Influenza a 2009 pandemic h1n1 polypeptide fragments comprising endonuclease activity and their use

Abstract

The present invention relates to polypeptide fragments comprising an amino-terminal fragment of the PA subunit of a viral RNA-dependent RNA polymerase possessing endonuclease activity, wherein said PA subunit is from Influenza A 2009 pandemic H1N1 virus or is a variant thereof. This invention also relates to (i) crystals of the polypeptide fragments which are suitable for structure determination of said polypeptide fragments using X-ray crystallography and (ii) computational methods using the structural coordinates of said polypeptide to screen for and design compounds that modulate, preferably inhibit the endonucleolytically active site within the polypeptide fragment. In addition, this invention relates to methods identifying compounds that bind to the PA polypeptide fragments possessing endonuclease activity and preferably inhibit said endonucleolytic activity, preferably in a high throughput setting. This invention also relates to compounds which are able to modulate, preferably to inhibit, the endonuclease activity of the PA subunit polypeptide fragment or variant thereof of the present invention and pharmaceutical compositions comprising said compounds for the treatment of disease conditions caused by viral infections with viruses of the Orthomyxoviridae family, Bunyaviridae family and/or Arenviridae family, preferably caused by viral infections with Influenza A 2009 pandemic H1N1 virus. Preferably, said compounds are identifiable by the methods disclosed herein or said pharmaceutical compositions are producible by the methods disclosed herein.

1 - 45 . (canceled) 
     
     
         46 . A polypeptide fragment comprising an amino-terminal fragment of the PA subunit of a viral RNA-dependent RNA polymerase possessing endonuclease activity, wherein said PA subunit is from Influenza A 2009 pandemic H1N1 virus according to SEQ ID NO: 2 or is a variant thereof, wherein said variant comprises the amino acid serine at an amino acid position 186 according to SEQ ID NO: 2 or at an amino acid position corresponding thereto. 
     
     
         47 . The polypeptide fragment of  claim 46 , which is soluble. 
     
     
         48 . The polypeptide fragment of  claim 46 , which is crystallizable. 
     
     
         49 . The polypeptide fragment of  claim 46 , wherein the amino terminal fragment corresponds to at least amino acids 1 to 190 of the PA subunit of the RNA-dependent RNA polymerase of Influenza A 2009 pandemic H1N1 virus according to SEQ ID NO: 2. 
     
     
         50 . The polypeptide fragment of  claim 46 , wherein said polypeptide fragment is purified to an extent to be suitable for crystallization. 
     
     
         51 . The polypeptide fragment of  claim 46  to which two divalent cations are bound, wherein the divalent cation is preferably manganese and/or magnesium. 
     
     
         52 . The polypeptide fragment of  claim 46 , wherein the N-terminus is identical to or corresponds to amino acid position 1 and the C-terminus is identical to or corresponds to an amino acid at a position selected from positions 190 to 198 of the amino acid sequence of the PA subunit according to SEQ ID NO: 2, or is a variant thereof, wherein said variant comprises the amino acid serine at an amino acid position 186 according to SEQ ID NO: 2 or at an amino acid position corresponding thereto and retains the endonuclease activity. 
     
     
         53 . The polypeptide fragment of  claim 46 , which
 (a) consists of amino acids 1 to 198 of the amino acid sequence set forth in SEQ ID NO: 2 and optionally of an amino-terminal linker having the amino acid sequence MGSGMA (SEQ ID NO: 3) and which has the structure defined by (i) the structure coordinates as shown in  FIG. 1 , (ii) the structure coordinates as shown in  FIG. 2 , (iii) the structure coordinates as shown in  FIG. 3 , (iv) the structure coordinates as shown in  FIG. 4 , or (v) the structure coordinates as shown in  FIG. 5 , or   (b) consists of amino acids 1 to 198 of the amino acid sequence set forth in SEQ ID NO: 2 with amino acids 52 to 64 replaced by the amino acid glycine and optionally of an amino-terminal linker having the amino acid sequence MGSGMA (SEQ ID NO: 3) and which has the structure defined by (vi) the structure coordinates as shown in  FIG. 15 , or (vii) the structure coordinates as shown in  FIG. 16 .   
     
     
         54 . The polypeptide fragment of  claim 53 , wherein the polypeptide fragment having the structure defined by (i) has a crystalline form with space group C2 and unit cell dimensions of a=26.36 nm±0.5 nm, b=6.62 nm±0.3 nm, c=6.63 nm±0.3 nm, α=90 deg, β=96±2 deg. γ=90 deg, (ii) to (v) has a crystalline form with space group P2 1 2 1 2 1  and unit cell dimensions of a=5.46±0.3 nm, b=12.25±0.4 nm, c=13.0±0.3 nm, α=90 deg, β=90 deg, γ=90 deg, (vi) has a crystalline form with space group P6 2 22 and unit cell dimensions of a=7.50 nm±0.3 nm, b=7.50 nm±0.3 nm, c=12.00 nm±0.5 nm, α=90 deg, β=90 deg, γ=120 deg, or (vii) has a crystalline form with space group P6 4 22 and unit cell dimensions of a=9.99 nm±0.5 nm, b=9.99 nm±0.5 nm, c=8.27 nm±0.3 nm, α=90 deg, β=90 deg, γ=120 deg. 
     
