Thermostabilization of proteins

Abstract

Provided are compositions comprising a cocaine esterase (CocE) and a compound that thermostabilizes the CocE. Also provided are methods of thermostabilizing a cocaine esterase. Additionally provided are methods of treating a mammal undergoing a cocaine-induced condition. Methods of determining whether a compound is a thermostabilizing agent for a protein are also provided. Uses of the above-described compositions for the treatment of a cocaine-induced condition is additionally provided. Additionally provided is an isolated nucleic acid encoding a CocE polypeptide having the substitutions L169K and G173Q, and the CocE polypeptide encoded by that nucleic acid, and pharmaceutical compositions thereof. Further provided is the use of that composition for the manufacture of a medicament for the treatment of a cocaine-induced condition and for the treatment of a cocaine-induced condition.

1 . A composition comprising:
 a cocaine esterase (CocE) polypeptide; and   at least one thermostabilization compound,   wherein the CocE in the presence of the compound is more thermostable than the CocE in the absence of the compound.   
     
     
         2 . The composition of  claim 1 , wherein the CocE comprises:
 (i) an amino acid sequence of SEQ ID NO: 1;   (ii) an amino acid sequence at least 90% identical to SEQ ID NO: 1 having esterase activity; or   (iii) an amino acid sequence at least 90% identical to SEQ ID NO: 1 with one or more substitutions selected from the group consisting of L163V, V225I, I218L, A310D, A149S, S159A, S265A, S56G, W220A, S140A, F189L, A193D, T254R, N42V, V262L, L508G, Y152H, V160A, T172R, Y532F, T74S, W285T, L146P, D533S, A194R, G173Q, C477T, K531A, R41I, L119A, K46A, F84Y, T172R/G173Q, L169K, F189A, N197K, R182K, F189K, V190K, Q191K, A194K, and L169K/G173Q, or a combination thereof; and having esterase activity.   
     
     
         3 . The composition of  claim 2 , wherein the CocE has an amino acid sequence of SEQ ID NO: 1 with one or more substitutions selected from the group consisting of: T172R, S159A, N197K, L169K, F189K, G173Q, and T172R/G173Q. 
     
     
         4 . The composition of  claim 2 , wherein the CocE has an amino acid sequence of SEQ ID NO:1 with the substitution L169K/G173Q. 
     
     
         5 . The composition  claim 1 , wherein the CocE is a pegylated CocE. 
     
     
         6 . The composition of  claim 1 , wherein the one or more thermostabilization compounds are selected from: 
       
         
           
           
               
               
           
         
       
     
     
         7 . The composition of  claim 1 , wherein the at least one thermostabilization compound is selected from: 
       
         
           
           
               
               
           
         
       
     
     
         8 . The composition of  claim 1 , wherein the at least one thermostabilization compound is: 
       
         
           
           
               
               
           
         
       
     
     
         9 . The composition of  claim 1 , wherein the at least one thermostabilization compound is: 
       
         
           
           
               
               
           
         
       
     
     
         10 . The composition of  claim 1  further comprising a pharmaceutically acceptable carrier. 
     
     
         11 . The composition of  claim 1 , wherein
 the CocE comprises
 (i) an amino acid sequence of SEQ ID NO: 1; 
 (ii) an amino acid sequence at least 90% identical to SEQ ID NO: 1 having esterase activity; or 
 (iii) an amino acid sequence at least 90% identical to SEQ ID NO: 1 with one or more substitutions selected from the group consisting of L163V, V225I, I218L, A310D, A149S, S159A, S265A, S56G, W220A, S140A, F189L, A193D, T254R, N42V, V262L, L508G, Y152H, V160A, T172R, Y532F, T74S, W285T, L146P, D533S, A194R, G173Q, C477T, K531A, R41I, L119A, K46A, F84Y, T172R/G173Q, L169K, F189A, N197K, R182K, F189K, V190K, Q191K, A194K, and L169K/G173Q, or a combination thereof; and having esterase activity; 
   the one or more thermostabilization compounds are selected from:   
       
         
           
           
               
               
           
         
         and the composition further comprises a pharmaceutically acceptable carrier. 
       
