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 or variants thereof possessing endonuclease activity, wherein said PA subunit is from a virus belonging to the Orthomyxoviridae family. 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 and pharmaceutical compositions comprising the identified compounds for the treatment of disease conditions due to viral infections caused by viruses of the Orthomyxoviridae family.

1 . 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 a virus belonging to the Orthomyxoviridae family 
     
     
         2 . The polypeptide fragment of  claim 1 , which is soluble. 
     
     
         3 . The polypeptide fragment of  claim 1 , which is crystallizable. 
     
     
         4 . The polypeptide fragment of  claim 3  which is crystallizable using a protein solution of 5 to 10 mg/ml in 20 mM Tris pH 8.0, 100 mM NaCl and 2.5 mM MnCl 2  and a reservoir solution consisting of 1.2 M Li 2 SO 4 100 mM MES pH 6.0, 10 mM magnesium acetate, and 3% ethylene glycol. 
     
     
         5 . The polypeptide fragment of  claim 1 , wherein the PA subunit is from Influenza A, B, or C virus or is a variant thereof. 
     
     
         6 . The polypeptide fragment of  claim 1 , wherein the amino terminal fragment corresponds to at least amino acids 1 to 196 of the PA subunit of the RNA-dependent RNA polymerase of Influenza A virus (SEQ ID NO: 2). 
     
     
         7 . The polypeptide fragment of  claim 1 , wherein said polypeptide fragment is purified to an extent to be suitable for crystallization. 
     
     
         8 . The polypeptide fragment of  claim 1  to which two divalent cations are bound, wherein the divalent cation is preferably manganese. 
     
     
         9 . The polypeptide fragment of  claim 1 , wherein
 (i) 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 196 to 209 of the amino acid sequence of the PA subunit according to SEQ ID NO: 2,   (ii) 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 195 to 206 of the amino acid sequence of the PA subunit according to SEQ ID NO: 4, or   (iii) 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 178 to 189 of the amino acid sequence of the PA subunit according to SEQ ID NO: 6,   and variants thereof, which retain the endonuclease activity.   
     
     
         10 . The polypeptide fragment of  claim 1  which consists of amino acids 1 to 209 of the amino acid sequence set forth in SEQ ID NO: 2 and optionally an amino-terminal linker having the amino acid sequence GMGSGMA (SEQ ID NO: 19) and which has the structure defined by the structure coordinates as shown in  FIG. 18 . 
     
     
         11 . The polypeptide fragment of  claim 1 , wherein said polypeptide fragment has a crystalline form with space group P4 3 2 1 2 and unit cell dimensions of a=b=6.71±0.2 nm, c=30.29 nm±0.4 nm. 
     
     
         12 . The polypeptide fragment of  claim 11 , wherein the crystal diffracts X-rays to a resolution of 2.5 Å or higher, preferably 2.1 Å or higher. 
     
     
         13 . An isolated polynucleotide coding for an isolated polypeptide of  claim 1 . 
     
     
         14 . A recombinant vector comprising said isolated polynucleotide of  claim 13 . 
     
     
         15 . A recombinant host cell comprising said isolated polynucleotide of  claim 13 . 
     
     
         16 . A method for identifying compounds which modulate the endonuclease activity of the PA subunit of a viral RNA-dependent RNA polymerase from the Orthomyxoviridae family, comprising the steps of
 (a) constructing a computer model of the active site defined by the structure coordinates of the polypeptide fragment of  claim 1  which consists of amino acids 1 to 209 of the amino acid sequence set forth in SEQ ID NO: 2 and optionally an amino-terminal linker having the amino acid sequence GMGSGMA (SEQ ID NO: 19) and which has the structure defined by the structure coordinates as shown in  FIG. 18 ;   (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.   
     
     
         17 . The method of  claim 16 , wherein said active site comprises amino acids corresponding to amino acids Asp108, Ile120, and Lys134 of the PA subunit according to SEQ ID NO: 2. 
     
     
         18 . The method of  claim 17 , wherein said active site further comprises amino acids corresponding to amino acids His41, Glu80, and Glu119 of the PA subunit according to SEQ ID NO: 2. 
     
     
         19 . The method of  claim 17 , wherein said active site further comprises the amino acids corresponding to amino acids Tyr24, Arg84, Leu106, Tyr130, Glu133, and Lys137 of the PA subunit according to SEQ ID NO: 2. 
     
     
         20 . The method of  claim 16 , wherein said active site is defined by the structure coordinates of the PA subunit SEQ ID NO: 2 amino acids Asp108, Ile120, and Lys134 according to  FIG. 18 . 
     
     
         21 . The method of  claim 20 , wherein said active site is further defined by the structure coordinates of the PA subunit SEQ ID NO: 2 amino acids His41, Glu80, and Glu119 according to  FIG. 18 . 
     
