Use of a cysteine protease of Plasmodium vivax

Abstract

A use of vivapain-4 (VX-4), which is a cysteine protease of Plasmodium vivax, showing pH-dependent switching of substrate specificity, is provided. More specifically, a method of treating a parasitic disease caused by Plasmodium vivax by inhibiting VX-4; a method of screening a protease inhibitor acting on VX-4, wherein the protease inhibitor is useful as an anti-malarial agent acting on Plasmodium species, for example, Plasmodium vivax; and a method of identifying the activity of VX-4, are provided.

1 . A method of screening a protease inhibitor acting on  Plasmodium vivax  cysteine protease (vivapain-4, SEQ ID NO: 1) comprising;
 providing  Plasmodium vivax  cysteine protease (vivapain-4, SEQ ID NO: 1) or a recombinant expression vector comprising a polynucleotide encoding the vivapain-4;   contacting the vivapain-4 to a candidate material or culturing the recombinant expression vector with a candidate material; and   examining glutamic acid at 180 th  position (Glu180) in vivapain-4 or the expressed vivapain-4, wherein the number of the amino acid position is initiated from the mature domain (SEQ ID NO: 2) of vivapain-4,   wherein the candidate material is determined as the anti-malarial agent against  Plasmodium vivax,  when Glu180 is deleted, substituted with an amino acid other than glutamate, or inactivated, in the vivapain-4 or the expressed vivapain-4 from the recombinant expression vector.   
     
     
         2 . The method of  claim 1 , wherein the amino acid other than glutamate is a basic or hydrophobic amino acid. 
     
     
         3 . The method of  claim 1 , wherein the amino acid other than glutamate is an amino acid selected from the group consisting of arginine, histidine, lysine, alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophane, valine, and cysteine. 
     
     
         4 . The method of  claim 1 , wherein the protease inhibitor has an anti-malarial activity. 
     
     
         5 . A method of screening a protease inhibitor acting on  Plasmodium vivax  cysteine protease (vivapain-4, SEQ ID NO: 1) comprising:
 providing  Plasmodium vivax  cysteine protease (vivapain-4, SEQ ID NO: 1) or a recombinant expression vector comprising a polynucleotide encoding the vivapain-4;   contacting the vivapain-4 to a candidate material or culturing the recombinant expression vector with a candidate material;   measuring at least one selected from the group consisting of:   (a) the degrading activity of the vivapain-4 or the expressed vivapain-4 from the recombinant expression vector against a dipeptidyl substrate favored by cathepsin L at pH 4.5 to 6.5, wherein the dipeptidyl substrate has at least one hydrophobic amino acid;   (b) the degrading activity of the vivapain-4 or the expressed vivapain-4 from the recombinant expression vector against a dipeptidyl substrate favored by cathepsin B at pH 6.6 to 9, wherein the dipeptidyl substrate has at least one hydrophilic amino acid;   (c) the degrading activity of the vivapain-4 or the expressed vivapain-4 from the recombinant expression vector against actin at pH 6.8 to 7.2;   (d) the degrading activity of the vivapain-4 or the expressed vivapain-4 from the recombinant expression vector against hemoglobin at pH 5.5 to 6; and   (e) the cleavage activity of the vivapain-4 or the expressed vivapain-4 from the recombinant expression vector against  P. vivax  plasmepsin at pH 5-7; and   comparing the activity of the case with the candidate material treatment with that of the case without the candidate material treatment,   wherein the candidate material is determined as the protease inhibitor, when the at least one activity of the case of the candidate material treatment is decreased compared with that of the case without the candidate material treatment.   
     
     
         6 . The method of  claim 5 , wherein the hydrophobic amino acid of (a) is selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophane, valine, and cysteine, 
     
     
         7 . The method of  claim 5 , wherein the hydrophilic amino acid of (b) is selected from aspartic acid, glutamic acid, arginine, histidine, and lysine. 
     
     
         8 . The method of  claim 5 , wherein the dipeptidyl substrate of (a) is benzyloxycarbonyl-L-phenylalanyl-L-arginine 4-methyl-coumaryl-7-amide (Z-Phe-Arg-MCA, or benzyloxycarbonyl-leucyl-L-arginine 4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA). 
     
     
         9 . The method of  claim 5 , wherein the dipeptidyl substrate of (b) is benzyloxycarbonyl-L-arginyl-L-arginine 4-methyl-coumaryl-7-amide (Z-RR-MCA). 
     
     
         10 . The method of  claim 5 , wherein the protease inhibitor has an anti-malarial activity. 
     
