respiratory syncytial virus replication

FIELD OF THE INVENTION

The present invention relates to small molecules inhibiting the replication of influenza A and B virus and respiratory syncytial virus (RSV), and the use of such compounds for treating influenza A and B and RSV infections, in humans, mammals and birds.

BACKGROUND OF THE INVENTION

Influenza viruses are negative-stranded RNA viruses that cause yearly epidemics as well as recurring pandemics, resulting in high numbers of human cases and severe economic burden. In addition to the well-known pandemic influenza A viruses (such as the 1918 "Spanish" flu or H5N1 ), both type A and B viruses contribute greatly to the annual recurring epidemics that cause the vast majority of human cases and medical cost. The WHO recommends an annual vaccination against circulating influenza A (FIuA) and B (FIuB) strains. However, current vaccines confer incomplete protection against epidemic influenza. To date, only the neuraminidase inhibitors oseltamivir (Tamiflu™) and zanamivir (Relenza™) are available as antiviral treatment against both virus types. However, there is a growing fear within the medical community about the rapidly growing emergence of influenza strains resistant to both drugs. The older adamantane drugs are not effective against FIuB and the global spread of influenza viruses resistant to oseltamivir demonstrate the limitations of the neuraminidase inhibitors. A recent epidemiological survey in the U.S. found 98.5% of the H l N l isolates tested resistant to oseltamivir.

Human respiratory syncytial virus (RSV) is a negative-sense, single-stranded RNA virus of the family Para myxovi rid a e, and is the major cause for respiratory tract illnesses during infancy and childhood such as bronchiolitis and pneumonia. There is currently no vaccine available. Treatment is mainly limited to supportive care, including oxygen. Palivizumab (Synagis™) is used as a prophylactic drug in prevention of respiratory RSV infections for infants with a high risk of infection. Ribavirin has been used for treating RSV infections, but showed limited effectiveness.

Thus, new improved and alternative antiviral agents against both influenza A and B virus types and RSV are urgently needed.

OBJECTS OF THE I NVENTION

One object of the invention is to provide new, improved and/or alternative influenza and RSV antiviral compounds.

Another object of the invention is to obviate or mitigate disadvantages of influenza antiviral agents and RSV antiviral agents known from the state of the art.

These and other objects are achieved by a compound as a medicament, a compound for treating influenza type A and/or influenza type B and/or RSV infections in humans, mammals and/or birds, the use of a compound for the manufacture of a medicament for the treatment of influenza type A and/or in- fluenza type B and/or RSV infections in humans, mammals and/or birds, and a pharmaceutical composition comprising such a compound, according to the independent claims. Advantageous embodiments are given in the dependent claims.

SUMMARY OF THE INVENTION

The 2,1 ,3-benzoxadiazole compounds as a medicament according to the invention are: 4-[(4-methoxybenzyl)thio]-7-nitro-2,l ,3-benzoxadiazole,

2-[(7-nitro-2,l ,3-benzoxadiazol-4-yl)thio]ethyl 4-methoxybenzene-l -sulfonate, 4-[(4-methylphenyl)thio]-7-nitro-2,l ,3-benzoxadiazole, 4-[(2,4-dichlorophenyl)thio]-7-nitro-2,l ,3-benzoxadiazole, 2-[(7-nitro-2,l ,3-benzoxadiazol-4-yl)thio]ethan-l -ol, 4-[(4-methylbeπzyl)thio]-7-πitro-2,l ,3-beπzoxadiazole, 4-[(4-fluorophenyl)thio]-7-nitro-2,l ,3-benzoxadiazole, 4-[(3-chlorophenyl)thio]-7-nitro-2,l ,3-benzoxadiazole, 2-[(7-nitro-2,l ,3-benzoxadiazol-4-yl)thio]ethyl-4-methoxybenzoate, 5-[4-(tert-butyl)-l ,3-thiazol-2-yl]-2,l ,3-benzoxadiazole, N-benzyl-4-nitro-2,l ,3-benzoxadiazol-5-amine, 4-πitro-7 -(phenyl methylsulfaπyl)-2,l ,3-beπzoxadiazole, 4-nitro-7 -(phenyl methylsulfonyl)-2,l ,3-benzoxadiazole,

2-(hydroxymethyl)-5-[6-[(4-nitro-2,l , 3-benzoxadiazol-7-yl)sulfanyl]purin-9-yl]oxolane-3,4-diole, and 2-[2- amino-6-[(4-nitro-2,l , 3-benzoxadiazol-7-yl)sulfanyl]purin-9-yl]-5-(hydroxymethyl) oxolane-3,4-diol, as well as physiologically tolerable salts, solvates, or physiologically functional derivatives thereof.

The above defined compounds according to the invention are particular advantageous for treating and/or preventing influenza type A and/or influenza type B infections in humans, mammals and/or birds; as well as for treating and/or preventing respiratory syncytial virus (RSV) infections in humans, mammals and/or birds.

The compound according to the invention can be used for the manufacture of a medicament for the treatment and/or prevention of influenza type A and/or influenza type B infections in humans, mammals and/or birds, and/or for the treatment and/or prevention of respiratory syncytial virus infections in humans, mammals and/or birds.

A pharmaceutical composition according to the invention comprises a compound according to the invention. Advantageously such a composition comprises one or more excipients.

Surprisingly, it was found that compounds in accordance with the present invention are able to inhibit protein-protein interaction of the PA and PBl subunits of the heterotrimeric viral RNA polymerase complex of both influenza virus types A and B, and thus are able to inhibit replication of influenza A and B virus. The viral polymerase subunit interaction domain turned out as an effective target for the new antiviral compounds, since correct assembly of the three viral polymerase subunits PBl , PB2 and PA is required for viral RNA synthesis and infectivity. Structural data for the entire trimeric complex is missing. Based on the crystal structure of a truncated FIuA PA in complex with the N-terminus of PBl it was established that the crucial PA interaction domain of PBl consists of a 3io-helix formed by amino acids (amino acids 5-1 1 ). The domain is highly conserved and virus type specific among both, influenza A and B viruses.

An Enzyme-Linked Immunosorbent Assay (ELISA) based screening assay and other assays are used to prescreen compounds according to the invention that show antiviral activity against influenza A and B viruses. Since they are effective against both virus types, such compounds represent an attractive alternative to neuraminidase inhibitors. Therefore, the present invention represents a major step toward a sorely needed, near-universal medicament against influenza virus, and one which, due to its protein- protein interaction domain target, will likely be less susceptible to the emergence of drug-resistant strains for which influenza is well known.

