Structural constraints affect the metabolism of 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) by carboxylesterases

7-Ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin [CPT-11 ( irinotecan)] is a water-soluble camptothecin-derived prodrug that is activa ted by esterases to yield the potent topoisomerase I poison SN-38. We ident ified a rabbit liver carboxylesterase (CE) that was very efficient at CPT-1 1 metabolism; however, a human homolog that was more than 81% identical to this protein activated the drug poorly. Recently, two other human CEs have been isolated that are efficient in the conversion of CPT-11 to SN-38, yet both demonstrate little homology to the rabbit protein. To understand this phenomenon, we have characterized a series of esterases from human and rabb it, including several chimeric proteins, for their ability to metabolize CP T-11. Computer predictive modeling indicated that the ability of each enzym e to activate CPT-11 was dependent on the size of the entrance to the activ e site. Kinetic studies with a series of nitrophenyl and naphthyl esters co nfirmed these predictions, indicating that activation of CPT-11 by a CE is constrained by size-limited access of the drug to the active site catalytic amino acid residues.