This material is available free of charge via the Internet at http://pubs.acs.org/.. and far-reaching effects on health, the legal system, and the family unit. Protein-based therapies for cocaine habit have received substantial attention in recent years.4 Of particular interest are catalytic antibodies and enzymes specific for the hydrolysis of the benzoyl ester of cocaine 1, returning the nonpsychoactive products benzoic acid and methyl ecgonine 2 (Plan 1). Several catalytic antibodies have been developed for this reaction,5 including GNL3A66 and GNL7A17 from our laboratory, as well as enzymes such as butyrylcholinesterase (BuChE)8 and the phage-displayed cocaine esterases cocE-pHI and -pIX.9 However, the efficacy of any treatment for cocaine addiction is dependent on limiting potential deleterious side reactions. Antibodies have long been touted as possessing exquisite specificity for his or her cognate antigens. Yet, while heteroclitic relationships are well known for binding antibodies, 11 no catalytic antibody has been reported to display activity for two unique substrates of medical relevance. With antibody therapies rapidly entering human being screening, combined with the aftermath of the recent TGN1412 medical trial disaster,12 potential therapies must be cautiously scrutinized for adverse medical effects. Open in a separate window Plan 1 The products of enzymatic hydrolysis of cocaine 1 and acetylcholine 3. Given that the common neurotransmitter acetylcholine 3 (ACh) and cocaine both contain a cationic nitrogen and reactive ester features, we became intrigued in the possibility that antibodies and enzymes showing cocaine esterase activity might also hydrolyze ACh. Support for this Vitamin D2 hypothesis can be attained by an examination of the low energy conformations of these two molecules; indeed, in both molecules the nitrogen atom and catalytically cleaved ester moieties are separated by ~5.1 ?. Furthermore, crystallographic studies have exposed that cation- relationships between aryl residues and the cationic tropane amine of cocaine are key elements of substrate binding for both GNL7A17 and cocE,10 suggesting the quaternary nitrogen of ACh may similarly bind to these enzymes inside a effective orientation. To test this hypothesis, binding studies were performed with GNL7A1 and cocE by determining the potential binding sites, followed by ligand docking in Vitamin D2 MPSim-Dock.13 Crystallographic studies suggest that the catalytic activity of GNL7A1 effects from a conformational modify, thereby stabilizing the generated tetrahedral intermediate.7 Thus, the computational model of GNL7A1 was first validated by comparing the docked cocaine transition state analog with the crystal structure (Number 1A & 1C); gratifyingly, these two molecules displayed a high degree of overlap (RMDS = 0.384 ?). To examine if the antibody would similarly stabilize the transition state for ACh hydrolysis, a tetrahedral approximation of the ACh transition state was docked into the crystal structure of 7A1 Fab bound to a cocaine transition state analog (accession code 2AJX). By Rabbit polyclonal to Vitamin K-dependent protein C using this model, ACh was found to bind at the same site within the catalytic antibody and in a very related conformation as cocaine (Number 1ACD). In the lowest-energy structure, the quaternary nitrogen of ACh makes cation- contacts to the same residues as cocaine, and docks only 1 1.3 ? away from the tropane nitrogen. Additional residues such as TyrL94 and TyrH50 interact with the tetrahedral intermediate of both ACh and cocaine. Open in a separate window Number 1 The docked (cyan) and crystal structure (green) of a cocaine transition state analog in 7A1 Fab demonstrated in Vitamin D2 surface (A) and ribbon (C) look Vitamin D2 at, and the structure of an ACh transition state analog in 7A1 Fab (B, D). Cocaine (E, G) and ACh (F, H) dock into the same site of cocE. As with GNL7A1, cocE also has been crystallized in multiple conformations.10 For our studies, we chose the crystal structure of cocE bound to its product, benzoic acid (accession code 1JU4), as no structural data for cocaine-bound cocE has been reported. As was previously observed in the catalytic antibody dockings, the lowest-energy structure of cocaine binds to the active site of cocE (Number 1E & 1G). The methyl and benzoyl esters of cocaine make hydrogen relationship contacts with Trpl66 and His287 respectively, and the tropane nitrogen makes a cation- connection with Tyr44. The catalytic Ser117 also is in proximity with the reactive benzoyl carbon. The validity of the docked structure is supported from the high degree of overlap between the benzoyl moiety of cocaine and benzoic acid of the crystal structure.10 Replacement of cocaine with acetylcholine revealed that these two molecules bind cocE in similar conformations, and make similar contacts to the enzyme (Number.