Furthermore it was found that compounds according to the invention are also able to inhibit replication of respiratory syncytial virus (RSV).

Thus the compounds according to the invention can be used as a medicament, particularly as an influ- enza virus and/or RSV replication inhibitor and an influenza and/or RSV preventive/therapeutic agent, respectively.

The object, characteristics, and advantages of the present invention as well as the idea thereof will be apparent to those skilled in the art from the descriptions given herein. It is to be understood that the embodiments and specific examples of the invention described herein below are to be taken as pre- ferred examples of the present invention. These descriptions are only for illustrative and explanatory purposes and are not intended to limit the invention to these embodiments or examples. Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures. It is further apparent to those skilled in the art that various changes and modifi- cations may be made based on the descriptions given herein within the intent and scope of the present invention disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

Influenza Type A and B: Therapeutic target

In the international patent application with the title "Influenza A and B virus replication-inhibiting peptides" No. PCT/EP2009/055632, filed on 8 May 2009, novel peptides containing for example ammo acid sequences from both virus types A and B, are described. The content of said application is hereby included by reference in its entirety. Surprisingly, it was found that those novel peptides bind to PA subunits of both types of influenza A and B. Among said novel peptides, chimeric peptides, containing ammo acid sequences from both virus types A and B, were identified which not only bind to both PA subunits, but also decrease the viral polymerase activity and the spread of virus in cell culture for both influenza A and B. In the following the findings concerning the binding of said novel peptides are described in order to further specify the inhibition target of the small molecules compounds according to the present invention.

It should be noted that all ammo acids are preferably indicated by the IUPAC one letter code in the present application. Whenever three letter codes are used, they are also in accordance with IUPAC. The letter X is used to indicate a wildcard/variable or other ammo acid at a certain position.

It has been found that the crucial PA interaction domain of PBl consists of a 3io-helιx formed by ammo acids X ₅ to Xn . This domain is highly conserved and type-specific among both influenza A and B viruses (Figure I a). Additionally, FIuB PBl was able to bind to FIuA PA when these 25 ammo acids were exchanged with the FIuA PBl sequence (Figure 2).

Table I a shows the inhibitory concentrations of FluA/FluB-derived peptides determined by competitive ELISA. Competitor peptides (0.048 to 300OnM) were mixed with cell extracts containing HA-tagged PA from either FIuA or FIuB. Table 1 lists 1 2 competitive peptides. The first peptide PBI i ₁₅ A is the FIuA wild type, the second row shows the FIuB wild type. For the peptides of rows 3 to 8 letters indicate FIuB specific ammo acids. Rows 9 to 1 2 list further competitive peptides with ammo acids at position 6 being neither FIuA nor FIuB specific. Standard deviation is indicated in parenthesis. Asterisks indicate highest concentrations of peptides used without reaching 50% inhibition. Further competitive peptides which are not listed in the table but have effectively reached 50% inhibition at low peptide concentrations are PBl i-i ₅ A _T6 i , PBl i-i ₅ A _T6L and PB 1 ₁ _i ₅ A _T6 v ■ Peptides with slightly lower inhibition activity are PB 1 H ₅ A _T 6 _A and PBl i-i ₅ A _T6M which are also not shown in Table I a.

Table I a: inhibitory concentrations of FluA/FluB-derived peptides determined by competitive ELISA

Competitive peptide PA (RuA) PA (FIuB)

PBI MDVNFΓLLPLKVPAQ 43.32 (+/-531) >3000' PBI .NX..VF. -XD..X. >3000* 450 {+/-12.5) PBI S Ao≥N vSt.Lii: 6.69 {+/- 1.73; >3000' PBI 5 As.iOt.Ki-iO >3000' >3000 ^Λ PBi 5 AθΛ \ 3! 12.96 {+/- 3.98S >3000' PBi S Ayev i_7f 7.51 (+/-0.71) 3450 (+/-815} PBi S ^JJV 21.64{+/- 1.48) 107.1 (+/-31.3) PBI •5 A _T ≥f .F. 2.84 {+/- Q 48) 7504 {+/-2496) PBI S AΪSW .W. 3.4G(+/-0.51i 6283 <V-3S9.-n PBI S Av ₆ H .H .. 292.16 {+/- 34.04) X3O0O ^* PBI ς ATSC .C. 43.58 {+/- 567) >3Q0Q"

' highest concentration of competitive peptide used

A comprehensive and qualitative overview on further peptides with high inhibitory activity is provided in Table 1 b. In the table the amino acid sequences at positions X ₅ to Xi ₀ of wild type A mutants are indicated.

Table I b: qualitative overview of further preferred peptides

In Table I c the amino acid sequences at amino acid residues X ₅ to Xi ₀ of wild type A mutants are indicated. Said peptides exhibit lower activities than the above mentioned peptides according to Tables I a and 1 b.

Table I c: qualitative overview of further peptides

Based on the above presented information and results, it is clear for the person skilled in the art, that the synthesized or isolated influenza virus replication-inhibiting peptides interacting with the inhibition target for the small molecules compounds according to the invention comprise an ammo acid sequence of X ₅ X ₆ X ₇ XsXgXi _O , wherein X ₅ is P; X ₆ is T, Y, F, W, H, C, I, L, V, A or M; X ₇ is L or F; X ₈ is L, I, F or M; X ₉ is 5 F, Y, W, H, L, R or S, and Xi ₀ is L, I or Y. Said ammo acid sequence is at least 60 %, preferably at least 70%, more preferably at least 80% or 90% identical to the polypeptide according to the wild type PBI i _{1 1} A which is M DVN PTLLFLK. Within the aforementioned group of peptides, those peptides are preferred which comprising the ammo acid sequence of X ₆ X ₇ X ₈ XgXi _O , wherein X ₆ is T, Y, F, W, H, C, I, L or V; X ₇ is L or F; X ₈ is L or I; Xg is F, Y or W and Xi ₀ is L Even more preferred according to certain embodi- i o ments are peptides that comprise the ammo acid sequence of X ₆ X ₇ , wherein X ₆ is T, Y, F, W, H, C, I, L or V and X ₇ is L or F.

