Wilson-Smith/1933(H1N1) ID:4 BBL; Arranz et al, 2012; Influenza virus B/HongKong/CUHK-24964/2004ID:3TJ 0; Ng et al, 2012). As can be seen in figure 7, all viruses tested showed nuclear retention of newly synthesized vRNP with a reduction in progeny virus titer of about 80-90%. These results demonstrate the dependence of influenza viruses on the Raf/MEK/ERK/RSK pathway.

Example 5: specific nuclear localization of RSK1 following influenza A infection

Previous experiments revealed different contributions of the homologous isomers RSK1 and RSK2 to the virus life cycle. RSK2 has an antiviral effect, whereas RSK1 apparently appears to play a virus supporting role. Once RSK1 is activated at the plasma membrane and cytosol, it translocates into the nucleus. If RSK1 is a kinase that phosphorylates NP, its cellular distribution should be altered during the viral life cycle, depending on the activation of the virus-induced Raf/MEK/ERK pathway. To address this hypothesis, A549 cells were mock infected or infected 3H, 6H, and 9H post-infection with WSN/H1N1 virus with an MOI of 5. The localization of NP and RSK1 was analyzed by immunofluorescence staining. Specifically, as shown in FIG. 8A, A549 cells were infected or mock-infected with WSN/H1N1(MOI 5). After the indicated time points, the cells were fixed and analyzed by epifluorescence microscopy for cellular localization of vRNP (NP-Alexa-488) and RSK1 (Alexa-561). Nuclei were stained with Dapi. The dashed squares represent the enlargement area. An epifluorescence micrograph of a representative single focal plane is shown. The results of one of three independent experiments are shown. The scale bar represents 50 μm. FIGS. 8B and C quantify cellular localization of NP and RSK1 in FIG. 8A. 10 epifluorescence micrographs of each sample were analyzed using the "intensity to nuclear cytoplasm tool" of ImageJ, based on NP and RSK1 localization. Results are expressed as mean ± SD of three independent experiments. Statistical significance analysis was performed separately for each time point by unpaired t-test and Welch correction (ns p > 0.05;. p < 0.01). As expected at later time points of infection, nuclear localization of RSK1 increased as shown in fig. 8A. Quantification of three independent experiments showed a significant change in RSK1 nuclear concentration from 35.58% ± 0.59% of mock infection to 57.73% ± 1.47% of viral infection after 9 hours (fig. 8B), especially when NP output occurred (fig. 8A, C). This indicates that the virus induced nuclear import of RSK 1.

Example 6: nonspecific nuclear localization of RSK2 following influenza A infection

After confirming that RSK1 enters the nucleus during the virus life cycle, the problem to be solved is whether the virus-induced activation of the Raf/MEK/ERK pathway leads to the nuclear localization of the antiviral RSK 2. In general, activation of the Raf/MEK/ERK pathway results in translocation of RSK1 and RSK2 into the nucleus. Thus, the same experiment as described in example 5 was performed, but the localization of RSK2 was analyzed. Specifically, as shown in FIG. 9A, A549 cells were infected or mock-infected with WSN/H1N1(MOI 5). After the indicated time points, the cells were fixed and analyzed by epifluorescence microscopy for cellular localization of vRNP (NP-Alexa-488) and RSK2 (Alexa-561). Nuclei were stained with Dapi. The dashed squares represent the enlargement area. An epifluorescence micrograph of a representative single focal plane is shown. The results of one of three independent experiments are shown. The scale bar represents 50 μm. FIG. 9B, C quantifies the cellular localization of NP and RSK2 in FIG. 9A. 10 epifluorescence micrographs of each sample were analyzed using the "intensity to nuclear cytoplasm tool" of ImageJ, based on NP and RSK2 localization. Results are expressed as mean ± SD of three independent experiments. Statistical significance analysis was performed separately for each time point by unpaired t-test and Welch correction (ns p > 0.05;. p ≦ 0.05).

Compared to RSK1, no change in RSK2 subcellular distribution was found, independent of the time point of the viral infection phase (fig. 9A, B). The results show that the virus specifically induces translocation of virus-supporting RSK1 without affecting the cellular distribution of antiviral RSK 2.

Example 7: no effect on ERK1/2 localization after influenza A infection

After finding a specific translocation of RSK1, the question did arise whether the RSK upstream kinase ERK1/2 could possibly enter the nucleus due to viral pathway induction. Thus, the same experiment as in examples 5 and 6 was repeated using ERK. Specifically, as shown in FIG. 10A, A549 cells were infected or mock-infected with WSN/H1N1(MOI 5). After the indicated time points, the cells were fixed and analyzed by epifluorescence microscopy for cellular localization of vRNP (NP-Alexa-488) and ERK1/2 (Alexa-561). Nuclei were stained with Dapi. The dashed squares represent the enlargement area. An epifluorescence micrograph of a representative single focal plane is shown. The results of one of three independent experiments are shown. The scale bar represents 50 μm. FIG. 10B, C quantifies the cellular localization of NP and ERK1/2 in FIG. 10A. 10 epifluorescence micrographs of each sample were analyzed using the "intensity to nuclear cytoplasm tool" of ImageJ based on NP and ERK1/2 localization. Results are expressed as mean ± SD of three independent experiments. Statistical significance analysis was performed separately for each time point by unpaired t-test and Welch correction (ns p > 0.05). Surprisingly, no effect on ERK1/2 localization was observed after viral infection, independent of the time point of analysis (fig. 10A, B).

Example 8: stimulation with TPA leads to nuclear localization of RSK2 and ERK1/2

To exclude non-specific staining of RSK2 and ERK1/2, the Raf/MEK/ERK pathway was stimulated with TPA. It is known that incubation with TPA leads to activation of the Raf/MEK/ERK pathway, leading to nuclear localization of ERK1/2 and RSK within a few minutes after stimulation. As shown in fig. 11A, C, a549 cells were stimulated with TPA (200 nM). The solvent DMSO served as a negative control. After 1 hour, the cells were fixed and analyzed by epifluorescence microscopy for cell localization of RSK2 or ERK1/2 (Alexa-561). Nuclei were stained with Dapi. The dashed squares represent the enlargement area. An epifluorescence micrograph of a representative single focal plane is shown. The results of one of three independent experiments are shown. The scale bar represents 50 μm. As shown in FIGS. 11B and D, the cellular localization of RSK2 or ERK1/2 in 15 fluorescence micrographs of each sample was quantified using the "intensity ratio nuclear cytoplasm tool" of ImageJ based on the localization of RSK 2. Results are expressed as mean ± SD of three independent experiments. Statistical significance was analyzed using the Welch-corrected unpaired t-test (. p < 0.01). After 1 hour incubation with 100nM TPA, both kinases were found in the nucleus, confirming that the staining was specific (fig. 11A, C). Quantification showed that the nuclear accumulation of ERK1/2 changed from 27.99% + -1.97% in the unstimulated sample to 48.02% + -2.25% in the TPA-stimulated sample. The effect on RSK2 kinase was less pronounced than for ERK1/2, 29.37% ± 1.15% in the unstimulated samples and 34.89% ± 0.67% in the TPA stimulated samples (fig. 11B, D).

Example 9: long-term treatment with MEK-or RSK-inhibitors CI-1040 or BI-D1870 does not introduce resistance in WSN/H1N1

To compare the ability of antiviral drugs that either directly target the virions (oseltamivir, balomavir) or exert antiviral effects via inhibition of the Raf/MEK/ERK/RSK pathway (CI-1040, BI-D1870) to induce resistant viral variants, a549 cells were infected with WSN/H1N1(MOI 0.01) and treated with MEK inhibitor CI-1040, RSK inhibitor BI-D1870, viral NA-inhibitor oseltamivir acid, or the polymerase subunit PA Baloxavir Marboxil, a viral cap-dependent endonuclease inhibitor. Progeny virus titers were determined by standard plaque methods 24h post-infection. In the next few rounds, fresh a549 cells were infected with collected supernatant (MOI0.01) and treated with different concentrations of inhibitor. The runs in which the concentration remained constant are marked with arrows. The solvent DMSO (1%) served as a negative control. Relative viral titers are shown. The titer of the DMSO control was arbitrarily set at 100% (marked with dashed line). The data shown in figure 12A represent the mean ± SD of two independent experiments. Each experiment was performed in triplicate. Statistical significance (. times.p < 0.001) was calculated by two-way analysis of variance (two-way ANOVA) followed by bonveroni post-test (Bonferroni post-test). Inhibitor concentrations were increased to enhance the inhibitory effect and cause mutagenesis. Increases in the concentrations of CI-1040 and BI-D1870 result in decreased viral titers. Compared to the DMSO control, in the first round, the titer of CI-1040 or BI-D1870 decreased by 64.75% + -0.32% and 35.08% + -3.20%, respectively, when 1 μ M inhibitor was used. With increasing inhibitor concentration, the titer further decreased until round 4. Both inhibitors were used at a concentration of 8 μ M at this time. From round 5 to round 12, CI-1040 and BI-D1870 were used at a concentration of 10. mu.M. Mean titers for CI-1040 treatments were calculated over 5 to 12 rounds, 8.45% ± 3.41% compared to DMSO control. The mean titer of BI-D1870 treatment was calculated to be 12.27% + -6.36% over rounds 5 to 12. At any time point within 12 rounds, no constant increase in viral titer was found, nor was a change in plaque morphology found, indicating that no mutations introduced resistance occurred. Complete resistance was found after 5 rounds of oseltamivir treatment. At this time, the inhibitor was used at a concentration of 16. mu.M. At round 9, the titer began to decrease to 48.30% ± 11.42%. This effect is accompanied by a progressive reduction in plaque size. The mean titer of the baroxavir treatment was calculated to be 8.08% ± 4.10% from round 1 to round 9. The trend of increasing titre started in round 10, with a mean titre of 28.39% ± 1.88%. At round 12, mean titers further increased to 46.94% ± 1.03% (fig. 12A). Figure 12B shows the inhibitor concentration profile used for each round. The concentration remained constant at round 5 (CI-1040, BI-D1870) or round 6 (Oseltamivir, Balosavir).

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Association of functional influenza viral proteins and RNAs with nuclear chromatin and sub-chromatin structure[Artikel]/Verf.Takizawa N.,Watanabe,K.,Nouno,K.,Kobayashi,N.,Nagata,K.,-2006.-8.-S.823-833.