     
         22 . The method of  claim 20 , wherein said active site is further defined by the structure coordinates of the PA subunit SEQ ID NO: 2 amino acids Tyr24, Arg84, Leu106, Tyr130, Glu133, and Lys137 according to  FIG. 18 . 
     
     
         23 . The method of  claim 21 , wherein the active site of a PA subunit polypeptide fragment variant has a root mean square deviation from the backbone atoms of the amino acids Asp 108, Ile120, and Lys134 of said active site of not more than 2.5 Å. 
     
     
         24 . The method of any of  claim 16  comprising the further 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). 
 
     
     
         25 . The method of  claim 24  comprising the further step of
 (f) contacting said compound and said polypeptide fragment of  claim 1  and determining the ability of said compound to modulate the endonuclease activity of said PA subunit polypeptide fragment. 
 
     
     
         26 . A compound identifiable by the method of  claim 16 , wherein said compound is able to modulate the endonuclease activity of the PA subunit or variant thereof. 
     
     
         27 . A compound identifiable by the method of  claim 16 , wherein said compound is able to inhibit the endonuclease activity of the PA subunit polypeptide fragment of 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 a virus belonging to the Orthomyxoviridae family, or variant thereof. 
     
     
         28 . A method for identifying compounds which modulate the endonuclease activity of the PA subunit or polypeptide variants thereof, comprising the steps of (i) contacting said polypeptide fragment of  claim 1  with a test compound and (ii) analyzing the ability of said test compound to modulate the endonuclease activity of said PA subunit polypeptide fragment. 
     
     
         29 . The method of  claim 28 , wherein the ability of said test compound to inhibit the endonuclease activity of said PA subunit polypeptide fragment is analyzed. 
     
     
         30 . The method of  claim 28  performed in a high-throughput setting. 
     
     
         31 . The method of any of  claim 28 , wherein said test compound is a small molecule. 
     
     
         32 . The method of any of  claim 28 , wherein said test compound is a peptide or protein. 
     
     
         33 . The method of  claim 16 , 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). 
     
     
         34 . A pharmaceutical composition producible according to the method of  claim 24 . 
     
     
         35 . A compound identifiable by the method of  claim 28 , wherein said compound is able to modulate the endonuclease activity of the PA subunit or variant thereof. 
     
     
         36 . A compound identifiable by the method of  claim 28 , wherein said compound is able to inhibit the endonuclease activity of the PA subunit polypeptide fragment of 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 a virus belonging to the Orthomyxoviridae family, or a variant thereof. 
     
     
         37 . An antibody directed against the active site of the PA subunit or variant thereof. 
     
     
         38 . The antibody of  claim 37 , 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 Tyr24, His41, Glu80, Arg84, Leu106, Asp108, Glu119, Ile120, Tyr130, Glu133, Lys134, and Lys137. 
     
     
         39 . Use of a compound according to  claim 26  or a pharmaceutical composition thereof for the manufacture of a medicament for treating, ameliorating, or preventing disease conditions caused by viral infections with viruses of the Orthomyxoviridae family 
     
     
         40 . The use of  claim 39 , wherein said disease condition is caused by a virus selected from the group consisting of Influenza A virus, Influenza B virus, and Influenza C virus. 
     
     
         41 . The use of an antibody according to  claim 37  for the manufacture of a medicament for treating, ameliorating, or preventing disease conditions caused by viral infections with viruses of the Orthomyxoviridae family. 
     
     
         42 . The use of the antibody according to  claim 41 , wherein said disease condition is caused by a virus selected from the group consisting of Influenza A virus, Influenza B virus and Influenza C virus. 
     
     
         43 . The method of  claim 28 , 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). 
     
     
         44 . A pharmaceutical composition producible according to the method of  claim 33 . 
     
     
         45 . A compound identifiable by the method of  claim 33 , wherein said compound is able to modulate the endonuclease activity of the PA subunit or variant thereof. 
     
     
         46 . A compound identifiable by the method of  claim 33 , wherein said compound is able to inhibit the endonuclease activity of the PA subunit polypeptide fragment of 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 a virus belonging to the Orthomyxoviridae family, or a variant thereof. 
     
     
         47 . Use of a compound according to  claim 27  or a pharmaceutical composition thereof for the manufacture of a medicament for treating, ameliorating, or preventing disease conditions caused by viral infections with viruses of the Orthomyxoviridae family 
     
     
         48 . Use of a compound according to  claim 35 , or a pharmaceutical composition thereof for the manufacture of a medicament for treating, ameliorating, or preventing disease conditions caused by viral infections with viruses of the Orthomyxoviridae family 
     
     
         49 . Use of a compound according to  claim 36 , or a pharmaceutical composition thereof for the manufacture of a medicament for treating, ameliorating, or preventing disease conditions caused by viral infections with viruses of the Orthomyxoviridae family 
     
     
         50 . Use of a pharmaceutical composition according to  claim 34 , for the manufacture of a medicament for treating, ameliorating, or preventing disease conditions caused by viral infections with viruses of the Orthomyxoviridae family