Effective peptides advantageously comprise at least 1 1 residues Xm , whereby preferably the proteins comprise the ammo acid sequence M DVN PX6X7 LFLKVPAQ wherein X6 is selected from the group: T, Y, F, W, H. C, A, I, L, V or M and X7 is selected from the group L or F. A preferred peptide comprises an 15 ammo acid sequence elected from the group: MDVNPYFLFLKVPAQ, MDVNPYLLFLKVPAQ, MDVNPWLLFLKVPAQ or MDVNPFLLFLKVPAQ. According to further preferred embodiments the peptides comprise at least 1 5 residues Xi _{] 5} according to the wild type PBl i _{] 5} A but not the wild type sequence MDVN PTLLFLKVPAQ.

Table 2 shows the 50%-ιnhιbιtory concentrations (IC ₅ o) of FluA-derived PBl peptides determined by 20 competitive ELISA. Peptide PBI i ₂₅ A was immobilized on microwell plates and incubated with increasing concentrations of competitor peptides and cell extract containing HA-tagged PA of FIuA. Bound PA was detected by HA-specific antibodies as described above. Standard deviation is shown in parenthesis. Asterisks indicate highest concentrations of peptides used without detectable inhibitory effect. Grey boxes highlight ammo acids that are part of the 3i o-helιx, which comprises the core PA-binding region of PBl . 25 Ammo acids known to form hydrogen bonds with PA residues are represented in bold. The systematic truncation of the 25mer peptide comprising the PA-binding domain of PBl at the N- and C- terminus showed - based on the ELISA assay results - that ι) the 25mer peptide can be truncated at the C- termmus until the first 14 or even 1 3 N-terminal ammo acids remain without losing ability to i nhibit the bound peptide-PA interaction. Truncation at the C-terminus down to the first 1 2 or even 1 1 ammo acids resulted in peptides which still showed considerable activity. The systematic truncation showed further that ii) N-terminal truncation is not possible without major loss in inhibitory activity of the peptide.

Table 2: Inhibitory concentrations (IC ₅₀ ) of FluA-derived PBl peptides

FIuA-PBI peptides (aa) IC ₅₀ (nM)

3-ιr-helιx

1 -25 MDVNFELLFi _^ KVPAQNAISTTFPYT 1.80 (+/- 0.49)

3 -25 — VNIΦΣAF&KVF AQNAI STTFPY T 661.77 (+/- 22.08}

5 _25 2 ^< KLLFl,ϊfVP AQNAI STTFPYT 483 20 (+/- 51 9S)

7 -25 LLFLKVPAONAISTTFPYT >3000*

9 -25 FLKVPAONAISTTFPYT >3000*

11 -25 KVPAQNAISTTFPYT >3000'

1 -20 MDVNl LLfI ₁ SVPAQNAIST 33.80 (+/- 5.53}

1 -15 MDVNITS&F&RVFAQ 43 32 (+/- 5.31 )

1 - 14 MDVN2ΪXLF&RVFA 34 53 (+/- 2.19} i -13 MDVN3*O&£Ϊ»KVP 138.17 (+/- 7.88)

1 -12 MDVNPTi ₁ LFi ₁ KV 643.93 (+1- 180.75}

1 -11 MDVNTOlΛFjys 899.53 (+/- 54 31 )

1-10 MDVNPTLLFL >3000' i-9 MDVNPTLLF >3000'

1-8 MDVNPTLL >3000'

1-7 MDVNPTL >30QCT

1-6 MDVNPT >3000*

* highest concentration of competitive peptide used

Table 3 illustrates the inhibitory concentrations (IC ₅₀ ) of FluA-derived competitor peptides determined by ELISA. Peptide PBI _{1 25} A was again immobilized on microwell plates and incubated with increasing concentrations of competitor peptide and cell extract containing HA-tagged PA of FIuA. HA-specific antibodies detected bound PA. Standard deviations are shown in parenthesis. Asterisks indicate highest concentrations of peptides used without detectable inhibitory effect. Table 3: Inhibitory concentrations (IC ₅₀ ) of FluA-derived PBl peptides

Competitive peptide 1C50 in nM

PBI _M5 A MDVNPTLLFLKVPAQ 43.32 (+/- 5.31)

PBI1-15AMIA ADVNPTLLFLKVPAQ 460.30 (+/- 27.85}

PBI _M5 A D2A MAVNPTLLFLKVPAQ 209.17 (+/-44.62)

PBI ₁₁₅ A V3A MDANPTLLFLKVPAO 154.93(+/- 18.18)

PB1 _M5 AN4A MDVAPTLLFLKVPAO >3000 ^x

PBI _M5 APSA MDVNATLLFLKVPAQ 2728.67 (+/-133.43)

PBI ₁ - _I5 ATeA MDVNPALLFLKVPAQ 701.87 (+/-20.59)

PBI _M5 A L7A MDVNPTALFLKVPAQ >3000*

PB1i-«AL8A MDVNPTLAFLKVPAQ >30G0 ^*

PBI _M5 A F9A MDVNPTLLALKVPAQ >3000'

PBI _M5 A LIOA MDVNPTLLFAKVPAQ >3000"

PBI _M5 A KHA MDVKPTLLFLAVPAO 1290.33 (+/-210.37)

PB1 ₁₁₅ AV12A MDVNPTLLFLKAPAO 707.87 (+/-168.54)

PBI _M5 A P13A MDVHPTLLFLKVAAO 257.93 (+/- 36.76)

PBI _M5 AMID DDVKPTLLFLKVPAQ 1375.67 (+/-268.11}

PB1 _M5 AVSD MDDNPTLLFLKVPAQ >3000"

PBI _M5 A N4D MDVDPTLLFLKVPAQ >3000'

PBI _M5 A PSD MDVNDTLLFLKVPAO >3000 ^x

PBI _M5 A T6D MDVNPDLLFLKVPAQ 2067.67 (+/- 584.98)

PBI ₁₁₅ ALJD MDVNPTDLFLKVPAQ >3000'

PBI _M5 A LSD MDVNPTLDFLKVPAQ >3000 ^*

PBI _M5 A F9D MDVNPTLLDLKVPAQ ^OOO"

PBI _M5 A LIOD MDVNPTLLFDKVPAO >3000"

