RT Journal Article
SR Electronic
T1 Catalytic Antibodies that Hydrolyze (−)-Cocaine Obtained by a High-Throughput Procedure
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP 952
OP 961
VO 293
IS 3
A1 Cashman, John R.
A1 Berkman, Clifford E.
A1 Underiner, Gail E.
YR 2000
UL http://jpet.aspetjournals.org/content/293/3/952.abstract
AB Antibodies to a 2β-carboxamido-2β-phosphonate transition-state analog of (−)-cocaine benzoate ester hydrolysis were elicited in mice. A large number of hybridoma cell lines were propagated, and the catalytic activity of culture fluid was determined with a high-throughput photometric assay using cocaine benzoyl thioester as substrate. Binding avidity of the hybridoma supernatants to the phosphonate hapten was also determined. The initial rate constants for cocaine benzoyl thioester hydrolysis and binding avidity for a large number of hybridoma supernatants elicited to the phosphonate hapten did not always correlate. The lack of correlation of substrate hydrolysis with the binding affinity of 70 catalytic antibodies was also observed for (−)-cocaine hydrolysis using derivatization and HPLC analysis of methyl ecgonine as meta-nitrococaine. Thekcat values for cocaine benzoyl thioester hydrolysis by monoclonal antibodies 3, 5, and 12 were 38, 4.2, and 0.6 min−1, respectively. For monoclonal antibody 5, the selectivity ratios (Ki value divided by theKm value for the hydrolysis of cocaine benzoyl thioester) with ecgonine benzoyl ester, ecgonine methyl ester, norcocaine, and ecgonine were 101, 25, 9.4, and 4, respectively. Three active esterolytic monoclonal antibodies identified with the high-throughput assay procedure were examined in detail for their ability to hydrolyze (−)-cocaine. The kcatvalues for the hydrolysis of (−)-cocaine with monoclonal antibodies 3, 5, and 12 were 6.6, 0.4, and 0.1 min−1, respectively. Hydrolysis of (−)-cocaine by monoclonal antibody 3 approached thekcat value for that of human butyrylcholinesterase. Cocaine esterolytic catalytic antibodies that approach or exceed the catalytic efficiency of human butyrylcholinesterase may represent a new pharmacological intervention approach to the treatment of cocaine abuse, and the high-throughput process described here represents an advance in the effort to develop clinically useful antibodies. The American Society for Pharmacology and Experimental Therapeutics