     
         11 . A method of identifying the activity of  Plasmodium vivax  cysteine protease (vivapain-4, SEQ ID NO: 1) comprising at least one step of:
 (a) measuring the degrading activity of the vivapain-4 or the expressed vivapain-4 from the recombinant expression vector against a dipeptidyl substrate favored by cathepsin L at pH 4.5 to 6.5, wherein the dipeptidyl substrate has at least one hydrophobic amino acid;   (b) measuring the degrading activity of the vivapain-4 or the expressed vivapain-4 from the recombinant expression vector against a dipeptidyl substrate favored by cathepsin B at pH 6.6 to 9, wherein the dipeptidyl substrate has at least one hydrophilic amino acid;   (c) measuring the degrading activity of the vivapain-4 or the expressed vivapain-4 from the recombinant expression vector against actin at pH 6.8 to 7.2;   (d) measuring the degrading activity of the vivapain-4 or the expressed vivapain-4 from the recombinant expression vector against hemoglobin at pH 5.5 to 6; and   (e) measuring the cleavage activity of the vivapain-4 against plasmepsin at pH 5-7, and   (f) identifying the presence of glutamic acid at 180 th  position (Glu180) in vivapain-4, wherein the number of the amino acid position is initiated from the mature domain (SEQ ID NO: 2) of vivapain-4.   
     
     
         12 . The method of  claim 11 , wherein the hydrophobic amino acid of (a) is selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophane, valine, and cysteine, 
     
     
         13 . The method of  claim 11 , wherein the hydrophilic amino acid of (b) is selected from aspartic acid, glutamic acid, arginine, histidine, and lysine. 
     
     
         14 . The method of  claim 11 , wherein the dipeptidyl substrate of (a) is benzyloxycarbonyl-L-phenylalanyl-L-arginine 4-methyl-coumaryl-7-amide (Z-Phe-Arg-MCA) or benzyloxycarbonyl-leucyl-L-arginine 4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA). 
     
     
         15 . The method of  claim 11 , wherein the dipeptidyl substrate of (b) is benzyloxycarbonyl-L-arginyl-L-arginine 4-methyl-coumaryl-7-amide (Z-RR-MCA). 
     
     
         16 . A method of treating malaria caused by  Plasmodium vivax,  comprising:
 administering a therapeutically effective amount of an inhibitor against  Plasmodium vivax  cysteine protease (vivapain-4, SEQ ID NO: 1) to a patient in need of the treatment,   wherein the inhibitor is capable of   inhibiting the expression of glutamic acid at 180 th  position (Glu180) in vivapain-4, substituting the Glu180 with an amino acid other than glutamate, or inactivating the Glu180, wherein the number of the amino acid position is initiated from the mature domain (SEQ ID NO: 2) of vivapain-4 or   decreasing at least one selected from the group consisting of:   (a) the degrading activity of the vivapain-4 or the expressed vivapain-4 from the recombinant expression vector against a dipeptidyl substrate favored by cathepsin L at pH 4.5 to 6.5, wherein the dipeptidyl substrate has at least one hydrophobic amino acid;   (b) the degrading activity of the vivapain-4 or the expressed vivapain-4 from the recombinant expression vector against a dipeptidyl substrate favored by cathepsin B at pH 6.6 to 9, wherein the dipeptidyl substrate has at least one hydrophilic amino acid;   (c) the degrading activity of the vivapain-4 or the expressed vivapain-4 from the recombinant expression vector against actin at pH 6.8 to 7.2;   (d) the degrading activity of the vivapain-4 or the expressed vivapain-4 from the recombinant expression vector against hemoglobin at pH 5.5 to 6; and   (e) the cleavage activity of the vivapain-4 or the expressed vivapain-4 from the recombinant expression vector against  P. vivax  plasmepsin at pH 5-7.   
     
     
         17 . The method of  claim 16 , wherein the inhibitor is capable of substituting the Glu180 with a basic or hydrophobic amino acid. 
     
     
         18 . The method of  claim 16 , wherein the inhibitor is capable of substituting the Glu180 with an amino acid selected from the group consisting of arginine, histidine, lysine, alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophane, valine, and cysteine. 
     
     
         19 . The method of  claim 16 , wherein the hydrophobic amino acid of (a) is selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophane, valine, and cysteine, 
     
     
         20 . The method of  claim 16 , wherein the hydrophilic amino acid of (b) is selected from aspartic acid, glutamic acid, arginine, histidine, and lysine. 
     
     
         21 . A cysteine protease having a modified amino acid sequence of SEQ ID NO: 2, wherein glutamic acid at 180 th  position (Glu180) is deleted, or substituted with an amino acid selected from the group consisting of arginine, histidine, lysine, alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophane, valine, and cysteine.