Baloxavir Marboxil for uncomplicated influenza in adults and adolescents[Journal]/Verf.Hayden F.G.,Sugaya,N.,Hirotsu,N.,Lee,N.,de Jong,M.D.,Hurt,A.C.,Ishida,T.,Sekino,H.,Yamada,K.,Portsmouth,S.,Kawaguchi,K.,Shishido,T.,Arai,M.,Tsuchiya,K.,Uehara,T.,and Watanabe,A.,//N Engl J Med.-2018.-379.-S.913-923.

Characterization of influenza virus variants induced by treatment with the endonclease inhibitor baloxavir marboxil[Journal]/Verf.Omoto S.,Speranzini,V.,Hashimoto,T.,Noshi,T.,Yamaguchi,H.,Kawai,M.,Kawaguchi,K.,Uehara,T.,Shishido,T.,Naito,A.,and Cusack S.//Scientific Reports.-2018.-S.8:9633.

Chromatin docking and exchange activity enhancement of RCC1 by Histones H2A and H2B[Journal]/Verf.Nemergut M.E.,Mizzen,C.A.,Stukenberg,T.,Allis,C.D.,and Macara,I.G.,//Science.-2001.-292(5521).-S.1540-1543.

Comparison of the clinical effectiveness of zanamivir and lanamivir octanoate for children with influenza A(H3N2)and B in the 2011-2012 season[Journal]/Verf.Koseki N.,Kaiho,M.,Kikuta,H.,Oba,K.,Togashi,T.,Ariga,T.,Ishiguro,N.,//Influenza Other Respir Viruses.-2014.-8(2).-S.151-158.

Comparison of the clinical effectivness of Oseltamivir and Zanamivir against influenza virus infection in children[Journal]/Verf.Sugaya N.,Tamura,D.,Yamazaki,M.,Ichikawa,M.,Kawakami,C.,Kawaoka,Y.,and Mitamura,K.,//Clin Infect Dis.-2008.-47(3).-S.339-345.

Disruption of the virus-host cell interaction and cell signaling pathways as an anti-viral approach against influenza virus infection[Journal]/Verf.Ludwig S.,.-2011.-392.-S.837-847.

Facilitated nucleocytoplasmic shuttling of the Ran binding protein RanBP1[Journal]/Verf.Plafker K.,and Macara,I.G.,//Molecular and cellular biology.-2000.-10:Bd.20.-S.3510-3521.

Identification of substrate recognition determinants for human ERK1 and ERK2 protein kinases[Journal]/Verf.Gonzalez F.A.,Raden,D.L.,Davis,R.J.,//J Biol Chem.-1991.-33:Bd.266.-S.22159-63.

Incidence of adamantane resistance among influenza A(H3N2)viruses isolated worldwide from 1994 to 2005:a cause of concern[Journal]/Verf.Bright R.A.,Medina,M.J.,Xu,X.,Perez-Oronaz,G.,Wallis,R.T.,Davis,T.M.,Povinelli,L.,Cox,N.J.,and Klimov,A.I..-2005.-S.67338-2.

Influenza virus propagation is impaired by inhibition of the Raf/MEK/ERK signalling cascade.[Journal]/Verf.Pleschka S.,Wolff,T.,Ehrhardt,C.,Hobom,G.,Planz,O.,Rapp,U.R.,and Ludwig,S.,//NatCell Biol..-2001.-3(3).-S.301-305.

Influenza virus ribonucleoprotein complexes gain preferential acces to cellular export machinery through chromatin targeting [Journal]/Verf.Chase G.P.,Rameix-Welti,M.A.,Zvirbliene,A.,Zvirblis,G.,V.,Wolff,T.,Naffakh,N.,and Schwemmle,M.,//PLoSPathogens.-2011.-7(9).-S.e1002187.

Influenza virus-specific RNA and protein syntheses in cells infected with temperature-sensitive mutants defective in the genome segment encoding nonstructural proteins[Journal]/Verf.Wolstenholme A.J.,Barrett,T.,Nichol,S.T.,and Mahy,B.W.J.,//JVirol..-1980.-35(1).-S.1-7.

Influenza-virus-induced signaling cascades:targets for antiviral therapy？[Journal]/Verf.Ludwig S.,Planz,O.,Pleschka,S.,and Wolff,T.,.-2003.-9(2).-S.46-52.

Inhibition of nuclear export of ribonucleoprotein complexes of the influenza virus by leptomycin B[Journal]/Verf.Watanabe K.,Takizawa,N.,Katoh,M.,Hoshida,K.,Kobayashi,N.,and Nagata,K.,//Virus Research.-2001.-77(1).-S.31-42.

Interaction of the influenza virus nucleoprotein with the cellular CRM1-mediated nuclear export pathway[Journal]/Verf.Elton D.,Simpson-Holley,M.,Archer,K.,Medcalf,L.,Hallam,R.,McCauley,J.,and Digard,P.,//J Virol..-2001.-75(1).-S.408-419.

Late activation of the Raf/MEK/ERK pathway is required for translocation of the respiratory syncytial virus F protein to the plasma membrane and efficient viral replication[Journal]/Verf.Preugschas H.F.,Hrincius,E.R.,Mewis,C.,Tran,G.V.Q.,Ludwig,S.,and Ehrhardt,C.,//Cellular microbiology.-2018.-S.e12955.

Lower clinical effectivness of Oseltamivir against influenza B contrasted with influenza A infection in children[Journal]/Verf.Sugaya N.,Mitamura,K.,Yamazaki,M.,Tamura,D.,Ichikawa,M.,Kimura,K.,Kawakami,C.,Kiso,M.,Ito,M.,Hatakeyama,S.,and Kawaoka,Y.,//Clin Infect Dis.-2007.-44(2).-S.197-202.

MEK inhibition impairs influenza B virus propagation without emergence of resistant variants.[Journal]/Verf.Ludwig S.,Wolff,T.,Ehrhardt,C.,Wurzer,W.J.,Reinhardt,J.,Planz,O.,and Pleschka,S.,//FEBS Lett..-2004.-561(1-3).-S.37-43.

Mitogen-activated protein kinase-activated kinase RSK2 plays a role in innate immune responses to influenza virus infection.[Journal]/Verf.Kakugawa S.,Shimojima,M.,Goto,H.,Horimoto,T.,Oshimori,N.,Neumann,G.,Yamamoto,T.,Kawaoka,Y.,//J Virol.-2009.-6:Bd.83.-S.2510-7.

Molecular characterization of the complete genome of human influenza H5N1 virus isolates from Thailand[Journal]/Verf.Puthavathana P.,Auewarakul,P.,Charoenying,P.C.,Sangsiriwut,K.,Pooruk,P.,Boonak,K.,Khanyok,R.,Thawachsupa,P.,Kijphati,R.,and Sawanpanyalert,P.//Journal of General Virology.-2005.-S.423-433.

Molecular mechanisms of influenza virus resistance to neuraminidase inhibitors[Journal]/Verf.Gubareva L.V.//Virus Research.-2004.-S.199-203.

Nuclear export of influenza virus ribonucleoproteins:Identification of an export intermediate at the nuclear periphery [Artikel]/Verf.Ma K.,Roy,A.M.M.,Whittaker,G.R.,.-2001.-282.-S.215-220.

Nuclear transport of influenza virus ribonucleoproteins:The viral matrix protein(M1)promotes export and inhibits import[Journal]/Verf.Martin K.,and Helenius,A.,//Cell.-1991.-67(1).-S.117-130.

Overview of the 3rd isirv-antiviral group conference-advances in clinical management.[Journal]/Verf.Hurt A.C.,Hui,D.S.,Hay,A.,and Hayden,F.G.,//Influenza Other Respir Viruses.-2015.-9(1).-S.20-31.

Regulated production of an influenza virus spliced mRNA mediated by virus-specific products[Journal]/Verf.Smith D.B.,and Inglis,S.C.,.-1985.-4(9).-S.2313-2319.

Regulation and function of the RSK family of protein kinases[Journal]/Verf.Romeo Y.,Zhang,X.,Roux,P.P.,//Biochem J..-2012.-2:Bd.441.-S.553-69.

Role of the influenza virus M1 protein in nuclear export of viral ribonucleoproteins[Journal]/Verf.Bui M.,Wills,E.G.,Helenius,A.,and Whittaker,G.R.,.-2000.-74(4).-S.1781-1786.

Signal Transduction through MAP Kinase cascades[Journal]/Verf.Lewis T.S.,Shapiro,P.S.,and Ahn,N.G.,//Advances in Cancer Research.-1998.-74.-S.49-139.

The clinically approved MEK inhibitor Trametinib efficiently blocks influenza A virus propagation and cytokine expression[Artikel]/Verf.T.,Dudek,S.E.,Schreiber,A.,Ehrhardt,C.,Planz,O.,Ludwig,S.,.-2018.-157.-S.80-92.

The extracellular signal-regulated kinase:Multiple substrates regulate diverse cellular functions[Journal]/Verf.Yoon S.,and Seger,R.,//Growth Factors.-2006.-24(1).-S.21-44.

The mechansim of resistance to favipiravir in influenza [Journal]/Verf.Goldhill D.H.,te Velthuis,A.J.W.,Fletcher,R.A.,Langat,P.,Zambon,M.,Lackenby,A.,and Barclay,W.S.,//PNAS.-2018.-115(45).-S.11613-11618.

The MEK-inhibitor CI-1040 displays a broad anti-influenza virus activity in vitro and provides a prolonged treatment window compared to standard of care in vivo[Journal]/Verf.Haasbach E.,Müller,C.,Ehrhardt,C.,Schreiber,A.,Pleschka,S.,Ludwig,S.,and Planz,O.,//Antiviral Research.-2017.-142.-S.178-184.

The MEK-inhibitor CI-1040 displays a broad anti-influenza virus activity in vitro and provides a prolonged treatment window compared to standard of care in vivo[Journal]/Verf.Haasbach E.,Müller,C.,Ehrhardt,C.,Schreiber,A.,Pleschka,S.,Ludwig,S.,Planz,O.,//Antiviral Research.-2017.-142.-S.178-184.

The nuclear export protein of H5N1 influenza A viruses recruits Matrix 1(M1)protein to the viral ribonucleoprotein to mediate nuclear export.[Journal]/Verf.Brunotte L.,Flies,J.,Bolte,H.,Reuther,P.,Vreede,F.,and Schwemmle,M.,//J Biol Chem..-2014.-289(29).-S.20067-77.

The potential impact of neuraminidase inhibitor resistant influenza[Journal]/Verf.Lackenby A.,Thompson,C.,and Democratis,J.,//Current Opinion in Infectious Diseases.-2008.-21(6).-S.626-638.

