Isolation and characterization of a cDNA encoding a horse liver butyrylcholinesterase - Evidence for CPT-11 drug activation

Butyrylcholinesterases (BuChEs; acylcholine acylhydrolase; EC 3.1.1.8) have been demonstrated to convert the anticancer agent CPT-11 (irinotecan, 7-et hyl-10-[-4(1-piperidino)-1-piperidino]carbonyloxy- camptothecin) into its a ctive metabolite SN-38 (7-ethyl-10-hydroxycamptothecin). In addition, signi ficant differences in the extent of drug metabolism have been observed with BuChEs derived from different species. In an attempt to understand these d ifferences, we have isolated the cDNA encoding a horse BuChE. Based upon th e NH2-terminal amino acid sequence of a purified horse BuChE, we designed d egenerate primers to amplify the coding sequence from horse liver cDNA. Fol lowing polymerase chain reaction and rapid amplification of the cDNA ends, we generated an 1850-bp DNA fragment, containing an 1806-bp open reading fr ame. The cDNA encodes a protein of 602 amino acid residues, including a 28- amino acid NH2-terminal signal peptide. Furthermore, the DNA sequence and t he deduced amino acid sequence revealed extensive homology to butyrylcholin esterase genes from several other species. In vitro transcription-translati on of the cDNA produced a 66-kDa protein, identical to the size of native h orse serum BuChE following removal of carbohydrate residues with endoglycos idase F. Additionally, transient expression of the cDNA in Cos-7 cells yiel ded extracts that exhibited cholinesterase activity and demonstrated a K-m value for butyrylthiocholine of 106 +/- 9 nM. This extract converted the an ticancer drug CMPT-11 into SN-38, demonstrating that this drug can be activ ated by enzymes other than carboxylesterases, BIOCHEM PHARMACOL 59;7:713-78 1, 2000. (C) 2000 Elsevier Science Inc.