Characterization of inhibitors of specific carboxylesterases: Development of carboxylesterase inhibitors for translational application Kyoung Jin P. Yoon, 1 Janice L. Hyatt, 1 Christopher L. Morton, 1 Richard E. Lee, 2 Philip M. Potter, 1 and Mary K. Danks 1 1 Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, Tennessee and 2 Department of Pharmaceutical Science, University of Tennessee Health Science Center, Memphis, Tennessee Abstract Carboxylesterases, expressed at high levels in human liver and intestine, are thought to detoxify xenobiotics. The anticancer prodrug 7-ethyl-10-[4-1-piperidino)-1-piperidi- no]carbonyloxycamptothecin (CPT-11) is also metabolized by carboxylesterases to produce the active drug 7-ethyl- 10-hydroxycamptothecin. Activation of CPT-11 by human intestinal carboxylesterase (hiCE) in the human intestine may contribute to delayed onset diarrhea, a dose-limiting side effect of this drug. The goal of this study was to develop small molecule inhibitors selective for hiCE to circumvent or treat the toxic side effects of CPT-11. A secondary goal was to develop molecules that specifically inhibit activation of CPT-11 by a rabbit liver carboxyles- terase (rCE). rCE is the most efficient CPT-11 – activating enzyme thus far identified, and this enzyme is being devel- oped for viral-directed enzyme prodrug therapy applica- tions. Based on in vitro assays with partially purified hiCE and rCE proteins and on growth inhibition assays using U373MG human glioma cells transfected to express hiCE or rCE (U373pIREShiCE or U373pIRESrCE), we identified specific inhibitors of each enzyme. Lead compounds are derivatives of nitrophenol having 4-(furan-2-carbonyl)- piperazine-1-carboxylic acid or 4-[(4-chlorophenyl)-phenyl- methyl]-piperazine-1-carboxylic acid substitutions in the p position. Kinetic analysis of each compound for hiCE compared with rCE showed that the K i values of the most selective of these inhibitors differed by 6- to 10-fold. In growth inhibition assays, nontoxic, low micromolar con- centrations of these inhibitors increased the EC 50 of CPT- 11 for U373pIREShiCE or U373pIRESrCE cells by 13- to >1,500-fold. The four compounds characterized in this study will serve as lead compounds for a series of inhibi- tors to be constructed using a combinatorial approach. [Mol Cancer Ther 2004;3(8):903 – 9] Introduction Carboxylesterases comprise a family of enzymes ubiqui- tously expressed in animals and mammalian tissues (1). These enzymes are associated with metabolism of xeno- biotics such as pesticides, nerve gasses, heroin, and many drugs including some prodrugs used in chemotherapy (1, 2). Esterases are usually classified according to the sub- strates they metabolize, but there is significant overlap in substrate and inhibitor ‘‘specificity’’ among esterase fam- ilies. In cancer patients treated with 7-ethyl-10-[4-1-piper- idino)-1-piperidino]carbonyloxycamptothecin (CPT-11), carboxylesterases convert CPT-11 to its active form 7-ethyl-10-hydroxycamptothecin (SN-38); SN-38 is a potent inhibitor of topoisomerase I (3). The purpose of this study was to develop small molecule inhibitors of specific carbo- xylesterases that activate and therefore mediate the anti- tumor efficacy and toxicity of the antitumor agent CPT-11. Three carboxylesterases have been identified that acti- vate CPT-11 relatively efficiently (4 – 7). This study focuses on the two carboxylesterases that are most likely to impact on the clinical use of CPT-11. The first of these is human intestinal carboxylesterase (hiCE), which is expressed at high levels in the small intestine and at f 10-fold lower levels in the liver (also called hCE2; refs. 5, 7). Because of the high level of expression of hiCE in the intestine, we postulate that this enzyme is likely to be at least partly responsible for producing high local levels of SN-38 and to contribute to the gastrointestinal toxicity seen in pa- tients treated with CPT-11. The second enzyme known to activate CPT-11 is a rabbit liver carboxylesterase (rCE) that is being developed for viral-directed enzyme prodrug therapy approaches to chemotherapy (8 – 10). rCE activates CPT-11 most efficiently of all carboxylesterases thus far characterized. Because of the inevitable variability in endo- genous (hiCE) or exogenous (viral-directed enzyme pro- drug therapy with rCE) levels of enzymes, small molecules that specifically inhibit the activity of the enzyme(s) respon- sible for CPT-11 activation might be used to ameliorate or circumvent toxic effects of CPT-11 therapy. Therefore, our long-term goal is to develop small molecule inhibitors of hiCE or rCE. The study described in this article confirms that it is possible to develop small molecules with such specificity and identifies lead compounds that selectively inhibit hiCE or rCE. Received 1/7/04; revised 4/6/04; accepted 5/27/04. Grant support: NIH Cancer Center Core grant CA21765 and American Lebanese Syrian Associated Charities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Mary K. Danks, Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. Phone: 901-495-3440; Fax: 901-495-4293. E-mail: mary.danks@stjude.org Copyright C 2004 American Association for Cancer Research. Molecular Cancer Therapeutics 903 Mol Cancer Ther 2004;3(8). August 2004 on April 12, 2016. © 2004 American Association for Cancer Research. mct.aacrjournals.org Downloaded from