PBI _M5 AKHD MDVNPTLLFLDVPAO >3000*

PBI _M5 A V12D MDVNPTLLFLKDPAQ 2302.67 (+/- 280.39)

PBI _M5 A PI 3D MDVNPTLLFLKVPAQ 1097.47 (+/-217.54)

highest concentration of competitive peptide used

Figure 1 shows binding and inhibitory activity of PB11-25AT6Y. Based on Figure 1 the binding and inhibitory activity of peptides binding to the inhibition target with a focus on the preferred protein PBl]. ₂₅ A _T6 γshall be illustrated in the following part of the description. Figure Ia shows in the upper panel the alignment of the consensus sequence of the N-terminal 25 amino acids of FIuA and FIuB PBl, wherein the dotted box indicates the 3io-helix comprising the core PA-binding domain of PBl and the FIuA- specific and FluB-specific amino acids are printed in bold letters. Middle and lower panels show the alignment of the N-terminal 25 amino acids of all available FIuA and FIuB sequences derived from PBl full length sequences provided by the NCBI influenza virus database. The binding of HA-tagged PA subunits from cell extracts to the immobilized peptides corresponding to the domains of FIuA PBl (PBl _{1 25} A), FIuB PBl (PBl _{1 25} B) or FIuA PBl T6Y (PBl _{1 25} A _τ6 γ) determined by ELISA is shown in Figure I b. Signals using the cognate peptide and lysate were normalized to 1 . Binding of the PA subunits to the control peptides was not observed. Upper panels: Western blot of the PA- 5 containing cell extracts used. Molecular weights shown in kilodaltons.

Figure I c provides graphic information on the structure of FIuA PBl _{1 1 5} bound to FIuA PA. T6 forms a hydrogen bond to a water molecule. Molecular modeling suggests that the aromatic side chain in the mutant T6Y fits into a hydrophobic pocket and displaces the water molecule. The polymerase inhibitory activity of PBl _{1 25} -derιved CFP fusion proteins in FIuA and FIuB polymerase reconstitution assays is i o shown in Figure I d. The activity in experiments containing all viral plasmids and Flag-CFP was set to 1 00%.

Figure I e shows a plaque reduction assay using PB1 _{1 25} A-Tat; PBl _{1 25} A _T6 γ-Tat; PX-Tat (control peptide) with FIuA, FIuB and VSV (vesicular stomatitis virus). A H ₂ O control was used to standardize the assay to 1 00%. Note that PB1 _{1 25} B-Tat could not be tested due to insolubility. Error bars represent standard de- 15 viations.

Virus type-specific interaction of PA with PBl is illustrated in Figure 2. Figure 2a shows A/SC35M- and B/Yamagata/73-derιved PBl chimeras used in tests according to Figure 2b. Note that all PBl proteins were expressed with C-terminal HA-tags. Figure 2b shows human 293T cells which were transfected with expression plasmids coding for the indicated PBl proteins and the C-terminally hexahistidme-tagged PA

20 of FIuA (FIUAPAH _IS ). Cell lysates were prepared 24 hours post transfection and subjected to immunopre- cipitation (IP) using antι-HA (aHA) agarose. Precipitated material was separated by SDS-PACE and analyzed by Western blot for the presence of either His- or HA-tagged polymerase subunits using appropriate antibodies. Protein expression was controlled by analyzing equal amounts of cell lysate. Molecular weights are shown in kilodaltons. The 25-mer peptide, PBl _{1 25} A, comprising a helical domain inhibits

25 the polymerase activity and replication of FIuA, whereas the activity of FIuB polymerase is not affected.

In Figure 3 dual-binding properties of the FluA/B peptide chimera PBl _{1 25} A _T6 γ are illustrated in comparison to PBl _{1 25} A and PBl _{1 25} B. The Lower panels show peptides PBl _{1 25} A, PBl _{1 25} B or PBl _{1 25} A _T6 γ immobilized on microwell plates and incubated with increasing concentrations of cell extract containing the indicated PA-HA from FIuA or FIuB strains. Bound PA-HA was detected by HA-specific antibodies and peroxidase-labeled secondary antibodies. Binding efficiency was quantified by measuring substrate conversion at 405 nm. Standard deviations are indicated by error bars. Experiments were repeated in triplicates. Upper panels show analysis of corresponding amounts of cell lysate by Western blot controlled protein expression. Molecular weights are shown in kilodaltons.

Figure 4a shows CFP-PBl fusion proteins used in tests according to Figure 4b. The complex formation of PBl _{1 25} -derιved CFP fusion proteins and HA-tagged PA of FIuA and FIuB is shown in Figure 4b. Indicated proteins were expressed in human 293T cells and binding of the CFP fusion proteins was analyzed by immunoprecitation (IP) of PA using antι-HA agarose and subsequent immunoblotting (IB). Precipitated material was analyzed by Western blot using the indicated antibodies for the presence of either HA- tagged PA or CFP. Molecular weights are shown in kilodaltons.

Influenza Type A and B: Materials and Methods

Virus strains: For the infection experiments A/WSN/33 (Hl N l ) according to Chanem et al. (2007) and A/Thaιland/l (Kan-l )/2004 according to Chockephaibulkit et al. (2005), B/Yamagat/73 according to Norton (1987) and VSV (serotype Indiana) as described in Schwemmle (1995) were used.