Sequence listing

<110> Home therapy Co., Ltd

<120> RSK inhibitors for the treatment of viral diseases

<130> OLP16685LU

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 735

<212> PRT

<213> human

<400> 1

Met Pro Leu Ala Gln Leu Lys Glu Pro Trp Pro Leu Met Glu Leu Val

1 5 10 15

Pro Leu Asp Pro Glu Asn Gly Gln Thr Ser Gly Glu Glu Ala Gly Leu

20 25 30

Gln Pro Ser Lys Asp Glu Gly Val Leu Lys Glu Ile Ser Ile Thr His

35 40 45

His Val Lys Ala Gly Ser Glu Lys Ala Asp Pro Ser His Phe Glu Leu

50 55 60

Leu Lys Val Leu Gly Gln Gly Ser Phe Gly Lys Val Phe Leu Val Arg

65 70 75 80

Lys Val Thr Arg Pro Asp Ser Gly His Leu Tyr Ala Met Lys Val Leu

85 90 95

Lys Lys Ala Thr Leu Lys Val Arg Asp Arg Val Arg Thr Lys Met Glu

100 105 110

Arg Asp Ile Leu Ala Asp Val Asn His Pro Phe Val Val Lys Leu His

115 120 125

Tyr Ala Phe Gln Thr Glu Gly Lys Leu Tyr Leu Ile Leu Asp Phe Leu

130 135 140

Arg Gly Gly Asp Leu Phe Thr Arg Leu Ser Lys Glu Val Met Phe Thr

145 150 155 160

Glu Glu Asp Val Lys Phe Tyr Leu Ala Glu Leu Ala Leu Gly Leu Asp

165 170 175

His Leu His Ser Leu Gly Ile Ile Tyr Arg Asp Leu Lys Pro Glu Asn

180 185 190

Ile Leu Leu Asp Glu Glu Gly His Ile Lys Leu Thr Asp Phe Gly Leu

195 200 205

Ser Lys Glu Ala Ile Asp His Glu Lys Lys Ala Tyr Ser Phe Cys Gly

210 215 220

Thr Val Glu Tyr Met Ala Pro Glu Val Val Asn Arg Gln Gly His Ser

225 230 235 240

His Ser Ala Asp Trp Trp Ser Tyr Gly Val Leu Met Phe Glu Met Leu

245 250 255

Thr Gly Ser Leu Pro Phe Gln Gly Lys Asp Arg Lys Glu Thr Met Thr

260 265 270

Leu Ile Leu Lys Ala Lys Leu Gly Met Pro Gln Phe Leu Ser Thr Glu

275 280 285

Ala Gln Ser Leu Leu Arg Ala Leu Phe Lys Arg Asn Pro Ala Asn Arg

290 295 300

Leu Gly Ser Gly Pro Asp Gly Ala Glu Glu Ile Lys Arg His Val Phe

305 310 315 320

Tyr Ser Thr Ile Asp Trp Asn Lys Leu Tyr Arg Arg Glu Ile Lys Pro

325 330 335

Pro Phe Lys Pro Ala Val Ala Gln Pro Asp Asp Thr Phe Tyr Phe Asp

340 345 350

Thr Glu Phe Thr Ser Arg Thr Pro Lys Asp Ser Pro Gly Ile Pro Pro

355 360 365

Ser Ala Gly Ala His Gln Leu Phe Arg Gly Phe Ser Phe Val Ala Thr

370 375 380

Gly Leu Met Glu Asp Asp Gly Lys Pro Arg Ala Pro Gln Ala Pro Leu

385 390 395 400

His Ser Val Val Gln Gln Leu His Gly Lys Asn Leu Val Phe Ser Asp

405 410 415

Gly Tyr Val Val Lys Glu Thr Ile Gly Val Gly Ser Tyr Ser Glu Cys

420 425 430

Lys Arg Cys Val His Lys Ala Thr Asn Met Glu Tyr Ala Val Lys Val

435 440 445

Ile Asp Lys Ser Lys Arg Asp Pro Ser Glu Glu Ile Glu Ile Leu Leu

450 455 460

Arg Tyr Gly Gln His Pro Asn Ile Ile Thr Leu Lys Asp Val Tyr Asp

465 470 475 480

Asp Gly Lys His Val Tyr Leu Val Thr Glu Leu Met Arg Gly Gly Glu

485 490 495

Leu Leu Asp Lys Ile Leu Arg Gln Lys Phe Phe Ser Glu Arg Glu Ala

500 505 510

Ser Phe Val Leu His Thr Ile Gly Lys Thr Val Glu Tyr Leu His Ser

515 520 525

Gln Gly Val Val His Arg Asp Leu Lys Pro Ser Asn Ile Leu Tyr Val

530 535 540

Asp Glu Ser Gly Asn Pro Glu Cys Leu Arg Ile Cys Asp Phe Gly Phe

545 550 555 560

Ala Lys Gln Leu Arg Ala Glu Asn Gly Leu Leu Met Thr Pro Cys Tyr

565 570 575

Thr Ala Asn Phe Val Ala Pro Glu Val Leu Lys Arg Gln Gly Tyr Asp

580 585 590

Glu Gly Cys Asp Ile Trp Ser Leu Gly Ile Leu Leu Tyr Thr Met Leu

595 600 605

Ala Gly Tyr Thr Pro Phe Ala Asn Gly Pro Ser Asp Thr Pro Glu Glu

610 615 620

Ile Leu Thr Arg Ile Gly Ser Gly Lys Phe Thr Leu Ser Gly Gly Asn

625 630 635 640

Trp Asn Thr Val Ser Glu Thr Ala Lys Asp Leu Val Ser Lys Met Leu

645 650 655

His Val Asp Pro His Gln Arg Leu Thr Ala Lys Gln Val Leu Gln His

660 665 670

Pro Trp Val Thr Gln Lys Asp Lys Leu Pro Gln Ser Gln Leu Ser His

675 680 685

Gln Asp Leu Gln Leu Val Lys Gly Ala Met Ala Ala Thr Tyr Ser Ala

690 695 700

Leu Asn Ser Ser Lys Pro Thr Pro Gln Leu Lys Pro Ile Glu Ser Ser

705 710 715 720

Ile Leu Ala Gln Arg Arg Val Arg Lys Leu Pro Ser Thr Thr Leu

725 730 735

<210> 2

<211> 2208

<212> DNA

<213> human

<400> 2

atgccgctcg cccagctcaa ggagccctgg ccgctcatgg agctagtgcc gctggacccg 60

gagaatggac agacctcagg ggaagaagct ggacttcagc cgtccaagga tgagggcgtc 120

ctcaaggaga tctccatcac gcaccacgtc aaggctggct ctgagaaggc tgatccatcc 180

catttcgagc tcctcaaggt tctgggccag ggatcctttg gcaaagtctt cctggtgcgg 240

aaagtcaccc ggcctgacag tgggcacctg tatgctatga aggtgctgaa gaaggcaacg 300

ctgaaagtac gtgaccgcgt ccggaccaag atggagagag acatcctggc tgatgtaaat 360

cacccattcg tggtgaagct gcactatgcc ttccagaccg agggcaagct ctatctcatt 420

ctggacttcc tgcgtggtgg ggacctcttc acccggctct caaaagaggt gatgttcacg 480

gaggaggatg tgaagtttta cctggccgag ctggctctgg gcctggatca cctgcacagc 540

ctgggtatca tttacagaga cctcaagcct gagaacatcc ttctggatga ggagggccac 600

atcaaactca ctgactttgg cctgagcaaa gaggccattg accacgagaa gaaggcctat 660

tctttctgcg ggacagtgga gtacatggcc cctgaggtcg tcaaccgcca gggccactcc 720

catagtgcgg actggtggtc ctatggggtg ttgatgtttg agatgctgac gggctccctg 780

cccttccagg ggaaggaccg gaaggagacc atgacactga ttctgaaggc gaagctaggc 840

atgccccagt ttctgagcac tgaagcccag agcctcttgc gggccctgtt caagcggaat 900

cctgccaacc ggctcggctc cggccctgat ggggcagagg aaatcaagcg gcatgtcttc 960

tactccacca ttgactggaa taagctatac cgtcgtgaga tcaagccacc cttcaagcca 1020

gcagtggctc agcctgatga caccttctac tttgacaccg agttcacgtc ccgcacaccc 1080

aaggattccc caggcatccc ccccagcgct ggggcccatc agctgttccg gggcttcagc 1140

ttcgtggcca ccggcctgat ggaagacgac ggcaagcctc gtgccccgca ggcacccctg 1200

cactcggtgg tacagcaact ccatgggaag aacctggttt ttagtgacgg ctacgtggta 1260

aaggagacaa ttggtgtggg ctcctactct gagtgcaagc gctgtgtcca caaggccacc 1320

aacatggagt atgctgtcaa ggtcattgat aagagcaagc gggatccttc agaagagatt 1380

gagattcttc tgcggtatgg ccagcacccc aacatcatca ctctgaaaga tgtgtatgat 1440

gatggcaaac acgtgtacct ggtgacagag ctgatgcggg gtggggagct gctggacaag 1500

atcctgcggc agaagttctt ctcagagcgg gaggccagct ttgtcctgca caccattggc 1560

aaaactgtgg agtatctgca ctcacagggg gttgtgcaca gggacctgaa gcccagcaac 1620

atcctgtatg tggacgagtc cgggaatccc gagtgcctgc gcatctgtga ctttggtttt 1680

gccaaacagc tgcgggctga gaatgggctc ctcatgacac cttgctacac agccaacttt 1740

gtggcgcctg aggtgctgaa gcgccagggc tacgatgaag gctgcgacat ctggagcctg 1800

ggcattctgc tgtacaccat gctggcagga tatactccat ttgccaacgg tcccagtgac 1860

acaccagagg aaatcctaac ccggatcggc agtgggaagt ttaccctcag tgggggaaat 1920

tggaacacag tttcagagac agccaaggac ctggtgtcca agatgctaca cgtggatccc 1980

caccagcgcc tcacagctaa gcaggttctg cagcatccat gggtcaccca gaaagacaag 2040

cttccccaaa gccagctgtc ccaccaggac ctacagcttg tgaagggagc catggctgcc 2100

acgtactccg cactcaacag ctccaagccc accccccagc tgaagcccat cgagtcatcc 2160

atcctggccc agcggcgagt gaggaagttg ccatccacca ccctgtga 2208

<210> 3

<211> 740

<212> PRT

<213> human

<400> 3

Met Pro Leu Ala Gln Leu Ala Asp Pro Trp Gln Lys Met Ala Val Glu

1 5 10 15

Ser Pro Ser Asp Ser Ala Glu Asn Gly Gln Gln Ile Met Asp Glu Pro

20 25 30