     
     
         12 . A method of thermostabilizing a cocaine esterase (CocE), the method comprising combining a CocE polypeptide with one or more thermostabilization compounds selected from: 
       
         
           
           
               
               
           
         
       
     
     
         13 . The method of  claim 12 , wherein the CocE polypeptide comprises:
 (i) an amino acid sequence of SEQ ID NO: 1;   (ii) an amino acid sequence at least 90% identical to SEQ ID NO: 1 having esterase activity; or   (iii) an amino acid sequence at least 90% identical to SEQ ID NO: 1 with one or more substitutions selected from the group consisting of L163V, V225I, I218L, A310D, A149S, S159A, S265A, S56G, W220A, S140A, F189L, A193D, T254R, N42V, V262L, L508G, Y152H, V160A, T172R, Y532F, T74S, W285T, L146P, D533S, A194R, G173Q, C477T, K531A, R41I, L119A, K46A, F84Y, T172R/G173Q, L169K, F189A, N197K, R182K, F189K, V190K, Q191K, A194K, and L169K/G173Q, or a combination thereof; and having esterase activity.   
     
     
         14 . The method of  claim 12 , wherein combining the CocE polypeptide with one or more thermostabilization compounds occurs (i) in vivo in a mammal, (ii) in vitro, (iii) during purification of CocE, (iv) during storage of CocE, or a combination thereof. 
     
     
         15 . A method of treating a cocaine-induced condition, the method comprising:
 administering to a mammalian subject in need thereof a composition selected from:
 (i) a composition according to  claim 1 ; or 
 (ii) composition comprising an isolated cocaine esterase (CocE) polypeptide having (a) an amino acid sequence of SEQ ID NO:1, except for substitutions L169K and G173Q; or (b) an amino acid sequence having at least 85% sequence identity with SEQ ID NO:1, having substitutions L169K and G173Q; esterase activity; and increased thermostability at 37° C. as compared to wild-type CocE; 
   wherein the subject is diagnosed with, or at risk for, a cocaine-induced condition selected from the group consisting of cocaine overdose, cocaine toxicity, cocaine addiction, or cocaine dependence.   
     
     
         16 . The method of  claim 15 , wherein the CocE polypeptide comprises:
 (i) an amino acid sequence of SEQ ID NO: 1;   (ii) an amino acid sequence at least 90% identical to SEQ ID NO: 1 having esterase activity; or   (iii) an amino acid sequence at least 90% identical to SEQ ID NO: 1 with one or more substitutions selected from the group consisting of L163V, V225I, I218L, A310D, A149S, S159A, S265A, S56G, W220A, S140A, F189L, A193D, T254R, N42V, V262L, L508G, Y152H, V160A, T172R, Y532F, T74S, W285T, L146P, D533S, A194R, G173Q, C477T, K531A, R41I, L119A, K46A, F84Y, T172R/G173Q, L169K, F189A, N197K, R182K, F189K, V190K, Q191K, A194K, and L169K/G173Q, or a combination thereof; and having esterase activity.   
     
     
         17 . The method of  claim 15 , wherein the at least one thermostabilization compound is selected from: 
       
         
           
           
               
               
           
         
       
     
     
         18 . The method of  claim 15 , wherein the composition is administered intravenously or via a mini-infusion pump. 
     
     
         19 . A method for identifying a thermostabilization compound, the method comprising:
 measuring thermostability of a cocaine esterase (CocE);   measuring thermostability of the CocE in combination with a candidate compound;   determining whether the candidate compound increases thermostability of the CocE.   
     
     
         20 . A composition comprising:
 (i) an isolated cocaine esterase (CocE) polypeptide, the CocE polypeptide comprising:
 (a) an amino acid sequence of SEQ ID NO:1, except for substitutions L169K and G173Q; or 
 (b) an amino acid sequence having at least 85% sequence identity with SEQ ID NO:1, wherein the encoded CocE polypeptide has substitutions L169K and G173Q and esterase activity with increased thermostability at 37° C. as compared to wild-type CocE; or 
   (ii) an isolated nucleic acid encoding the polypeptide of (i); and   (iii) optionally, a pharmaceutically acceptable carrier.