Plasmid constructions: Plasmids pCA-Flag-CFP and pCA-PBl _{1 25} A-CFP, pCA-PBl -HA, the FIuA minireph- con plasmids and the expression plasmids for the FIuB minireplicon are described in Chanem (2007), Mayer (2007) and Pleschka (1996). The FIuB minigenome expression plasmid, pPoll-lucRT_B, was ob- tamed by cloning the firefly luciferase ORF (inverse orientation) flanked by the non-coding region of the segment 8 of the B/Yamagata/73 into the Sapl-digested plasmid pPoll-Sapl-Rib according to Pleschka (1996). For the construction of pCA-PBl _{1 25} B-CFP, a linker containing the first 25 codons of PBl (B/Yamagata/73) was cloned into the EcoRI/Notl sites of pCA-Flag-CFP plasmid, replacing the Flag- coding sequence with PBI _{1 25} B. Site directed mutagenesis was carried out with pCA-PBl _{1 25} A-CFP to create the plasmid pCA-PBl _{1 25} A _T6 rGFP. The ORFs of PBl (B/Yamagata/73) and PA (A/SC35M, A/Thaιland/1 (KAN-I )/04, A/Vιetnam/1 203/04, B/Yamagata/73, B/Lee/40) were PCR amplified with sense primers containing an Notl site (FIuA strains) or a EcoRI site (FIuB strains) upstream of the initiation codon and antisense primers with a deleted stop codon followed by an Xmal site, a coding sequence for an HA-tag and a Xhol site. The PCR products were cloned into a modified pCACCsvector (Schneider, 2003) digested either with EcoRI/Xhol or Notl/Xhol, resulting in pCA-PBl -HA or pCA-PA- HA plasmids, coding for C-terminal tagged versions of the polymerase subunits. To obtain the pCA- PA _/ yscss _M -His plasmid, pCA-PA _/ yscss _M -HA was digested with Xmal/Xhol and the HA coding sequence was replaced by a 6xHis-l inker. The A/B-chimeric expression plasmids were obtained by assembly PCR using the pCAPBl -HA plasmids of SC35M and B/Yamagata/73 and by cloning the resulting PCR product in pCA-PBl _B /γ _amagata / ₇₃ -HA digested with EcoRI/EcoRV.

Reconstitution of the influenza virus polymerase activity: HEK293T cells were transiently transfected with a plasmid mixture containing either FIuA- or FluB-derived PBl-, PB2-, PA- and NP-expression plasmids, polymerase I (Pol l)-driven plasmid transcribing an influenza A or influenza B virus-like RNA coding for the reporter protein firefly luciferase to monitor viral polymerase activity and with expression plasmids coding for the indicated CFP fusion proteins. Both minigenome RNAs were flanked by non-coding sequences of segment 8 of FIuA and FIuB, respectively. The transfection mixture also contained a plas- mid constitutively expressing Renilla luciferase, which served to normalize variation in transfection efficiency. The reporter activity was determined 24h post transfection and normalized using the Dual-Clu® Lufierase Assay System (Promega). The activity observed with transfection reactions containing Flag-CFP were set to 100%.

Peptide synthesis: The solid-phase synthesis of the peptides was carried out on a Pioneer automatic peptide synthesizer (Applied Biosystems, Foster City, USA) employing Fmoc chemistry with TBTU/diisopropylethyl amine activation. Side chain protections were as follows: Asp, CIu, Ser, Thr and Tyr: t-Bu; Asn, CIn and His: Trt; Arg: Pbf; Lys and Trp: Boc. Coupling time was 1 h. Double couplings were carried out if a difficult coupling was expected according to the program Peptide Companion (Coshi- Soft/PeptiSearch, Tucson, USA). All peptides were generated as carboxyl amides by synthesis on Rapp S RAM resin (Rapp Polymere, Tubingen, Germany). Biotin was incorporated at the C-terminus of indicated peptides with Fmoc- Lys(Biotin)-OH (NovaBiochem/Merck, Nottingham, UK) and TBTU/diisopropylethylamine activation for 18h, followed by coupling of Fmoc-β-Ala-OH for I h. Peptides were cleaved from the resin and deprotected by a 3h treatment with TFA containing 3% triisobutylsi- lane and 2% water (10ml/g resin). After precipitation with t-butylmethylether, the resulting crude pep- tides were purified by preparative HPLC (RP-18) with water/acetonitrile gradients containing 0.1 % TFA and characterized by analytical HPLC and MALDI-MS. Some peptides were synthesized by pep- tides&elephants (Nuthetal, Germany) and subsequently purified and characterized as described above.

lmmunoprecipitation experiments: HEK293T cells were transfected with the indicated plasmids in 6- well plates using Metafectene (Biontex, Martinsried, Germany). Cells were incubated 24h post transfec- tion with lysis buffer (2OmM Tris pH7.5, 10OmM NaCI, 0.5mM EDTA, 0.5% NP-40, 1 % Protease inhibitor Mix G, (Serva, Heidelberg, Germany), I mM DTT) for 1 5 min on ice. After centrifugation by 1 3.000 rpm at 4°C supernatant was incubated with anti HA-specific antibodies coupled to agarose beads (Sigma) for 1 h at 4°C. After three washes with 1 ml of washing buffer (lysis buffer without protease inhibitor mix), bound material was eluted under denaturing conditions and separated on SDSPAGE gels and transferred to PVDF membranes. Viral polymerase subunits and GFP fusion proteins were detected with antibodies directed against the HA-tag (Covance, Berkeley, California) or His-tag (Qiagen) or GFP- tag (Santa Cruz Biotechnology).

Plaque reduction assay: The experiments were carried out as described by Schmidke (2001 ) with modi- fications. Confluent MDCK cells were infected with I OOPFU of A/WSN/33, B/Yamagata/73, A/KAN-] , or VSV/lndiana in PBS containing BSA at room temperature. After removal of the inoculum, cells were overlaid with medium (DMEM with 2OmM Hepes, 0.01 % DEAE Dextran, 0.001 % NaHC03) containing 1 % Oxoidagar and candidate peptides or small molecule compounds at the indicated concentrations. After incubation for 24h (VSV), 48h (A/WSN/33, A/KAN-1 ) at 37°C with 5% CO2, or 72h at 33°C with 5% CO2 (B/Yamagata/73) respectively, cells were fixed with formaldehyde and stained with crystal violet. Plaques were counted and mean plaque number of the water control was set to 100%.

Enzyme-Linked Immunosorbent Assay (ELISA): For the ELISA microwell plates (Pierce) were incubated with saturating concentrations of peptides at room temperature, washed and subsequently incubated at room temperature with HA-tagged PA. To obtain PA-HA, 293T cells were seeded into 94mm-dishes, transfected with the respective plasmid and treated with lysis buffer 24h post transfection as described in detail by Mayer et al. (2007). After washing the microwell plates, the wells were incubated with an HA-specific primary antibody (Covance), followed by three washes and an incubation with a peroxidase- coupled secondary antibody (Jackson lmmuno Research, Newmarket, UK) for further 30min. After the final wash step, ABTS-substrate (Sigma, ready-to-use solution) was added and the optical density was determined at 405nm.

The competition ELISA was carried out as described above with the exception that the candidate peptide or small molecule competitor compound were added to wells of the plate with bound peptides prior to addition of the cell extract containing HA-tagged PA subunits.