Met Gly Glu Glu Glu Ile Asn Pro Gln Thr Glu Glu Val Ser Ile Lys

35 40 45

Glu Ile Ala Ile Thr His His Val Lys Glu Gly His Glu Lys Ala Asp

50 55 60

Pro Ser Gln Phe Glu Leu Leu Lys Val Leu Gly Gln Gly Ser Phe Gly

65 70 75 80

Lys Val Phe Leu Val Lys Lys Ile Ser Gly Ser Asp Ala Arg Gln Leu

85 90 95

Tyr Ala Met Lys Val Leu Lys Lys Ala Thr Leu Lys Val Arg Asp Arg

100 105 110

Val Arg Thr Lys Met Glu Arg Asp Ile Leu Val Glu Val Asn His Pro

115 120 125

Phe Ile Val Lys Leu His Tyr Ala Phe Gln Thr Glu Gly Lys Leu Tyr

130 135 140

Leu Ile Leu Asp Phe Leu Arg Gly Gly Asp Leu Phe Thr Arg Leu Ser

145 150 155 160

Lys Glu Val Met Phe Thr Glu Glu Asp Val Lys Phe Tyr Leu Ala Glu

165 170 175

Leu Ala Leu Ala Leu Asp His Leu His Ser Leu Gly Ile Ile Tyr Arg

180 185 190

Asp Leu Lys Pro Glu Asn Ile Leu Leu Asp Glu Glu Gly His Ile Lys

195 200 205

Leu Thr Asp Phe Gly Leu Ser Lys Glu Ser Ile Asp His Glu Lys Lys

210 215 220

Ala Tyr Ser Phe Cys Gly Thr Val Glu Tyr Met Ala Pro Glu Val Val

225 230 235 240

Asn Arg Arg Gly His Thr Gln Ser Ala Asp Trp Trp Ser Phe Gly Val

245 250 255

Leu Met Phe Glu Met Leu Thr Gly Thr Leu Pro Phe Gln Gly Lys Asp

260 265 270

Arg Lys Glu Thr Met Thr Met Ile Leu Lys Ala Lys Leu Gly Met Pro

275 280 285

Gln Phe Leu Ser Pro Glu Ala Gln Ser Leu Leu Arg Met Leu Phe Lys

290 295 300

Arg Asn Pro Ala Asn Arg Leu Gly Ala Gly Pro Asp Gly Val Glu Glu

305 310 315 320

Ile Lys Arg His Ser Phe Phe Ser Thr Ile Asp Trp Asn Lys Leu Tyr

325 330 335

Arg Arg Glu Ile His Pro Pro Phe Lys Pro Ala Thr Gly Arg Pro Glu

340 345 350

Asp Thr Phe Tyr Phe Asp Pro Glu Phe Thr Ala Lys Thr Pro Lys Asp

355 360 365

Ser Pro Gly Ile Pro Pro Ser Ala Asn Ala His Gln Leu Phe Arg Gly

370 375 380

Phe Ser Phe Val Ala Ile Thr Ser Asp Asp Glu Ser Gln Ala Met Gln

385 390 395 400

Thr Val Gly Val His Ser Ile Val Gln Gln Leu His Arg Asn Ser Ile

405 410 415

Gln Phe Thr Asp Gly Tyr Glu Val Lys Glu Asp Ile Gly Val Gly Ser

420 425 430

Tyr Ser Val Cys Lys Arg Cys Ile His Lys Ala Thr Asn Met Glu Phe

435 440 445

Ala Val Lys Ile Ile Asp Lys Ser Lys Arg Asp Pro Thr Glu Glu Ile

450 455 460

Glu Ile Leu Leu Arg Tyr Gly Gln His Pro Asn Ile Ile Thr Leu Lys

465 470 475 480

Asp Val Tyr Asp Asp Gly Lys Tyr Val Tyr Val Val Thr Glu Leu Met

485 490 495

Lys Gly Gly Glu Leu Leu Asp Lys Ile Leu Arg Gln Lys Phe Phe Ser

500 505 510

Glu Arg Glu Ala Ser Ala Val Leu Phe Thr Ile Thr Lys Thr Val Glu

515 520 525

Tyr Leu His Ala Gln Gly Val Val His Arg Asp Leu Lys Pro Ser Asn

530 535 540

Ile Leu Tyr Val Asp Glu Ser Gly Asn Pro Glu Ser Ile Arg Ile Cys

545 550 555 560

Asp Phe Gly Phe Ala Lys Gln Leu Arg Ala Glu Asn Gly Leu Leu Met

565 570 575

Thr Pro Cys Tyr Thr Ala Asn Phe Val Ala Pro Glu Val Leu Lys Arg

580 585 590

Gln Gly Tyr Asp Ala Ala Cys Asp Ile Trp Ser Leu Gly Val Leu Leu

595 600 605

Tyr Thr Met Leu Thr Gly Tyr Thr Pro Phe Ala Asn Gly Pro Asp Asp

610 615 620

Thr Pro Glu Glu Ile Leu Ala Arg Ile Gly Ser Gly Lys Phe Ser Leu

625 630 635 640

Ser Gly Gly Tyr Trp Asn Ser Val Ser Asp Thr Ala Lys Asp Leu Val

645 650 655

Ser Lys Met Leu His Val Asp Pro His Gln Arg Leu Thr Ala Ala Leu

660 665 670

Val Leu Arg His Pro Trp Ile Val His Trp Asp Gln Leu Pro Gln Tyr

675 680 685

Gln Leu Asn Arg Gln Asp Ala Pro His Leu Val Lys Gly Ala Met Ala

690 695 700

Ala Thr Tyr Ser Ala Leu Asn Arg Asn Gln Ser Pro Val Leu Glu Pro

705 710 715 720

Val Gly Arg Ser Thr Leu Ala Gln Arg Arg Gly Ile Lys Lys Ile Thr

725 730 735

Ser Thr Ala Leu

740

<210> 4

<211> 2223

<212> DNA

<213> human

<400> 4

atgccgctgg cgcagctggc ggacccgtgg cagaagatgg ctgtggagag cccgtccgac 60

agcgctgaga atggacagca aattatggat gaacctatgg gagaggagga gattaaccca 120

caaactgaag aagtcagtat caaagaaatt gcaatcacac atcatgtaaa ggaaggacat 180

gaaaaggcag atccttccca gtttgaactt ttaaaagtat tagggcaggg atcatttgga 240

aaggttttct tagttaaaaa aatctcaggc tctgatgcta ggcagcttta tgccatgaag 300

gtattgaaga aggccacact gaaagttcga gaccgagttc ggacaaaaat ggaacgtgat 360

atcttggtag aggttaatca tccttttatt gtcaagttgc attatgcttt tcaaactgaa 420

gggaagttgt atcttatttt ggattttctc aggggaggag atttgtttac acgcttatcc 480

aaagaggtga tgttcacaga agaagatgtc aaattctact tggctgaact tgcacttgct 540

ttagaccatc tacatagcct gggaataatt tatagagact taaaaccaga aaatatactt 600

cttgatgaag aaggtcacat caagttaaca gatttcggcc taagtaaaga gtctattgac 660

catgaaaaga aggcatattc tttttgtgga actgtggagt atatggctcc agaagtagtt 720

aatcgtcgag gtcatactca gagtgctgac tggtggtctt ttggtgtgtt aatgtttgaa 780

atgcttactg gtacactccc tttccaagga aaagatcgaa aagaaacaat gactatgatt 840

cttaaagcca aacttggaat gccacagttt ttgagtcctg aagcgcagag tcttttacga 900

atgcttttca agcgaaatcc tgcaaacaga ttaggtgcag gaccagatgg agttgaagaa 960

attaaaagac attcattttt ctcaacgata gactggaata aactgtatag aagagaaatt 1020

catccgccat ttaaacctgc aacgggcagg cctgaagata cattctattt tgatcctgag 1080

tttactgcaa aaactcccaa agattcacct ggcattccac ctagtgctaa tgcacatcag 1140

ctttttcggg ggtttagttt tgttgctatt acctcagatg atgaaagcca agctatgcag 1200

acagttggtg tacattcaat tgttcagcag ttacacagga acagtattca gtttactgat 1260

ggatatgaag taaaagaaga tattggagtt ggctcctact ctgtttgcaa gagatgtata 1320

cataaagcta caaacatgga gtttgcagtg aagattattg ataaaagcaa gagagaccca 1380

acagaagaaa ttgaaattct tcttcgttat ggacagcatc caaacattat cactctaaag 1440

gatgtatatg atgatggaaa gtatgtgtat gtagtaacag aacttatgaa aggaggtgaa 1500

ttgctggata aaattcttag acaaaaattt ttctctgaac gagaggccag tgctgtcctg 1560

ttcactataa ctaaaaccgt tgaatatctt cacgcacaag gggtggttca tagagacttg 1620

aaacctagca acattcttta tgtggatgaa tctggtaatc cggaatctat tcgaatttgt 1680

gattttggct ttgcaaaaca gctgagagcg gaaaatggtc ttctcatgac tccttgttac 1740

actgcaaatt ttgttgcacc agaggtttta aaaagacaag gctatgatgc tgcttgtgat 1800

atatggagtc ttggtgtcct actctataca atgcttaccg gttacactcc atttgcaaat 1860

ggtcctgatg atacaccaga ggaaatattg gcacgaatag gtagcggaaa attctcactc 1920

agtggtggtt actggaattc tgtttcagac acagcaaagg acctggtgtc aaagatgctt 1980

catgtagacc ctcatcagag actgactgct gctcttgtgc tcagacatcc ttggatcgtc 2040

cactgggacc aactgccaca ataccaacta aacagacagg atgcaccaca tctagtaaag 2100

ggtgccatgg cagctacata ttctgctttg aaccgtaatc agtcaccagt tttggaacca 2160

gtaggccgct ctactcttgc tcagcggaga ggtattaaaa aaatcacctc aacagccctg 2220

tga 2223

<210> 5

<211> 733

<212> PRT

<213> human

<400> 5

Met Asp Leu Ser Met Lys Lys Phe Ala Val Arg Arg Phe Phe Ser Val

1 5 10 15

Tyr Leu Arg Arg Lys Ser Arg Ser Lys Ser Ser Ser Leu Ser Arg Leu

20 25 30

Glu Glu Glu Gly Val Val Lys Glu Ile Asp Ile Ser His His Val Lys

35 40 45

Glu Gly Phe Glu Lys Ala Asp Pro Ser Gln Phe Glu Leu Leu Lys Val

50 55 60

Leu Gly Gln Gly Ser Tyr Gly Lys Val Phe Leu Val Arg Lys Val Lys

65 70 75 80

Gly Ser Asp Ala Gly Gln Leu Tyr Ala Met Lys Val Leu Lys Lys Ala

85 90 95