     
         55 . The polypeptide fragment of  claim 53 , wherein the crystal diffracts X-rays to a resolution of 2.6 Å, 2.1 Å, or 1.9 Å or higher. 
     
     
         56 . An isolated polynucleotide coding for an isolated polypeptide fragment of  claim 46 . 
     
     
         57 . A recombinant vector comprising said isolated polynucleotide of  claim 56 . 
     
     
         58 . A recombinant host cell comprising said isolated polynucleotide of  claim 56 . 
     
     
         59 . A method for identifying compounds, which modulate the endonuclease activity of the PA subunit of a RNA-dependent RNA polymerase from Influenza A 2009 pandemic H1N1 virus or a variant thereof comprising the steps of:
 (a) constructing a computer model of the active site defined by (i) the structure coordinates of the polypeptide fragment of  claim 53  as shown in  FIG. 1 , (ii) the structure coordinates of the polypeptide fragment of  claim 53  as shown in  FIG. 2 , (iii) the structure coordinates of the polypeptide fragment of  claim 53  as shown in  FIG. 3 , (iv) the structure coordinates of the polypeptide fragment of  claim 53  as shown in  FIG. 4 , (v) the structure coordinates of the polypeptide fragment of  claim 53  as shown in  FIG. 5 , (vi) the structure coordinates of the polypeptide fragment of  claim 53  as shown in  FIG. 15 , or (vii) the structure coordinates of the polypeptide fragment of  claim 53  as shown in  FIG. 16 ,   (b) selecting a potential modulating compound by a method selected from the group consisting of:
 (i) assembling molecular fragments into said compound, 
 (ii) selecting a compound from a small molecule database, and 
 (iii) de novo ligand design of said compound; 
   (c) employing computational means to perform a fitting program operation between computer models of the said compound and the said active site in order to provide an energy-minimized configuration of the said compound in the active site; and   (d) evaluating the results of said fitting operation to quantify the association between the said compound and the active site model, whereby evaluating the ability of said compound to associate with the said active site.   
     
     
         60 . The method of  claim 59 , wherein said active site comprises amino acids Glu80, Glu119, Asp108, Ile120, and His41 of the PA subunit according to SEQ ID NO: 2 or amino acids corresponding thereto. 
     
     
         61 . The method of  claim 60 , wherein said active site further comprises amino acids Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, and Arg124 of the PA subunit according to SEQ ID NO: 2 or amino acids corresponding thereto. 
     
     
         62 . The method of  claim 60 , wherein said active site further comprises amino acids Glu26, Lys134, Leu106, and Lys137 of the PA subunit according to SEQ ID NO: 2 or amino acids corresponding thereto. 
     
     
         63 . The method of  claim 59 , wherein said active site is defined by the structure coordinates of the PA subunit SEQ ED NO: 2 amino acids Glu80, Glu119, Asp108, Ile120, and His41 according to  FIGS. 1 to 5  or by the structure coordinates of the PA subunit amino acids Glu, Glu, Asp, Ile, and His corresponding to amino acids Glu80, Glu119, Asp108, Ile120, and His41 of SEQ ID NO: 2, respectively, according to  FIG. 15 or 16 . 
     
     
         64 . The method of  claim 63 , wherein said active site is further defined by the structure coordinates of the PA subunit SEQ ID NO: 2 amino acids Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, and Arg124 according to  FIGS. 1 to 5  or by the structure coordinates of the PA subunit amino acids Lys, Tyr, Arg, Phe, Tyr, Ile, and Arg corresponding to amino acids Lys34, Tyr24, Arg84, Phe105, Tyr130, Ile38, and Arg124 of SEQ ID NO: 2, respectively, according to  FIG. 15 or 16 . 
     
     
         65 . The method of  claim 63 , wherein said active site is further defined by the structure coordinates of the PA subunit SEQ ID NO: 2 amino acids Glu26, Lys134, Leu106, and Lys137 according to  FIGS. 1 to 5  or by the structure coordinates of the PA subunit amino acids Glu, Lys, Leu, and Lys corresponding to amino acids Glu26, Lys134, Leu106, and Lys137 of SEQ ID NO: 2, respectively, according to  FIG. 15 or 16 . 
     
     
         66 . The method of  claim 59  further comprising the step of:
 (e) synthesizing said compound and optionally formulating said compound or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable excipient(s) and/or carrier(s). 
 
