Butyrylcholinesterase variant polypeptides with increased catalytic efficiency and methods of use

Abstract

The invention provides a butyrylcholinesterase variant having increased cocaine hydrolysis activity as well as the corresponding encoding nucleic acid. The invention further provides methods of hydrolyzing a cocaine-based butyrylcholinesterase substrate as well as methods of treating a cocaine-induced condition with the invention variant.

1 - 12 . (canceled)  
     
     
         13 . A butyrylcholinesterase variant polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42.  
     
     
         14 . The butyrylcholinesterase variant polypeptide of  claim 13 , wherein said amino acid sequence is SEQ ID NO: 22.  
     
     
         15 . The butyrylcholinesterase variant polypeptide of  claim 13 , wherein said amino acid sequence is SEQ ID NO: 24.  
     
     
         16 . The butyrylcholinesterase variant polypeptide of  claim 13 , wherein said amino acid sequence is SEQ ID NO: 26.  
     
     
         17 . The butyrylcholinesterase variant polypeptide of  claim 13 , wherein said amino acid sequence is SEQ ID NO: 28.  
     
     
         18 . The butyrylcholinesterase variant polypeptide of  claim 13 , wherein said amino acid sequence is SEQ ID NO: 30.  
     
     
         19 . The butyrylcholinesterase variant polypeptide of  claim 13 , wherein said amino acid sequence is SEQ ID NO: 32.  
     
     
         20 . The butyrylcholinesterase variant polypeptide of  claim 13 , wherein said amino acid sequence is SEQ ID NO: 34.  
     
     
         21 . The butyrylcholinesterase variant polypeptide of  claim 13 , wherein said amino acid sequence is SEQ ID NO: 36.  
     
     
         22 . The butyrylcholinesterase variant polypeptide of  claim 13 , wherein said amino acid sequence is SEQ ID NO: 38.  
     
     
         23 . The butyrylcholinesterase variant polypeptide of  claim 13 , wherein said amino acid sequence is SEQ ID NO: 40.  
     
     
         24 . The butyrylcholinesterase variant polypeptide of  claim 13 , wherein said amino acid sequence is SEQ ID NO: 42.  
     
     
         25 . A nucleic acid encoding a butyrylcholinesterase variant polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42.  
     
     
         26 . A nucleic acid encoding a butyrylcholinesterase variant polypeptide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41.  
     
     
         27 . A method of treating a cocaine-induced condition comprising administering to an individual an effective amount of a butyrylcholinesterase variant polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42, exhibiting increased cocaine hydrolysis activity compared to butyrylcholinesterase.  
     
     
         28 . The method of  claim 27 , wherein said cocaine-based substance is cocaine.  
     
     
         29 . The method of  claim 27 , wherein said individual is symptomatic of a cocaine-overdose.  
     
     
         30 . The method of  claim 27 , wherein said individual is symptomatic of cocaine addiction.  
     
     
         31 . A method of hydrolyzing a cocaine-based butyrylcholinesterase substrate comprising contacting said butyrylcholinesterase substrate with a butyrylcholinesterase variant polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42, under conditions that allow hydrolysis of cocaine into metabolites, wherein said butyrylcholinesterase variant polypeptide exhibits a two-fold or more increase in cocaine hydrolysis activity compared to butyrylcholinesterase.