Thr Leu Lys Val Arg Asp Arg Val Arg Ser Lys Met Glu Arg Asp Ile

100 105 110

Leu Ala Glu Val Asn His Pro Phe Ile Val Lys Leu His Tyr Ala Phe

115 120 125

Gln Thr Glu Gly Lys Leu Tyr Leu Ile Leu Asp Phe Leu Arg Gly Gly

130 135 140

Asp Leu Phe Thr Arg Leu Ser Lys Glu Val Met Phe Thr Glu Glu Asp

145 150 155 160

Val Lys Phe Tyr Leu Ala Glu Leu Ala Leu Ala Leu Asp His Leu His

165 170 175

Ser Leu Gly Ile Ile Tyr Arg Asp Leu Lys Pro Glu Asn Ile Leu Leu

180 185 190

Asp Glu Glu Gly His Ile Lys Ile Thr Asp Phe Gly Leu Ser Lys Glu

195 200 205

Ala Ile Asp His Asp Lys Arg Ala Tyr Ser Phe Cys Gly Thr Ile Glu

210 215 220

Tyr Met Ala Pro Glu Val Val Asn Arg Arg Gly His Thr Gln Ser Ala

225 230 235 240

Asp Trp Trp Ser Phe Gly Val Leu Met Phe Glu Met Leu Thr Gly Ser

245 250 255

Leu Pro Phe Gln Gly Lys Asp Arg Lys Glu Thr Met Ala Leu Ile Leu

260 265 270

Lys Ala Lys Leu Gly Met Pro Gln Phe Leu Ser Gly Glu Ala Gln Ser

275 280 285

Leu Leu Arg Ala Leu Phe Lys Arg Asn Pro Cys Asn Arg Leu Gly Ala

290 295 300

Gly Ile Asp Gly Val Glu Glu Ile Lys Arg His Pro Phe Phe Val Thr

305 310 315 320

Ile Asp Trp Asn Thr Leu Tyr Arg Lys Glu Ile Lys Pro Pro Phe Lys

325 330 335

Pro Ala Val Gly Arg Pro Glu Asp Thr Phe His Phe Asp Pro Glu Phe

340 345 350

Thr Ala Arg Thr Pro Thr Asp Ser Pro Gly Val Pro Pro Ser Ala Asn

355 360 365

Ala His His Leu Phe Arg Gly Phe Ser Phe Val Ala Ser Ser Leu Ile

370 375 380

Gln Glu Pro Ser Gln Gln Asp Leu His Lys Val Pro Val His Pro Ile

385 390 395 400

Val Gln Gln Leu His Gly Asn Asn Ile His Phe Thr Asp Gly Tyr Glu

405 410 415

Ile Lys Glu Asp Ile Gly Val Gly Ser Tyr Ser Val Cys Lys Arg Cys

420 425 430

Val His Lys Ala Thr Asp Thr Glu Tyr Ala Val Lys Ile Ile Asp Lys

435 440 445

Ser Lys Arg Asp Pro Ser Glu Glu Ile Glu Ile Leu Leu Arg Tyr Gly

450 455 460

Gln His Pro Asn Ile Ile Thr Leu Lys Asp Val Tyr Asp Asp Gly Lys

465 470 475 480

Phe Val Tyr Leu Val Met Glu Leu Met Arg Gly Gly Glu Leu Leu Asp

485 490 495

Arg Ile Leu Arg Gln Arg Tyr Phe Ser Glu Arg Glu Ala Ser Asp Val

500 505 510

Leu Cys Thr Ile Thr Lys Thr Met Asp Tyr Leu His Ser Gln Gly Val

515 520 525

Val His Arg Asp Leu Lys Pro Ser Asn Ile Leu Tyr Arg Asp Glu Ser

530 535 540

Gly Ser Pro Glu Ser Ile Arg Val Cys Asp Phe Gly Phe Ala Lys Gln

545 550 555 560

Leu Arg Ala Gly Asn Gly Leu Leu Met Thr Pro Cys Tyr Thr Ala Asn

565 570 575

Phe Val Ala Pro Glu Val Leu Lys Arg Gln Gly Tyr Asp Ala Ala Cys

580 585 590

Asp Ile Trp Ser Leu Gly Ile Leu Leu Tyr Thr Met Leu Ala Gly Phe

595 600 605

Thr Pro Phe Ala Asn Gly Pro Asp Asp Thr Pro Glu Glu Ile Leu Ala

610 615 620

Arg Ile Gly Ser Gly Lys Tyr Ala Leu Ser Gly Gly Asn Trp Asp Ser

625 630 635 640

Ile Ser Asp Ala Ala Lys Asp Val Val Ser Lys Met Leu His Val Asp

645 650 655

Pro His Gln Arg Leu Thr Ala Met Gln Val Leu Lys His Pro Trp Val

660 665 670

Val Asn Arg Glu Tyr Leu Ser Pro Asn Gln Leu Ser Arg Gln Asp Val

675 680 685

His Leu Val Lys Gly Ala Met Ala Ala Thr Tyr Phe Ala Leu Asn Arg

690 695 700

Thr Pro Gln Ala Pro Arg Leu Glu Pro Val Leu Ser Ser Asn Leu Ala

705 710 715 720

Gln Arg Arg Gly Met Lys Arg Leu Thr Ser Thr Arg Leu

725 730

<210> 6

<211> 2202

<212> DNA

<213> human

<400> 6

atggacctga gcatgaagaa gttcgccgtg cgcaggttct tctctgtgta cctgcgcagg 60

aagtcgcgct ccaagagctc cagcctgagc cggctcgagg aagaaggtgt cgtgaaggag 120

atagacatca gccatcatgt gaaggagggc tttgagaagg cagatccttc ccagtttgag 180

ctgctgaagg ttttaggaca aggatcctat ggaaaggtgt tcctggtgag gaaggtgaag 240

gggtccgacg ctgggcagct ctacgccatg aaggtcctta agaaagccac cctaaaagtt 300

cgggaccgag tgagatcgaa gatggagaga gacatcttgg cagaagtgaa tcaccccttc 360

attgtgaagc ttcattatgc ctttcagacg gaaggaaagc tctacctgat cctggacttc 420

ctgcggggag gggacctctt cacccggctc tccaaagagg tcatgttcac ggaggaggat 480

gtcaagttct acctggctga gctggccttg gctttagacc atctccacag cctggggatc 540

atctacagag atctgaagcc tgagaacatc ctcctggatg aagaggggca cattaagatc 600

acagatttcg gcctgagtaa ggaggccatt gaccacgaca agagagcgta ctccttctgc 660

gggacgatcg agtacatggc gcccgaggtg gtgaaccggc gaggacacac gcagagtgcc 720

gactggtggt ccttcggcgt gctcatgttt gagatgctca cggggtccct gccgttccag 780

gggaaggaca ggaaggagac catggctctc atcctcaaag ccaagctggg gatgccgcag 840

ttcctcagtg gggaggcaca gagtttgctg cgagctctct tcaaacggaa cccctgcaac 900

cggctgggtg ctggcattga cggagtggag gaaattaagc gccatccctt ctttgtgacc 960

atagactgga acacgctgta ccggaaggag atcaagccac cgttcaaacc agcagtgggc 1020

aggcctgagg acaccttcca ctttgacccc gagttcacag cgcggacgcc cacagactct 1080

cctggcgtcc ccccgagtgc aaacgctcat cacctgttta gaggattcag ctttgtggcc 1140

tcaagcctga tccaggagcc ctcacagcaa gatctgcaca aagtcccagt tcacccaatc 1200

gtgcagcagt tacacgggaa caacatccac ttcaccgatg gctacgagat caaggaggac 1260

atcggggtgg gctcctactc agtgtgcaag cgatgtgtgc ataaagccac agacaccgag 1320

tatgccgtga agatcattga taagagcaag agagacccct cggaagagat tgagatcctc 1380

ctgcggtacg gccagcaccc gaacatcatc accctcaagg atgtctatga tgatggcaag 1440

tttgtgtacc tggtaatgga gctgatgcgt ggtggggagc tcctggaccg catcctccgg 1500

cagagatact tctcggagcg cgaagccagt gacgtcctgt gcaccatcac caagaccatg 1560

gactacctcc attcccaggg ggttgttcat cgagacctga agccgagtaa catcctgtac 1620

agggatgagt cggggagccc agaatccatc cgagtctgcg acttcggctt tgccaagcag 1680

ctgcgcgcgg ggaacgggct gctcatgaca ccctgctaca cggccaattt cgtggccccg 1740

gaggtcctga agcgtcaagg ctatgatgcg gcgtgtgaca tctggagttt ggggatcctg 1800

ttgtacacca tgctggcagg atttacccct tttgcaaatg ggccagacga tacccctgag 1860

gagattctgg cgcggatcgg cagtgggaag tatgcccttt ctgggggaaa ctgggactcg 1920

atatctgacg cagctaaaga cgtcgtgtcc aagatgctcc acgtggaccc tcatcagcgc 1980

ctgacggcga tgcaagtgct caaacacccg tgggtggtca acagagagta cctgtcccca 2040

aaccagctca gccgacagga cgtgcacctg gtgaagggcg cgatggccgc cacctacttt 2100

gctctaaaca gaacacctca ggccccgcgg ctggagcccg tgctgtcgtc caacctggct 2160

cagcgcagag gcatgaagag actcacgtcc acgcggttgt ag 2202

<210> 7

<211> 745

<212> PRT

<213> human

<400> 7

Met Leu Pro Phe Ala Pro Gln Asp Glu Pro Trp Asp Arg Glu Met Glu

1 5 10 15

Val Phe Ser Gly Gly Gly Ala Ser Ser Gly Glu Val Asn Gly Leu Lys

20 25 30

Met Val Asp Glu Pro Met Glu Glu Gly Glu Ala Asp Ser Cys His Asp

35 40 45

Glu Gly Val Val Lys Glu Ile Pro Ile Thr His His Val Lys Glu Gly

50 55 60

Tyr Glu Lys Ala Asp Pro Ala Gln Phe Glu Leu Leu Lys Val Leu Gly

65 70 75 80

Gln Gly Ser Phe Gly Lys Val Phe Leu Val Arg Lys Lys Thr Gly Pro

85 90 95

Asp Ala Gly Gln Leu Tyr Ala Met Lys Val Leu Lys Lys Ala Ser Leu

100 105 110

Lys Val Arg Asp Arg Val Arg Thr Lys Met Glu Arg Asp Ile Leu Val

115 120 125

Glu Val Asn His Pro Phe Ile Val Lys Leu His Tyr Ala Phe Gln Thr

130 135 140

Glu Gly Lys Leu Tyr Leu Ile Leu Asp Phe Leu Arg Gly Gly Asp Val

145 150 155 160

Phe Thr Arg Leu Ser Lys Glu Val Leu Phe Thr Glu Glu Asp Val Lys

165 170 175