Fluorescence Polarization (FP) Assay: The test sample includes a known binding pair of proteins or protein subunits including a fluorescent label, which can be analyzed according to a preferred embodiment of the present invention by fluorescence polarization. Here, we use the interaction of Influenza A virus polymerase subunit PBl , represented by the first 25, N-terminal amino acids, and subunit PA. The test sample is then contacted with a candidate peptide or small molecule inhibitor compound and the resulting fluorescence polarization is determined. The ability of the compound to cause dissociation of or otherwise interfere with or prevent binding of the proteins or protein subunits is monitored by fluorescence polarization (FP). FP measurements allow for discrimination between fluorescently labeled bound and unbound proteins, peptides, subunits or fragments thereof. The FP of the fluorescently Ia- beled first fragment rotates rapidly in solution and, therefore, has randomized photo-selected distributions, which result in the small observed FP. When the fluorescently labeled first fragment of the first subunit interacts with the fragment of the second subunit, which is typically a larger, more slowly rotating molecule, the rotation of the fluorescently labeled first fragment slows and the fluorescence polarization increases. Accordingly, disruption of the subunit interaction by a test compound provides a de- crease in the fluorescence polarization, which is indicative of inhibition of the protein interactions. The FP measurements in the presence of a test compound can be compared with the FP measurements in the absence of the test compound. Comparison can be made manually by the operator or automatically by a computer, especially in high throughput assays using 384-well plates.

For protein purification influenza A virus polymerase subunit PA was cloned into a suitable expression vector with a C-terminally attached 6xHis-linker or hemagglutinine epitope (HA). Human 293T cells were transfected with the plasmid. Cell lysates were prepared 24 hours post transfection using lysis buffer (2OmM TrisHCI pH 7.5, 10OmM NaCI, 0.5mM EDTA, 0.5% NP-40, I mM DTT and 1 % Protase inhibitor mix) For purification from the lysate, PA subunit was bound to Ni- or anti-HA-agarose and washed with lysis buffer without protease mix. After elution with HA-peptide in 2OmM TrisHCI pH 7.5, 1 5OmM NaCI, 0.5mM EDTA, I mM DTT and 5% Glycerol, PA-protein was concentrated when necessary using Vivaspin20 5OK columns and frozen at -8O ⁰ C until further use. After thawing, the elution buffer was exchanged to low fluorescent grade reagents and any HA-peptide was removed simultaneously using 10-DC Bio-Cel columns.

Fluorescently labeled peptide corresponding to the 25 first N-terminal amino acids of Influenza A virus polymerase subunit PBl at 3 nM concentration was added to l OμM HA-PA in 2OmM TrisHCI pH 7.5, 1 5OmM NaCI, 0.5mM EDTA, I mM DTT, 5% Glycerol and l OOmg/ml bovine gamma globulin. The mix was distributed into black 384-well plates to a total volume of 20 μl per well and kept on ice. Test com- pounds solved in DMSO were added to a final concentration of 25μM. After incubation for 10 minutes at room temperature, plates were read using an Infinite F200 reader (Tecan). FP values of the wells containing test compounds were compared to wells without test compounds, without DMSO and with peptide only.

Sequence alignment: Alignments were performed with MUSCLE as described in Edgar (2004) using the full-length sequences provided from the public influenza virus database (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html).

Modelling: Manual docking of the mutated peptide into the PA(C)-PBl (N) crystal structure (He et al., 2008) and subsequent minimization was performed with Accelrys Discovery Studio.

Respiratory Syncytial Virus: Materials and Methods

Activity of compounds in reducing RSV induced cell death: HEp-2 cells (obtained from ATCC) were seeded in 96-well plates (1 .5 x 104 cells per well) and grown in MEM-alpha medium containing 10% FBS (Gibco-BRL) for 24 h. To infect cells, 500 pfu of RSV Long strain (obtained from ATCC) were added in 50 μl of OptiMEM (Gibco-BRL) for 1 h. Cells were then incubated in the presence of a serial dilution of compounds (from 100 to 0.14 μM) in MEM-alpha containing 2% FBS for 72 h. Cells were fixed in 3.7% formaldehyde and stained with 0.025% of crystal violet (Sigma). The integrity of the cellular monolayer was measured at 540 nm using a microplate reader. The activity of the compounds to reduce virus- induced cell death is expressed as the mean of three independent experiments each performed in triplicates.

Experimental Results

Protein-protein interactions (PPIs) are crucial to most, if not all, biological processes. Of the roughly 30,000 protein sequences that comprise the human proteome, only about 1 % have been successfully targeted with small-molecule drugs. Yet, most of the conventional targets in drug discovery fall into the same few structural or functional families such as enzymes or C protein-coupled receptors (CPCRs). They typically share the property that the natural substrates or ligands, with which they interact are themselves small organic molecules. Historically there has been notably little success in developing drug-like inhibitors of proteins whose natural ligands are other proteins. Designing a small molecule to bind to a protein-protein interface and inhibit the interaction poses several challenges, including the initial identification of suitable PPIs, the surface area of the interface, and the localization of "hot spots". Thus, small molecule inhibition of PPIs is a challenging area in drug discovery.

The present invention uses the fact that proteomes of many viruses and PPIs crucial for viral replication are described in the literature. For any proteome of interest, this data is according to the novel method supplemented with proteomic approaches for identification of PPIs like yeast two-hybrid or co-immuno precipitation screening in order to identify potential target regions for development of PPI inhibitors. Subsequently, a unique combination of phylogenetic analysis and structure prediction or structure analysis (where applicable) of the protein partners involved detects druggable protein-binding domains. Within the present disclosure, the term druggable denotes preferably protein-binding domains which can be blocked, altered or modified by small molecules in a way that the protein-protein interaction is inhibited or disrupted. The term small molecules denotes organic molecules, preferably synthetic organic molecules (not peptides), which have a molecular weight below 1 500, preferably below 1000 and most preferred below 500 u. It has been found, that these domains bear a couple of characteristic features: (i) helical structure, (ii) hydrophobic character and (iii) high conservation among all virus strains. It has been shown that they tend to be located at a terminal end of the protein or are located on their surface. The peptides corresponding to these potential binding domains are synthesized in an overlapping way and tested for their ability to bind the protein partner involved in the PPI. If peptides resembling short, (less than 20 amino acids) continuous binding domains are identified, these are used for the development of a binding assay, preferably an ELISA or fluorescence polarization (FP) assay, which is afterwards employed in a high-throughput screening campaign for small molecule and/or peptidic inhibitors of the PPI.

