Anti-cocaine compositions and treatment

Abstract

Embodiments of the invention disclosed herein generally relate to anti-cocaine therapeutics. Specifically, some embodiments of the invention relate to highly efficient, thermostable, and long-lasting cocaine esterase (CocE) mutants that can protect against the toxic and reinforcing effects of cocaine in subjects. Provided herein are mutant CocE polypeptides displaying thermostable esterase activity. Also provided are methods of treating cocaine-induced conditions in a subject in need via administration of mutant CocE as well as methods for high-throughput screening of candidate esterase polypeptides.

1 - 20 . (canceled) 
     
     
         21 . An isolated mutant polypeptide comprising SEQ ID NO: 1 comprising a mutation, esterase activity, and increased thermostability. 
     
     
         22 . The mutant polypeptide of  claim 21 , wherein increased thermostability comprises increased thermostability at 37° C. 
     
     
         23 . The mutant polypeptide of  claim 22 , wherein increased thermostability at 37° C. comprises increased thermostability at 37° C. of the mutant polypeptide as compared to a polypeptide consisting of SEQ ID NO: 1. 
     
     
         24 . The mutant polypeptide of  claim 23 , wherein the increase in thermostability of the mutant polypeptide over the polypeptide consisting of SEQ ID NO: 1 is about 2.0 kcal/mol or greater. 
     
     
         25 . The mutant polypeptide of  claim 21 , wherein esterase activity comprises cocaine esterase activity. 
     
     
         26 . The mutant polypeptide of  claim 25 , wherein the mutant polypeptide has at least about 10% of the esterase activity of the polypeptide consisting of SEQ ID NO: 1. 
     
     
         27 . The mutant polypeptide of  claim 21 , wherein the mutation comprises a substitution, addition or deletion. 
     
     
         28 . The mutant polypeptide of  claim 21 , wherein at least one mutation is selected from the group consisting of T172R, G173Q, L169K, and F189K. 
     
     
         29 . The mutant polypeptide of  claim 21 , comprising mutations T172R and G173Q. 
     
     
         30 . The mutant polypeptide of  claim 21 , comprising mutation L169K. 
     
     
         31 . The mutant polypeptide of  claim 21 , comprising mutations selected from the group consisting of T172R/G173Q, T172R/F189A, T172R/A193D, T172R/G173Q-1175-G-G-A186, and T172R/G173Q-T176-G-G-D185. 
     
     
         32 . A pharmaceutical composition comprising the mutant polypeptide of  claim 21  and a pharmaceutically acceptable carrier or excipient. 
     
     
         33 . An isolated mutant cocaine esterase (CocE) polypeptide comprising SEQ ID NO: 1 comprising a mutation selected from the group consisting of T172R, G173Q, L169K, and F189K. 
     
     
         34 . The CocE polypeptide of  claim 33 , comprising a mutation selected from the group consisting of T172R/G173Q, T172R/F189A, T172R/A193D, T172R/G173Q-1175-G-G-A186, and T172R/G173Q-T176-G-G-D185. 
     
     
         35 . The CocE polypeptide of  claim 33 , comprising mutations T172R and G173Q. 
     
     
         36 . The CocE polypeptide of  claim 33 , further comprising a mutation selected from the group consisting of R41I, N42V, K46A, S56G, T74S, F84Y, L119A, 5140A, L146P, A149S, Y152H, S159A, V160A, L163V, 1175-G-G-A186, T176-G-G-D18, R182K, F189A, F189L, V190K, Q191K, A193D, A194K, A194R, N197K, 1218L, W285T, W220A, V225I, T254R, V262L, S265A, A310D, C477T, L508G, K531A, Y532F, and D533S. 
     
     
         37 . The CocE polypeptide of  claim 33 , comprising at least 90% identity to SEQ ID NO: 1 and esterase activity. 
     
     
         38 . The CocE polypeptide of  claim 33 , comprising at least 95% identity to SEQ ID NO: 1 and esterase activity. 
     
     
         39 . The CocE polypeptide of  claim 33 , comprising at least 95% identity to SEQ ID NO: 1, esterase activity, and increased thermostability at 37° C. as compared to a polypeptide consisting of SEQ ID NO: 1. 
     
     
         40 . A pharmaceutical composition comprising the CocE polypeptide of  claim 33  and a pharmaceutically acceptable carrier or excipient. 
     
     
         41 . An isolated polypeptide comprising SEQ ID NO: 39 (double mutant). 
     
     
         42 . A pharmaceutical composition comprising the polypeptide of  claim 41  and a pharmaceutically acceptable carrier or excipient.