Phe Tyr Leu Ala Glu Leu Ala Leu Ala Leu Asp His Leu His Gln Leu

180 185 190

Gly Ile Val Tyr Arg Asp Leu Lys Pro Glu Asn Ile Leu Leu Asp Glu

195 200 205

Ile Gly His Ile Lys Leu Thr Asp Phe Gly Leu Ser Lys Glu Ser Val

210 215 220

Asp Gln Glu Lys Lys Ala Tyr Ser Phe Cys Gly Thr Val Glu Tyr Met

225 230 235 240

Ala Pro Glu Val Val Asn Arg Arg Gly His Ser Gln Ser Ala Asp Trp

245 250 255

Trp Ser Tyr Gly Val Leu Met Phe Glu Met Leu Thr Gly Thr Leu Pro

260 265 270

Phe Gln Gly Lys Asp Arg Asn Glu Thr Met Asn Met Ile Leu Lys Ala

275 280 285

Lys Leu Gly Met Pro Gln Phe Leu Ser Ala Glu Ala Gln Ser Leu Leu

290 295 300

Arg Met Leu Phe Lys Arg Asn Pro Ala Asn Arg Leu Gly Ser Glu Gly

305 310 315 320

Val Glu Glu Ile Lys Arg His Leu Phe Phe Ala Asn Ile Asp Trp Asp

325 330 335

Lys Leu Tyr Lys Arg Glu Val Gln Pro Pro Phe Lys Pro Ala Ser Gly

340 345 350

Lys Pro Asp Asp Thr Phe Cys Phe Asp Pro Glu Phe Thr Ala Lys Thr

355 360 365

Pro Lys Asp Ser Pro Gly Leu Pro Ala Ser Ala Asn Ala His Gln Leu

370 375 380

Phe Lys Gly Phe Ser Phe Val Ala Thr Ser Ile Ala Glu Glu Tyr Lys

385 390 395 400

Ile Thr Pro Ile Thr Ser Ala Asn Val Leu Pro Ile Val Gln Ile Asn

405 410 415

Gly Asn Ala Ala Gln Phe Gly Glu Val Tyr Glu Leu Lys Glu Asp Ile

420 425 430

Gly Val Gly Ser Tyr Ser Val Cys Lys Arg Cys Ile His Ala Thr Thr

435 440 445

Asn Met Glu Phe Ala Val Lys Ile Ile Asp Lys Ser Lys Arg Asp Pro

450 455 460

Ser Glu Glu Ile Glu Ile Leu Met Arg Tyr Gly Gln His Pro Asn Ile

465 470 475 480

Ile Thr Leu Lys Asp Val Phe Asp Asp Gly Arg Tyr Val Tyr Leu Val

485 490 495

Thr Asp Leu Met Lys Gly Gly Glu Leu Leu Asp Arg Ile Leu Lys Gln

500 505 510

Lys Cys Phe Ser Glu Arg Glu Ala Ser Asp Ile Leu Tyr Val Ile Ser

515 520 525

Lys Thr Val Asp Tyr Leu His Cys Gln Gly Val Val His Arg Asp Leu

530 535 540

Lys Pro Ser Asn Ile Leu Tyr Met Asp Glu Ser Ala Ser Ala Asp Ser

545 550 555 560

Ile Arg Ile Cys Asp Phe Gly Phe Ala Lys Gln Leu Arg Gly Glu Asn

565 570 575

Gly Leu Leu Leu Thr Pro Cys Tyr Thr Ala Asn Phe Val Ala Pro Glu

580 585 590

Val Leu Met Gln Gln Gly Tyr Asp Ala Ala Cys Asp Ile Trp Ser Leu

595 600 605

Gly Val Leu Phe Tyr Thr Met Leu Ala Gly Tyr Thr Pro Phe Ala Asn

610 615 620

Gly Pro Asn Asp Thr Pro Glu Glu Ile Leu Leu Arg Ile Gly Asn Gly

625 630 635 640

Lys Phe Ser Leu Ser Gly Gly Asn Trp Asp Asn Ile Ser Asp Gly Ala

645 650 655

Lys Asp Leu Leu Ser His Met Leu His Met Asp Pro His Gln Arg Tyr

660 665 670

Thr Ala Glu Gln Ile Leu Lys His Ser Trp Ile Thr His Arg Asp Gln

675 680 685

Leu Pro Asn Asp Gln Pro Lys Arg Asn Asp Val Ser His Val Val Lys

690 695 700

Gly Ala Met Val Ala Thr Tyr Ser Ala Leu Thr His Lys Thr Phe Gln

705 710 715 720

Pro Val Leu Glu Pro Val Ala Ala Ser Ser Leu Ala Gln Arg Arg Ser

725 730 735

Met Lys Lys Arg Thr Ser Thr Gly Leu

740 745

<210> 8

<211> 2238

<212> DNA

<213> human

<400> 8

atgctaccat tcgctcctca ggacgagccc tgggaccgag aaatggaagt gttcagcggc 60

ggcggcgcga gcagcggcga ggtaaatggt cttaaaatgg ttgatgagcc aatggaagag 120

ggagaagcag attcttgtca tgatgaagga gttgttaaag aaatccctat tactcatcat 180

gttaaggaag gctatgagaa agcagatcct gcacagtttg agttgctcaa ggttcttggt 240

caggggtcat ttggaaaggt ttttcttgtt agaaagaaga ccggtcctga tgctgggcag 300

ctctatgcaa tgaaggtgtt aaaaaaagcc tctttaaaag ttcgagacag agttcggaca 360

aagatggaga gggatatact ggtggaagta aatcatccat ttattgtcaa attgcactat 420

gcctttcaga ctgaagggaa actgtactta atactggatt ttctcagggg aggagatgtt 480

ttcacaagat tatccaaaga ggttctgttt acagaggaag atgtgaaatt ctacctcgca 540

gaactggccc ttgctttgga tcatctgcac caattaggaa ttgtttatag agacctgaag 600

ccagaaaaca ttttgcttga tgaaatagga catatcaaat taacagattt tggactcagc 660

aaggagtcag tagatcaaga aaagaaggct tactcatttt gtggtacagt agagtatatg 720

gctcctgaag tagtaaatag gagaggccat tcccagagtg ctgattggtg gtcatatggt 780

gttcttatgt ttgaaatgct tactggtact ctgccatttc aaggtaaaga cagaaatgag 840

accatgaata tgatattaaa agcaaaactt ggaatgcctc aatttcttag tgctgaagca 900

caaagtcttc taaggatgtt attcaaaagg aatccagcaa atagattggg atcagaagga 960

gttgaagaaa tcaaaagaca tctgtttttt gcaaatattg actgggataa attatataaa 1020

agagaagttc aacctccttt caaacctgct tctggaaaac cagatgatac tttttgtttt 1080

gatcctgaat ttactgcaaa aacacctaaa gattctcccg gtttgccagc cagtgcaaat 1140

gctcatcagc tcttcaaagg attcagcttt gttgcaactt ctattgcaga agaatataaa 1200

atcactccta tcacaagtgc aaatgtatta ccaattgttc agataaatgg aaatgctgca 1260

caatttggtg aagtatatga attgaaggag gatattggtg ttggctccta ctctgtttgc 1320

aagcgatgca tacatgcaac taccaacatg gaatttgcag tgaagatcat tgacaaaagt 1380

aagcgagacc cttcagaaga gattgaaata ttgatgcgct atggacaaca tcccaacatt 1440

attactttga aggatgtctt tgatgatggt agatatgttt accttgttac ggatttaatg 1500

aaaggaggag agttacttga ccgtattctc aaacaaaaat gtttctcgga acgggaggct 1560

agtgatatac tatatgtaat aagtaagaca gttgactatc ttcattgtca aggagttgtt 1620

catcgtgatc ttaaacctag taatatttta tacatggatg aatcagccag tgcagattca 1680

atcaggatat gtgattttgg gtttgcaaaa caacttcgag gagaaaatgg acttctctta 1740

actccatgct acactgcaaa ctttgttgca cctgaggttc ttatgcaaca gggatatgat 1800

gctgcttgtg atatctggag tttaggagtc cttttttaca caatgttggc tggctacact 1860

ccatttgcta atggccccaa tgatactcct gaagagatac tgctgcgtat aggcaatgga 1920

aaattctctt tgagtggtgg aaactgggac aatatttcag acggagcaaa ggatttgctt 1980

tcccatatgc ttcatatgga cccacatcag cggtatactg ctgaacaaat attaaagcac 2040

tcatggataa ctcacagaga ccagttgcca aatgatcagc caaagagaaa tgatgtgtca 2100

catgttgtta agggagcaat ggttgcaaca tactctgccc tgactcacaa gacctttcaa 2160

ccagtcctag agcctgtagc tgcttcaagc ttagcccagc gacggagcat gaaaaagcga 2220

acatcaactg gcctgtaa 2238

Detailed Description

As described above, viral infection is known to activate a variety of signaling processes in infected cells. Some of these activities are necessary for efficient viral replication. The dependence of viruses, particularly Influenza A Viruses (IAVs), on cellular signaling pathways opens the opportunity for new antiviral strategies by targeting host factors essential for viral replication. IAV infection induces the Raf/MEK/ERK kinase cascade for efficient nuclear export of newly synthesized viral ribonucleoproteins (vrnps), and this mechanism can be blocked with specific MEK inhibitors. This antiviral strategy reduces the likelihood of inducing viral resistance and enables further antiviral therapy for a longer period of time. However, the detailed mechanism of how this cellular kinase cascade promotes nuclear export of viral genomes remains a mystery. To this end, the present invention investigated the role of the Raf/MEK/ERK/RSK signaling pathway in the interaction of vRNP with viral M1 protein in chromatin by using specific MEK inhibitors (e.g., CI-1040) (Haasbach et al, (2017) antiviral. Res. 2017142: 178-184) and RSK inhibitors (e.g., BI-D1870).