The PPI inhibitors identified by the novel method according to the present invention, as opposed to conventional active site inhibitors, could offer a particular advantage when it comes to antivirals since it should be safe to assume that resistance development occurs at a much slower pace.

In order to identify chemical compounds that efficiently interrupt or disrupt the interaction between PBl and PA, for example by binding to the inhibition target on PA of FIuA and FIuB, the competitive ELISA assay described above for the influenza peptides was repeated with a number of small molecule compounds obtained from corresponding compound libraries from Maybridge Ltd., Cambridge, UK (www.maybridge.com) and from the Developmental Therapeutics Program NCI/NIH (http://www.dtp.nci.nih.gov) of the U.S. National Institutes of Health. The tested compounds are listed in Table 4, together with their systematic name, the source and the product code, and the found activity in the ELISA assay. The corresponding structures are shown in Figure 5.

Table 4: Compounds tested with competitive ELISA

Comp. ID Compound name Source* / Active in Product code ELISA

PKE060 4-[(4-methoxybenzyl)thιo]-7-nιtro-2,l ,3-benzoxadιazole M / KM06831 Yes

PKE061 2-[(7-nιtro-2,l ,3-benzoxadιazol-4-yl)thιo]ethyl 4- M / KM06890 Yes methoxybenzene-1 -sulfonate

PKE068 4-[(4-methylphenyl)thιo]-7-nιtro-2,l ,3-benzoxadιazole M / KM06815 Yes

PKE069 N4-(4-methoxyphenyl)-7-nιtro-2,l ,3-benzoxadιazol-4-amιne M / KM06816 No

PKE070 N4-(4-fluorophenyl)-7-nιtro-2,l ,3-benzoxadιazol-4-amιne M / KM06820 No

PKE071 N4-(3-fluorophenyl)-7-nιtro-2,l ,3-benzoxadιazol-4-amιne M / KM06822 No

PKE072 4-n ιtro-7-tetra hyd ro-1 H-pyrrol-l -yl-2,l ,3-benzoxadιazole M / KM06824 No

PKE073 N4-(2-thιenyl methyl )-7-nιtro-2,l ,3-benzoxadιazol-4-amιne M / KM06825 No

PKE074 N4-(4-methyl phenyl )-7-nιtro-2,l ,3-benzoxadιazol-4-amιne M / KM06826 No

PKE075 4-[(2,4-dιchlorophenyl)thιo]-7-nιtro-2,l ,3-benzoxadιazole M / KM06828 Yes

PKE076 2-[(7-nιtro-2,l ,3-benzoxadιazol-4-yl)thιo]ethan-l -ol M / KM06833 Yes

PKE077 4-[(4-methylbenzyl)thιo]-7-nιtro-2,l ,3-benzoxadιazole M / KM06835 Yes

PKE078 N4-(3-methyl phenyl )-7-nιtro-2,l ,3-benzoxadιazol-4-amιne M / KM06836 No

PKE079 4-[(4-fluorophenyl)thιo]-7-nιtro-2,l ,3-benzoxadιazole M / KM06837 Yes

PKE080 4-[(3-chlorophenyl)thιo]-7-nιtro-2,l ,3-benzoxadιazole M / KM06838 Yes

PKE081 2-[(7-nιtro-2,l ,3-benzoxadιazol-4-yl)thιo]ethyl 2,4- M / KM06863 No dichlorobenzoate PKE082 2-[(7-nιtro-2,l,3-benzoxadιazol-4-yl)thιo]ethyl-4 M/KM06867 Yes -methoxybenzoate

PKE083 2-[(7-nιtro-2,l ,3-benzoxadιazol-4-yl)thιo]ethyl benzoate M/KM06874 No

PKEl 07 4-nιtro-7-(phenylmethylsulfanyl)-2,l,3-benzoxadιazole N/NSC228147 Yes

PKEl 08 4-nιtro-7-(phenylmethylsulfonyl)-2,l,3-benzoxadιazole N/NSC228148 Yes

PKEIlO 6-[(4-nιtro-2,l,3-benzoxadιazol-7-yl)sulfanyl]-7H-puπn-2- amιne N/NSC348401 No

PKEl 18 4-nιtro-N-phenyl-2,l,3-benzoxadιazol-7-amιne N/NSC611541 No

PKEl 19 N-(4-methoxyphenyl)-4-nιtro-2,l,3-benzoxadιazol-7-amιne N/NSC611543 No

PKEl 20 N-(4-chlorophenyl)-4-nιtro-2,l,3-benzoxadιazol-7-amιne N/NSC611544 No

PKEl 30 N-[(4-methoxyphenyl)methyl]-4-nιtro-2,l,3-benzoxadιazol-7- amιne N/NSC240872 No

PKEl 31 N-(2-methylphenyl)-4-nιtro-2,l,3-benzoxadιazol-7-amιne N/NSC611542 No

PKEl 33 4-nιtro-l-oxιdo-7-[4-(phenylmethyl)pιperazιn-l-yl]-2,l,3 - N/NSC228099 No benzoxadιazol-1-ιum

PKEl 34 7-(4-butylpιperazιn-l-yl)-4-nιtro-l-oxιdo-2,l,3-benzoxad ιazol-l-ιum N/NSC228106 No

PKEl 35 4-nιtro-7-(4-phenylpιperazιn-l-yl)-2,l,3-benzoxadιazole N/NSC288659 No

PKEl 36 N,N-dιethyl-N'-(4-nιtro-l-oxιdo-2,l,3-benzoxadιazol-l-ι um-7- N/NSC288662 No yl)propane-l ,3-dιamιne

PKEl 37 2-(hydroxymethyl)-5-[6-[(4-nιtro-2,l , 3-benzoxadιazol- N/NSC335994 Yes 7-yl)sulfanyl]puπn-9-yl]oxolane-3,4-dιol

PKEl 38 2-[2-amιno-6-[(4-nιtro-2,l,3-benzoxadιazol-7-yl) sulfanyljpuπn- N/NSC348400 Yes 9-yl]-5-(hydroxymethyl)oxolane-3,4-dιol

