Inhibitors of human plasmin derived from the kunitz domains

Abstract

This invention provides: novel proteins, which are homologous to the first Kunitz domain (K1) of lipoprotein-associated coagulation inhibitor (LACI), and which are capable of inhibiting plasmin; uses of such novel proteins in therapeutic, diagnostic, and clinical methods; and polynucleotides that encode such novel proteins.

1 .- 11 . (canceled) 
     
     
         12 . A method of treating or preventing a condition selected from the group consisting of inappropriate fibrinolysis, inappropriate fibrinogenolysis, excessive bleeding associated with thrombolytics, post-operative bleeding and inappropriate angiogenesis, comprising administering, to a human or other animal subject, an inhibitory amount of a protein comprising a Kunitz domain having the formula:
 Xaa1-Xaa2-Xaa3-Xaa4-Cys-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Gly-Xaa13-Cys-Xaa15 -Xaa16-Xaa17-Xaa18-Xaa19-Arg-Xaa21-Xaa22-Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Cys-Xaa31-Xaa32-Phe-Xaa34-Xaa35-Xaa36-Gly-Cys-Xaa39-Xaa40-Xaa41-Xaa42-Xaa43-Xaa44-Xaa45- Xaa46-Xaa47-Xaa48-Xaa49-Xaa50-Cys-Xaa52-Xaa53-Xaa54-Cys-Xaa56-Xaa57-Xaa58, wherein   Xaa1, Xaa2, Xaa3, Xaa4, Xaa56, Xaa57 or Xaa58 may be absent;   Xaa10 is selected from Asp, Glu, Tyr and Gln;   Xaa11 is selected from Thr, Ala, Ser, Val and Asp;   Xaa13 is selected from Pro, Leu and Ala;   Xaa15 is selected from Lys and Arg;   Xaa16 is selected from Ala and Gly;   Xaa17 is selected from Arg, Lys and Ser;   Xaa18 is selected from Phe and Ile;   Xaa19 is selected from Glu, Gln, Asp, Pro, Gly, Ser and Ile;   Xaa21 is selected from Phe, Tyr and Trp;   Xaa22 is selected from Tyr and Phe;   Xaa23 is selected from Tyr or Phe;   Xaa31 is selected from Asp, Glu, Thr, Val, Gln and Ala;   Xaa32 is selected from Thr, Ala, Glu, Pro and Gln;   Xaa34 is selected from Val, Ile, Thr, Leu, Phe, Tyr, His, Asp, Ala and Ser;   Xaa35 is selected from Tyr and Trp;   Xaa36 is selected from Gly and Ser;   Xaa39 is selected from Glu, Gly, Asp, Arg, Ala, Gln, Leu, Lys and Met;   Xaa40 is selected from Gly and Ala;   Xaa43 is selected from Asn and Gly; and   Xaa45 is selected from Phe and Tyr.   
     
     
         13 . The method of  claim 12 , wherein:
 Xaa10 is selected from Asp and Glu;   Xaa11 is selected from Thr, Ala and Ser;   Xaa13 is Pro;   Xaa15 is Arg;   Xaa16 is Ala;   Xaa17 is Arg;   Xaa18 is Phe;   Xaa19 is selected from Glu and Asp;   Xaa21 is selected from Phe and Trp;   Xaa22 is selected from Tyr and Phe;   Xaa23 is selected from Tyr or Phe;   Xaa31 is selected from Asp and Glu;   Xaa32 is selected from Thr, Ala and Glu;   Xaa34 is selected from Val, Ile and Thr;   Xaa35 is Tyr;   Xaa36 is Gly;   Xaa39 is selected from Glu, Gly and Asp;   Xaa40 is selected from Gly and Ala;   Xaa43 is selected from Asn and Gly; and   Xaa45 is selected from Phe and Tyr.   
     
     
         14 . The method of  claim 12 , wherein the protein comprises a Kunitz domain comprising the formula: 
       
         
           
                 
               
                   (SEQ ID NO: 150) 
                 
                   Met-His-Ser-Phe-Cys-Ala-Phe-Lys-Ala-Xaa10-Xaa11- 
                 
                     
                 
                   Gly-Xaa13-Cys-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Arg- 
                 
                     
                 
                   Trp-Xaa22-Xaa23-Asn-Ile-Phe-Thr-Arg-Gln-Cys- 
                 
                     
                 
                   Xaa31-Xaa32-Phe-Xaa34-Xaa35-Gly-Gly-Cys-Xaa39- 
                 
                     
                 
                   Xaa40-Asn-Gln-Xaa43-Arg-Xaa45-Glu-Ser-Leu-Glu- 
                 
                     
                 
                   Glu-Cys-Lys-Lys-Met-Cys-Thr-Arg-Asp, 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         wherein 
         Xaa10 is selected from the group consisting of Asp, Glu, and Tyr; 
         Xaa11 is selected from the group consisting of Thr, Ala, Ser, Val, and Asp; 
         Xaa13 is selected from the group consisting of Pro, Leu, and Ala; 
         Xaa15 is selected from the group consisting of Arg and Lys; 
         Xaa16 is selected from the group consisting of Ala and Gly; 
         Xaa17 is selected from the group consisting of Arg, Lys, and Ser; 
         Xaa18 is selected from the group consisting of Phe and Ile; 
         Xaa19 is selected from the group consisting of Glu, Asp, Pro, Gly, Ser, and Ile; 
         Xaa22 is selected from the group consisting of Tyr and Phe; 
         Xaa23 is selected from the group consisting of Tyr and Phe; 
         Xaa31 is selected from the group consisting of Asp, Glu, Thr, Val, Gln, and Ala; 
         Xaa32 is selected from the group consisting of Thr, Ala, Glu, Pro, and Gln; 
         Xaa34 is selected from the group consisting of Val, Be, Thr, Leu, Phe, Tyr, His, Asp, Ala, and Ser; 
         Xaa35 is selected from the group consisting of Tyr and Trp; 
         Xaa39 is selected from the group consisting of Glu, Gly, Asp, Arg, Ala, Gln, Leu, Lys, and Met; 
         Xaa40 is selected from the group consisting of Gly and Ala; 
         Xaa43 is selected from the group consisting of Asn and Gly; and 
         Xaa45 is selected from the group consisting of Phe and Tyr. 
       