The object of the present invention is to provide a substance for use in the prophylaxis or treatment of viral diseases, particularly for use in the prophylaxis and/or treatment of minus-strand RNA viruses that replicate intracellularly and/or intranucleally, which substance does not directly target the function of the virus but selectively inhibits cellular enzymes, and by which selective action inhibits viral replication of the virus.

In the studies forming the present invention, the molecular mode of action of RSK was analyzed by considering the interaction of vRNP with the viral matrix protein (M1) on chromatin, using specific inhibitors against MEK (CI-1040) and RSK (BI-D1870) and siRNAs against ERK and RSK. To understand the nuclear distribution of viral proteins, chromatin fractionation analysis and random optical reconstruction microscopy (STORM) were used, as seen from the examples. To analyze the putative phosphorylation targets of this pathway in viral proteins, vRNP were purified after inhibitor treatment and their phosphorylation pattern was analyzed by mass spectrometry.

As exemplified by the undisturbed output of RanBP1, it can be seen that inhibition of this pathway specifically blocks vRNP output and does not generally affect nuclear output of other cellular proteins on which CRM1 depends. This suggests that viral proteins in vRNP can be modified by the Raf/MEK/ERK pathway to promote nuclear export. The location of the intranuclear vRNP complex of infected and inhibited cells was investigated and found to remain on chromatin. Immunopurification of the vRNP complex revealed a reduced binding capacity to M1 protein in the presence of kinase inhibitors. These results may be explained by the lack of phosphorylation of two serine residues 269 and 392 within the viral Nucleoprotein (NP), which under normal conditions may induce binding of the M1 protein to vRNP. It is further seen that the kinases RSK, downstream effectors of the Raf/MEK/ERK pathway, are mediators of output enhancing function. Inhibition of RSK resulted in a reduction in titer of all tested influenza viruses (including 2009 global swine influenza H1N1, highly pathogenic avian influenza viruses H5N1 and H7N9, and influenza B virus) due to nuclear retention of vRNP.

These results indicate that the Raf/MEK/ERK pathway is activated by influenza virus to successfully assemble vRNP nuclear export complexes on chromatin via RSK-dependent phosphorylation of NP protein and providing an interaction site for the viral M1 protein.

In example 1, the Raf/MEK/ERK pathway is shown to be dependent on phosphorylation of specific motifs within the nucleoprotein. Under physiological conditions, the Raf/MEK/ERK kinase cascade transmits extracellular signals within cells to promote cellular processes such as proliferation and differentiation. The signal is transmitted via sequential phosphorylation of protein kinases (Yoon, 2006). As shown in FIG. 3, it was found that two serine residues showed reduced phosphorylation after inhibition of this pathway with the MEK inhibitor CI-1040State. The crystal structure of vRNP binding to RNA indicates that the two serine residues 269 and 392 are in close proximity to each other. As can be seen in FIG. 3B, the recognition region (LILRGS) in the NP protein²⁶⁹V，AIRTRS³⁹²G) The consensus sequence with serine/threonine kinase ERK (Pro-Xaa-Ser/Thr-Pro) showed no similarity (Pro-Xaa-Ser/Thr-Pro) (Gonzalez, 1991). Thus, it is unlikely that the identified serine residues will be directly phosphorylated by ERK kinase.

In example 2, studies have shown that RSK1 acts as a link between the Raf/MEK/ERK pathway and the vRNP export. To elucidate whether RSK is a linker between the activation of the Raf/MEK/ERK signaling pathway and the nuclear export of the newly synthesized vRNP, its activation during the viral life cycle was analyzed. In the late stages of infection, not only ERK but also RSK and its downstream target GSK-3 β are phosphorylated and activated (fig. 4A). This activation could be blocked by incubation with RSK inhibitor BI-D1870, suggesting that virus-induced RSK activation is mediated by the Raf/MEK/ERK pathway (fig. 4B). In addition, there was no significant difference in progeny viral titers between CI-1040 treatment and BI-D1870 and CI-1040 combination treatment, further indicating that RSK is directly activated by the virus-induced Raf/MEK/ERK pathway (FIG. 4J). The inhibition of RSK by specific inhibitor BI-D1870 after virus infection results in the concentration-dependent reduction of GSK-3 beta phosphorylation, confirming the inhibition effect on RSK activation in the virus life cycle. Furthermore, we found that ERK activation increases after RSK inhibition, which can be explained by the inhibition of the negative feedback loop, which under normal conditions will prevent over-activation of this path (fig. 4K). Increasing the concentration of inhibitor BI-D1870 negatively affects vRNP nuclear output.

Furthermore, to rule out off-target effects of RSK inhibitors, RSK1 and RSK2 knockouts were introduced into a549 cells and their effect on the viral life cycle was analyzed (fig. 4). The RSK1 knockout resulted in newly synthesized vRNP nuclear retention, whereas the RSK2 knockout had no effect on nuclear export (FIGS. 4F-I). In multiplex replication assays, the RSK1 knockout had an antiviral effect, as shown by a reduction in viral titer. However, the RSK2 knockout appears to have proviral effects, as shown in figure 5, 2009 Kakugawa et al have described this supportive effect of the RSK2 knockout on viral replication. This suggests that the two RSK subtypes RSK1 and RSK2 have different roles in the influenza virus life cycle.

Furthermore, in example 3, MEK and RSK inhibitors were shown to specifically block vRNP export without interfering with the export of other proteins. Unlike leptomycin B, which blocks the nuclear export pathway very specifically, RSK and MEK inhibitors were found to have no general effect on the nuclear export pathway, but to be more specific for vRNP export, as shown in figure 6.

Finally, in example 4, RSK inhibitor BI-D1870 was found to have broad spectrum anti-influenza activity. Here different influenza a virus subtypes (including swine-derived pandemic virus and avian influenza virus) and influenza B virus were used to determine broad spectrum antiviral activity. All viruses tested showed nuclear retention of newly synthesized vRNP with a reduction in progeny virus titer of approximately 80-90%. These results demonstrate the dependence of influenza viruses on the Raf/MEK/ERK/RSK pathway (fig. 7), and the role of RSK as an effector in this cascade.

From these experiments it can be concluded that the kinases Rsk, in particular Rsk1(SEQ ID NO: 1 and 2), are downstream mediators of the Raf/MEK/ERK pathway, most likely directly phosphorylating the viral nucleoprotein, which is the main component of the vRNP complex. This post-translational modification appears to be required for vRNP export. Thus, Rsk inhibitors have antiviral activity by preventing export of the viral genome from the nucleus. This is an unexpected finding, as the literature suggests that Rsk inhibition, and in particular Rsk2 knock-out, exerts viral supporting effects (Kakugawa et al (2009) J Virol.; 83(6): 2510-7).

Surprisingly, it has been found that this object can be achieved by the RSK inhibitors of the invention, in particular by pharmaceutical compositions comprising RSK1 inhibitor compounds.

As mentioned above, human RSK (p90 ribosomal S6kinase) exists as four different isoforms, designated RSK1, RSK2, RSK3 and RSK 4. Alignment of the four isoforms, as shown at the amino acid level in FIG. 1 and at the nucleotide level in FIG. 2, indicates that the four forms are highly conserved, but also have specific regions of diversity. Although members of the RSK family are generally considered to be multifunctional effectors of the Ras/MAPK pathway, a single isoform appears to have both overlapping and specific functions. Although RSK1 and RSK2 play a role in cell growth and proliferation, RSK3 and RSK4 are associated with cell cycle arrest and apoptosis. In the present invention, it was found that inhibition of RSK1 alone has an antiviral effect, whereas inhibition of RSK2 alone has a pro-viral effect. However, when a general RSK inhibitor such as BI-D1870 or SL0101 is used, the inhibitory effect has an antiviral effect, and thus, it appears that the proviral activity of RSK1 dominates that of RSK 2. This is consistent with experimental evidence that only RSK1, but not RSK2, is involved in nuclear export of vRNP. Thus, both specific RSK1 inhibitors and general RSK inhibitors have antiviral effects.

These experiments show that the homologous isomers RSK1 and RSK2 have different contributions to the virus life cycle. RSK2 has an antiviral effect, whereas RSK1 apparently appears to play a virus supporting role. Once it is activated at the plasma membrane and cytosol, RSK1 translocates into the nucleus. If RSK1 is a kinase phosphorylating NP, its cellular distribution should change during the viral life cycle, depending on the activation of the virus-induced Raf/MEK/ERK pathway. To address this hypothesis, a549 cells were mock infected or infected with WSN/H1N1 virus and the localization of NP and RSK1 were analyzed by immunofluorescence staining as described in example 5. As expected, at a later time point of infection, an increase in nuclear localization of RSK1 was seen (fig. 8A). The results of three independent experiments quantitatively showed that after 9 hours, the nuclear concentration of RSK1 changed significantly from 35.58% ± 0.59% of mock infection to 57.73% ± 1.47% of viral infection (fig. 8B), especially when NP export occurred (fig. 8A, C). This indicates that the virus induced nuclear import of RSK 1.

After confirming that RSK1 enters the nucleus during the viral life cycle, the question to be solved is whether virus-induced Raf/MEK/ERK pathway activation would lead to nuclear localization of the antiviral RSK 2. In general, activation of the Raf/MEK/ERK pathway results in translocation of RSK1 and RSK2 into the nucleus. Thus, the same experiment as described in example 5 was performed, but the localization of RSK2 was analyzed, as described in example 6. In contrast to RSK1, no change in the subcellular distribution of RSK2 was found, independent of the time point during viral infection (fig. 9A, B). These results show that the virus specifically induces the translocation of virus-supported RSK1 without affecting the cellular distribution of antiviral RSK 2.

After finding a specific translocation of RSK1, the question did arise whether the RSK upstream kinase ERK1/2 could possibly enter the nucleus due to viral pathway induction. Surprisingly, as described in example 7, no effect on ERK1/2 localization was observed after viral infection, regardless of the time point of analysis (fig. 10A, B).