PKEl 39 4-nιtro-7-(7H-puπn-6-ylsulfanyl)-2,l,3-benzoxadιazole N/NSC348402 No

PKEl 40 5-[4-(tert-butyl)-l ,3-thιazol-2-yl]-2,l ,3-benzoxadιazole M/KM07316 Yes

PKEl 90 N-benzyl-4-nιtro-2,l,3-benzoxadιazol-5-amιne M/ BTBl 5221 Yes

PKE191 7-chloro-N,N-dιethyl-4-nιtro-2,l,3-benzoxadιazol-5-amιne M/ BTBl 5211 No

PKE217 8-phenylsulfanyl-6H-[l ,2,4]tπazolo[4,3-d][l ,2,4]tπazιn-5-one N/NSC360189 No

* M: Maybπdge Ltd., Cambridge, UK; N: Developmental Therapeutics Program NCI/NIH

For the compounds with positive ELISA prescreening the inhibitory concentrations (IC ₅ o) have been determined (Table 5), in a plaque reduction assay as described above for the influenza peptide studies or with a competitive ELISA assay as described above for the influenza peptide studies. In cases where the solubility was too low to reach the saturation region, the IC _5O value was calculated based on the inhibition on the maximum obtainable concentration. If an IC _5O value was not obtained, maximum ELISA inhibition at the highest concentration used (1000 μfvl) is given. Table 5: Influenza inhibitory concentrations (IC ₅ o) of compounds

IC ₅₀ [μM] IC ₅₀ [μM] Max. Inhibition

Compound ID (Plaque Red.) (ELISA) (ELISA) at lOOOμM

PKE060 1.00

PKE061 >1000 37%

PKE068 >1000 46%

PKE075 >1000 35%

PKE076 >woo 44%

PKE077 >1000 33%

PKE079 125

PKE080 60

PKE082 100

PKE107 125

PKEl 08 500

PKEl 37 200

PKEl 38 250

PKE140 >1000 34%

PKEl 90 >1000 37%

The compounds that have been found so far to be effective in binding to PA have a basic structure of 2,1 ,3,-benzoxadiazole. However, compound PKE060 has an IC _5O that is considerably lower than PKE079, PKE080, PKE082, PKEl 07, PKEl 08, PKEl 37, and PKEl 38. The IC ₅₀ of the other compounds is not sufficiently low to be physiologically acceptable.

A similar screening was carried out with the plaque reduction assay as described above, with influenza virus (A/WSN/33) and in addition also with RSV (Long strain) for the above-mentioned compounds, The results of the screening are given in Table 6, with the maximum inhibition obtained and, if deter- minable, the IC _5O value. If in an influenza pre-screening assay (competitive ELISA or other) the compound was found to be inactive or having a too high IC _5O , (IC50 ELISA > 100 μM) the influenza assay was not carried out for efficiency reasons.

The maximum inhibition of Influenza A & B activity was only determined for compounds PKE 060 and PKE 080. For compound PKE 060 the maximum inhibition was found to be 75% at 10 μM, and compound PKE 80 was found to be inactive in this assay. Table 6: Influenza and RSV inhibition of compounds

The assessed class of 2,1 ,3,-benzoxadiazole based compounds seems to be effective in the inhibition of replication of certain virus types. A number of compounds effectively inhibited the replication of influenza virus, particularly influenza A, namely compounds PKE 060, PKE 079, PKE 080, PKE 082, PKE 107, PKE 108, PKE 137, and PKE 138. Surprisingly it was found that a number of other compounds are also effective in the inhibition of RSV replication, namely compounds PKE 068, PKE 070, PKE 071 , PKE 072, PKE 073, PKE 075, PKE 078, PKE 079, PKE 080, PKE 081 , PKE 1 18, PKE 1 19, PKE 1 20, PKE 1 30, PKE 1 31 , and PKE 191 . A number of compounds inhibits the replication of both virus types.

Without being bound to any theory, it seems that the compounds according to the invention can be very effective broad band inhibitors of virus replication, and thus are a valuable source of effective new me- dicaments against certain types of the orthomyxoviridae and paramyxoviridae families.

The above-described embodiments of the present invention are intended to be examples only. Alterations, modifications and variations may be effected to the particular embodiments by those of skill in the art without departing from the scope of the invention, which is defined solely by the claims appended hereto. All references are herein incorporated by reference.

REFERENCES

1 . http://www.who.int/csr/disease/influenza/vaccinerecommendati onsl/en/.

2. Moscona, A. N Engl J Med 353, 1 363-1 373 (2005).

3. Davies, Wl. et al. Science 144, 862-863 (1 964).

4. Moscona, A. N Engl J Med 360, 953-956 (2009). 5. Dharan, NJ. et al. JAMA 301 , 1034-1041 (2009).

6. Pilger, B.D., Cui, C. & Coen, D.M. Chem Biol 1 1 , 647-654 (2004).

7. Brownlee, CC. & Sharps, Jl. J Virol 76, 7103-71 1 3 (2002).

8. Perales, B. & Ortin, J. J Virol 71 , 1 381 -1 385 (1997).

9. Fodor, E. et al. J Virol 76, 8989-9001 (2002). 10. He, X. et al. Nature 454, 1 123-1 1 26 (2008).

1 1 . Obayashi, E. et al. Nature 454, 1 1 27-1 1 31 (2008).

1 2. Chanem, A. et al. J Virol 81 , 7801 -7804 (2007).

1 3. Dostmann, W.R. et al. Proc Natl Acad Sci U S A 97, 14772-14777 (2000).

14. Chokephaibulkit, K. et al. Pediatr Infect Dis J 24, 1 62-1 66 (2005). 1 5. Norton, CP. et al. Virology 1 56, 204-21 3 (1987).

1 6. Schwemmle, M. et al., P. Virology 206, 545-554 (1995).

1 7. Mayer, D. et al. J Proteome Res 6, 672-682 (2007).

18. Pleschka, S. et al. J Virol 70, 4188-4192 (1996).

19. Schneider, U. et al., M. J Virol 77, 1 1 781 -1 1 789 (2003). 20. Schmidtke, M., et al., J Virol Methods 95, 1 33-143 (2001 ).

21 . Edgar, R.C. Nucleic Acids Res 32, 1 792-1 797 (2004).