     
     
         15 . The method of  claim 12 , wherein the protein comprises a Kunitz domain comprising the formula: 
       
         
           
                 
               
                   (SEQ ID NO: 150) 
                 
                   Met-His-Ser-Phe-Cys-Ala-Phe-Lys-Ala-Xaa10-Xaa11- 
                 
                     
                 
                   Gly-Xaa13-Cys-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Arg- 
                 
                     
                 
                   Trp-Xaa22-Xaa23-Asn-Ile-Phe-Thr-Arg-Gln-Cys- 
                 
                     
                 
                   Xaa31-Xaa32-Phe-Xaa34-Xaa35-Gly-Gly-Cys-Xaa39- 
                 
                     
                 
                   Xaa40-Asn-Gln-Xaa43-Arg-Xaa45-Glu-Ser-Leu-Glu- 
                 
                     
                 
                   Glu-Cys-Lys-Lys-Met-Cys-Thr-Arg-Asp, 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         wherein 
         Xaa10 is selected from the group consisting of Asp and Glu; 
         Xaa11 is selected from the group consisting of Thr, Ala, Ser, Val, and Asp; 
         Xaa13 is selected from the group consisting of Pro, Leu, and Ala; 
         Xaa15 is selected from the group consisting of Arg and Lys; 
         Xaa16 is selected from the group consisting of Ala and Gly; 
         Xaa17 is selected from the group consisting of Arg, Lys, and Ser; 
         Xaa18 is selected from the group consisting of Phe and Be; 
         Xaa19 is selected from the group consisting of Glu, Asp, Pro, Gly, Ser, and Ile; 
         Xaa22 is Phe; 
         Xaa23 is Phe; 
         Xaa31 is selected from the group consisting of Asp, Glu, Thr, Val, Gln, and Ala; 
         Xaa32 is selected from the group consisting of Thr, Ala, Glu, Pro, and Gln; 
         Xaa34 is selected from the group consisting of Val, Be, Thr, Leu, Phe, Tyr, His, Asp, Ala, and Ser; 
         Xaa35 is selected from the group consisting of Tyr and Trp; 
         Xaa39 is selected from the group consisting of Glu, Gly, Asp, Arg, Ala, Gln, Leu, Lys, and Met; 
         Xaa40 is selected from the group consisting of Gly and Ala; 
         Xaa43 is selected from the group consisting of Asn and Gly; and 
         Xaa45 is selected from the group consisting of Phe and Tyr. 
       
     
     
         16 . The method of  claim 12 , wherein Xaa15 is Arg, Xaa16 is Ala, Xaa17 is Arg, and Xaa18 is Phe. 
     
     
         17 . The method of  claim 12 , wherein Xaa13 is Leu. 
     
     
         18 . The method of  claim 12 , wherein Xaa16 is Gly. 
     
     
         19 . The method of  claim 12 , wherein Xaa17 is Ser. 
     
     
         20 . The method of  claim 12 , wherein Xaa18 is Phe. 
     
     
         21 . The method of  claim 12 , wherein Xaa19 is Glu or Gln. 
     
     
         22 . The method of  claim 12 , wherein Xaa31 is Gln. 
     
     
         23 . The method of  claim 12 , wherein Xaa32 is Gln. 
     
     
         24 . The method of  claim 12 , wherein the protein comprises a Kunitz domain having a sequence selected from the group consisting of:
 SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:57, SEQ ID NO:4, SEQ ID NO:11, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:39, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:125, and SEQ ID NO:126.   
     
     
         25 . The method of  claim 12 , wherein the protein comprises a Kunitz domain having a sequence selected from the group consisting of:
 SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:57, SEQ ID NO:4, SEQ ID NO:11.   
     
     
         26 . The method of  claim 12 , wherein the sequence of the Kunitz domain is identical at residues 10-21 and 31-39 and has five or fewer differences at residues 5-9, 22-30, and 40-55 as compared to a reference sequence selected from the group comprising
 SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:57, SEQ ID NO:4, and SEQ ID NO:11.   
     
     
         27 . The method of  claim 12 , wherein the protein comprises a Kunitz domain having a sequence selected from the group consisting of:
 SEQ ID NO:40; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49; SEQ ID NO:50; SEQ ID NO:51; SEQ ID NO:55; SEQ ID NO:58; SEQ ID NO:117; SEQ ID NO:118; and SEQ ID NO:119.   
     
     
         28 . The method of  claim 12 , wherein the protein comprises a Kunitz domain having a sequence selected from the group consisting of:
 SEQ ID NO:40; SEQ ID NO:117; SEQ ID NO:118; and SEQ ID NO:119.   
     
     
         29 . The method of  claim 12 , wherein the protein comprises a Kunitz domain having the sequence of SEQ ID NO:40. 
     
     
         30 . The method of  claim 12 , wherein the protein inhibits human plasmin with a K i  of 100 pM or less. 
     
     
         31 . The method of  claim 12 , wherein the protein inhibits human plasmin with a K i  of 300 pM or less.