To exclude non-specific staining of RSK2 and ERK1/2, the Raf/MEK/ERK pathway was stimulated with TPA as described in example 8. It is known that incubation with TPA leads to activation of the Raf/MEK/ERK pathway, leading to nuclear localization of ERK1/2 and RSK within a few minutes after stimulation. After 1 hour incubation with 100nM TPA, both kinases were found in the nucleus, confirming that the staining was specific (fig. 11A, C). Quantitative results showed that the nuclear accumulation of ERK1/2 increased from 27.99% + -1.97% in the unstimulated sample to 48.02% + -2.25% in the TPA-stimulated sample. The effect on RSK2 kinase was less pronounced than ERK1/2, with 29.37% ± 1.15% in the unstimulated sample and 34.89% ± 0.67% in the TPA stimulated sample (fig. 11B, D).

In summary, during the viral life cycle, RSK1 enters the nucleus of infected cells and most likely plays a role in viral support via phosphorylation of NP. This translocation is mediated through the activation of the Raf/MEK/ERK pathway. In addition, the virus appears to prevent nuclear localization of ERK1/2 and antiviral RSK 2.

The major problem with antiviral strategies that directly target viral particles is the introduction of resistant viral variants. Prolonged exposure to such antiviral drugs can cause mutations to occur, resulting in reduced drug sensitivity. One group of antiviral drugs is represented by neuraminidase inhibitors, such as oseltamivir. It binds to neuraminidase exposed on the surface of infected cells, leading to a conformational change in the active site by forming a binding pocket. Inhibition is reduced by mutations within neuraminidase, e.g. H275Y, E119D, I223R. The second group is polymerase inhibitors such as balosavir. This cap-dependent endonuclease inhibitor blocks the cap-grasping process of the PA subunit. The reduced sensitivity to Baloxavir marboxil may especially occur after the amino acid substitution I38T.

To compare the ability of antiviral drugs directly targeting viral particles (oseltamivir, barloxavir) or to exert antiviral effects via inhibition of the Raf/MEK/ERK/RSK pathway (CI-1040, BI-D1870) to induce resistant viral variants, a549 cells were infected with WSN/H1N1 virus using an MOI of 0.01 as described in example 9. Directly after infection, cells were treated with different inhibitors or DMSO and progeny virus titers at 24h post infection were determined by standard plaque titration. In the next few rounds, a549 cells were infected with the virus supernatant of the previous round (MOI0.01) and titers were determined 24h post-infection. The inhibitor concentration is increased to enhance the inhibitory effect and induce the occurrence of mutations. Increasing concentrations of CI-1040 and BI-D1870 resulted in decreased viral titers. In the first round, using 1 μ M inhibitor concentration, the titer of CI-1040 or BI-D1870 decreased by 64.75% + -0.32% and 35.08% + -3.20%, respectively, compared to the DMSO control. With increasing inhibitor concentration, the titer further decreased until round 4. In this case, a concentration of 8. mu.M was used for both inhibitors. From round 5 to round 12, CI-1040 and BI-D1870 were used at a concentration of 10. mu.M. Mean titers of CI-1040 treatment were calculated from round 5 to 12, 8.45% ± 3.41% relative to DMSO control. The mean titer from treatment with BI-D1870 in rounds 5 to 12 was calculated to be 12.27% + -6.36%. At any time point in round 12, neither a sustained increase in viral titer nor a change in plaque morphology was observed, indicating that no mutations introducing resistance occurred. Complete resistance was found after oseltamivir treatment round 5. At this time, the inhibitor concentration used was 16. mu.M. At round 9, the titer began to decrease to 48.30% ± 11.42%. This effect is accompanied by a progressive reduction in the size of the plaque. The mean titer for treatment with barbiturate from round 1 to round 9 was calculated to be 8.08% ± 4.10%. The trend of increasing titre started in round 10, with a mean titre of 28.39% ± 1.88%. At round 12, mean titers further increased to 46.94% ± 1.03% (fig. 12).

These results support the discovery that long-term treatment with MEK or RSK inhibitors does not cause viral resistance.

These and previous results in this study indicate that MEK and RSK inhibitors are suitable drugs for viral infections such as influenza virus infection. They are well tolerated by cells, have no cytotoxic effects, exhibit broad spectrum antiviral activity, and do not appear to promote the emergence of resistant viral variants.

Accordingly, in one aspect, the present invention provides a method for preventing and/or treating a viral disease, the method comprising administering an RSK inhibitor to a patient in need thereof.

In one aspect, the methods of the invention are used to prevent and/or treat viral diseases, which are infections caused by negative strand RNA viruses. More preferably, the viral disease is caused by influenza virus, even more preferably by influenza a or B virus. Influenza a viruses are for example H1N1, H3N2, H5N1, H7N7, H7N9 or H9N 2.

In another aspect, the methods of the invention are used to prevent and/or treat viral diseases, which are infections caused by positive-stranded RNA viruses, e.g., coronaviruses such as SARS-CoV, MERS-CoV or SARS-CoV-2, which cause respiratory infections such as SARS, MERS or Covid 19.

The RSK inhibitor of the invention is preferably selected from the group consisting of BI-D1870, SL0101, LJH685, LJI308, BIX 02565, FMK and a selective RSK1 inhibitor or a derivative, metabolite or pharmaceutically acceptable salt thereof.

Specifically, BI-D1870 is known to be an in vitro and in vivo potent and specific inhibitor of the p90 Ribosomal S6Kinase (RSK) isoform, which inhibits RSK1, RSK2, RSK3 and RSK4 in vitro (IC50 values of 31nM, 24nM, 18nM and 15nM, respectively). (GP Sapkota et al. BI-D1870 is a specific inhibitor of the p90 RSK (ribosol S6kinase) isoforms in vitro and in vivo. biochem. J.2007,401, 29-38).

SL-0101 is a cell-permeable kaempferol (catalog No. 420345) glycoside that targets the N-terminal kinase domain of p90 ribosomal S6kinase and inhibits RSK kinase activity (using 10 μ M ATP, IC50 ═ 89nM) in a selective, reversible and ATP-competitive manner (Ki ═ 1 μ M). SL-0101 was shown to inhibit the proliferation of MCF-7 but not normal breast cell line MCF-10A and to specifically block PDB-induced cellular phosphorylation of the RSK substrate p140, but not RSK or the kinase upstream of RSK even at concentrations up to 100. mu.M.

All known RSK inhibitors listed above inhibit all 4 homologous isomers of RSK. However, it is an object of the present invention to provide a specific RSK1 inhibitor which is capable of blocking RSK1 without affecting RSK2, RSK3 or RSK 4. One method of identifying inhibitors of RSK1 is to screen for substances that bind to RSK1(SEQ ID NOS: 1 and 2) but not to RSK2(SEQ ID NOS: 3 and 4), RSK3(SEQ ID NOS: 5 and 6), or RSK4(SEQ ID NOS: 7 and 8).

In the pharmaceutical use of the invention, the RSK inhibitor may be administered orally, intravenously, intrapleurally, intramuscularly, topically or via inhalation. Preferably, the RSK inhibitor is administered to the individual or patient via nasal inhalation or orally.

The subject or patient of the invention is a mammal or bird. Examples of suitable mammals include, but are not limited to, mice, rats, cows, goats, sheep, pigs, dogs, cats, horses, guinea pigs, dogs, hamsters, minks, seals, whales, camels, chimpanzees, rhesus monkeys, and humans. Examples of suitable birds include, but are not limited to, turkeys, chickens, geese, ducks, water ducks, green-headed ducks, \ 26891;, birds, northern long-tailed ducks, gulls, swans, guinea fowl, or waterfowls, to name a few. Human patients are a particular embodiment of the invention.

The pharmaceutical composition comprising the RSK inhibitor may be in the form of an oral suspension or tablet; nasal spray, sterile injectable preparation (intravenous, intrapleural, intramuscular), for example, as sterile injectable aqueous or oleaginous suspension or suppository. When administered orally as a suspension, these compositions are prepared according to techniques available in the art of pharmaceutical formulation and may contain microcrystalline cellulose for volume enhancement (dispersing bulk), alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweetening/flavoring agents known in the art. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, fillers, disintegrants, diluents and lubricants known in the art. Injectable solutions or suspensions may be formulated according to the known art using suitable non-toxic parenterally-acceptable diluents or solvents, for example mannitol, 1, 3-butanediol, water, ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting agents and suspending agents, for example sterile, mild, non-volatile oils, including synthetic mono-or diglycerides, and fatty acids, including oleic acid.

In the methods of treatment and pharmaceutical uses of the invention, the RSK inhibitor is administered in a therapeutically effective amount. The "therapeutically effective amount" may vary depending on factors including, but not limited to, the activity of the compound employed, the stability of the active compound in the patient, the severity of the condition to be alleviated, the total weight of the patient being treated, the route of administration, the ease with which the compound is absorbed, distributed, and excreted by the body, the age and sensitivity of the patient being treated, adverse events, and the like, as will be apparent to those skilled in the art. The amount administered may be adjusted depending on various factors over time.

In addition, the RSK inhibitor may be administered with a second anti-viral agent. The second antiviral agent can be administered prior to, concurrently with, or subsequent to administration of the RSK inhibitor. In addition, the RSK inhibitor and the second antiviral agent may be administered in one dosage form or in two separate dosage forms. It is also contemplated that the RSK inhibitor may be administered with two or more antiviral agents.

The second antiviral agent may be any known antiviral agent. The second antiviral agent may be selected from the group consisting of a ceramidase inhibitor, a polymerase complex inhibitor, an endonuclease inhibitor, a hemagglutinin inhibitor, a nucleoprotein inhibitor and a MEK inhibitor.

Preferred ceramidase inhibitors may be oseltamivir, oseltamivir phosphate, zanamivir, peramivir or ranimivir or a pharmaceutically acceptable salt thereof. Combinations with oseltamivir are considered a specific embodiment.

Preferred polymerase complex inhibitors may be Baloxavir, Baloxavir phosphate, Baloxavir Marboxil, fapivir or pimavavir or a pharmaceutically acceptable salt thereof.

Preferred MEK inhibitors may be CI-1040, PD-0184264, PLX-4032, AZD6244, AZD8330, AS-703026, GSK-1120212, RDEA-119, RO-5126766, RO-4987655, PD-0325901, GDC-0973, TAK-733, PD98059 or PD184352, or a pharmaceutically acceptable salt thereof. Combinations with CI-1040 or PD-0184264 are considered a specific embodiment.