     
     
         67 . The method of  claim 66  further comprising the step of:
 (f) contacting said compound and said polypeptide fragment or variant thereof of  claim 46  or said recombinant host cell according to  claim 58  and determine the ability of said compound to modulate the endonuclease activity of said PA subunit polypeptide fragment or variant thereof. 
 
     
     
         68 . A method for identifying compounds, which modulate the endonuclease activity of the PA subunit of a RNA-dependent RNA polymerase from Influenza A 2009 pandemic H1N1 virus or a variant thereof comprising the steps of:
 (i) contacting said polypeptide fragment or variant thereof of  claim 46  or said recombinant host cell of  claim 58  with a test compound, and   (ii) analyzing the ability of said test compound to modulate the endonuclease activity of said PA subunit polypeptide fragment or variant thereof.   
     
     
         69 . The method of  claim 68 , wherein the ability of said test compound to inhibit the endonuclease activity of said PA subunit polypeptide fragment or variant thereof is analyzed. 
     
     
         70 . The method of  claim 68 , wherein said method is performed in a high-throughput setting. 
     
     
         71 . The method of  claim 68 , wherein said test compound is a small molecule. 
     
     
         72 . The method of  claim 68 , wherein said test compound is a peptide or protein. 
     
     
         73 . The method of  claim 68 , wherein said method further comprises the step of formulating said compound or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable excipient(s) and/or carrier(s). 
     
     
         74 . A compound which is able to modulate, preferably to inhibit, the endonuclease activity of the PA subunit polypeptide fragment or variant thereof of  claim 46 . 
     
     
         75 . The compound of  claim 74 , wherein said compound is identifiable by the method of  claim 59  or by the method of  claim 68 . 
     
     
         76 . A pharmaceutical composition comprising the compound of  claim 74  or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipient(s) and/or carrier(s). 
     
     
         77 . The pharmaceutical composition of  claim 76 , wherein said pharmaceutical composition is producible according to the method of  claim 66  or  73 . 
     
     
         78 . An antibody directed against the active site of the PA subunit of the Influenza A 2009 pandemic H1N1 virus according to SEQ ID NO: 2 or a variant thereof, wherein said variant comprises the amino acid serine at an amino acid position 186 according to SEQ ED NO: 2 or at an amino acid position corresponding thereto. 
     
     
         79 . The antibody of  claim 78 , wherein said antibody recognizes a polypeptide fragment of a length between 5 and 15 amino acids of the amino acid sequence as set forth in SEQ ID NO: 2, wherein the polypeptide fragment comprises one or more amino acid residues selected from the group consisting of Lys34, Glu26, Ile38, Tyr24, His41, Glu80, Arg84, Leu106, Asp108, Glu119, Ile20, Tyr130, Lys134, Phe105, Lys137, and Arg124. 
     
     
         80 . The method of using a compound according to  claim 74 , a pharmaceutical composition according to  claim 76  or an antibody according to  claim 78  for the manufacture of a medicament for treating, ameliorating, or preventing disease conditions caused by viral infections with viruses of the Orthomyxoviridae family, Bunyaviridae family and/or Arenviridae family, preferably caused by viral infections with Influenza A 2009 pandemic H1N1 virus. 
     
     
         81 . The method of using 4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethyl)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoic acid (EMBL-R05-3), 4-[4-[(4-chlorophenyl)methyl]-1-(cyclohexylmethyl)-4-piperidinyl]-2-hydroxy-4-oxo-2-butenoic acid (EMBL-R05-2), 4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethylsulpho)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoic acid (EMBL-R05-1), or [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl]3,4,5-trihydroxybenzoate (EGCG) for the manufacture of a medicament for treating, ameliorating, or preventing disease conditions caused by viral infections with Influenza A 2009 pandemic H1N1 virus. 
     
     
         82 . A compound according to  claim 74 , a pharmaceutical composition according to  claim 76  or an antibody according to  claim 78  for treating, ameliorating, or preventing disease conditions caused by viral infections with viruses of the Orthomyxoviridae family, Bunyaviridae family and/or Arenviridae family, preferably caused by viral infections with Influenza A 2009 pandemic H1N1 virus. 
     
     
         83 . A compound comprising 4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethyl)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoic acid (EMBL-R05-3), 4-[4-[(4-chlorophenyl)methyl]-1-(cyclohexylmethyl)-4-piperidinyl]-2-hydroxy-4-oxo-2-butenoic acid (EMBL-R05-2), 4-[3-[(4-chlorophenyl)methyl]-1-(phenylmethylsulpho)-3-piperidinyl]-2-hydroxy-4-oxo-2-butenoic acid (EMBL-R05-1), or [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl]3,4,5-trihydroxybenzoate (EGCG) for treating, ameliorating, or preventing disease conditions caused by viral infections with Influenza A 2009 pandemic H1N1 virus.