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Carboxylesterases (CES) have important roles in pesticide and drug metabolism and contribute to the clearance of ester-containing xenobiotics in mammals. Tissues with the highest levels of CES expression are the liver and small intestine. In addition to xenobiotics, CES also harness their broad substrate specificity to hydrolyze endobiotics, such as cholesteryl esters and triacylglycerols. Here, we determined if two human CES isoforms, CES1 and CES2, hydrolyze the endocannabinoids 2-arachidonoylglycerol (2AG) and anandamide (AEA), and two prostaglandin glyceryl esters (PG-Gs), which are formed by COX-mediated oxygenation of 2AG. We show that recombinant CES1 and CES2 efficiently hydrolyze 2AG to arachidonic acid (AA) but not amide-containing AEA. Steady-state kinetic parameters for CES1- and CES2-mediated 2AG hydrolysis were, respectively, kcat, 59 and 43 min−1; Km, 49 and 46 μM; and kcat/Km, 1.2 and 0.93 μM−1min−1. kcat/Kmvalues are comparable to published values for rat monoacylglycerol lipase (MAGL)-catalyzed 2AG hydrolysis. Furthermore, we show that CES1 and CES2 also efficiently hydrolyze PGE2-G and PGF2α-G. In addition, when cultured human THP1 macrophages were treated with exogenous 2AG or PG-G (10 μM, 1 h), significant quantities of AA or PGs were detected in the culture medium; however, the ability of macrophages to metabolize these compounds was inhibited (60−80%) following treatment with paraoxon, the toxic metabolite of the insecticide parathion. Incubation of THP1 cell lysates with small-molecule inhibitors targeting CES1 (thieno[3,2-e][1]benzothiophene-4,5-dione or JZL184) significantly reduced lipid glyceryl ester hydrolase activities (40−50% for 2AG and 80−95% for PG-Gs). Immunodepletion of CES1 also markedly reduced 2AG and PG-G hydrolase activities. These results suggested that CES1 is in part responsible for the hydrolysis of 2AG and PG-Gs in THP1 cells, although it did not rule out a role for other hydrolases, especially with regard to 2AG metabolism since a substantial portion of its hydrolysis was not inactivated by the inhibitors. An enzyme (Mr31−32 kDa) of unknown function was detected by serine hydrolase activity profiling of THP1 cells and may be a candidate. Finally, the amounts of in situgenerated 2AG and PG-Gs in macrophages were enhanced by treating the cells with bioactive metabolites of OP insecticides. Collectively, the results suggest that in addition to MAGL and fatty-acid amide hydrolase (FAAH), which have both been documented to terminate endocannabinoid signaling, CES may also have a role. Furthermore, since PG-Gs have been shown to possess biological activities in their own right, CES may represent an important enzyme class that regulates their in vivo levels. [ABSTRACT FROM AUTHOR]

CPT-11 is an antitumor prodrug that is hydrolyzed by carboxylesterases (CE) to yield SN-38, a potent topoisomerase I poison. However, the dose limiting toxicity delays diarrhea that is thought to arise, in part, from activation of the prodrug by a human intestinal CE (hiCE). Therefore, we have sought to identify selective inhibitors of hiCE that may have utility in modulating drug toxicity. We have evaluated one such class of molecules (benzene sulfonamides) and developed QSAR models for inhibition of this protein. Using these predictive models, we have synthesized a panel of fluorene analogues that are selective for hiCE, demonstrating no cross reactivity to the human liver CE, hCE1, or toward human cholinesterases, and have Kivalues as low as 14 nM. These compounds prevented hiCE-mediated hydrolysis of the drug and the potency of enzyme inhibition correlated with the clogP of the molecules. These studies will allow the development and application of hiCE-specific inhibitors designed to selectively modulate drug hydrolysis in vivo. [ABSTRACT FROM AUTHOR]

Structures of mammalian carboxylesterases (CEs) reveal the presence of a ‘side door’ that is proposed to act as an alternative pore for the trafficking of substrates and products. p-Nitrobenzyl esterase (pnb CE) from Bacillus subtilis exhibits close structural homology and a similar side-door domain as mammalian CEs. We investigated the role of a specific ‘gate’ residue at the side door (i.e., Leu 362) during pnb CE-catalyzed hydrolysis of model esters, pesticides, and lipids. Recombinant pnb CE proteins containing mutations at position 362 demonstrated markedly lower kcat and kcat/ Km values. The mutation with the most significant impact on catalysis was the L362R mutant ( kcat/ Km was 22-fold lower). Moreover, the ability of the L362R mutant to be inhibited by organophosphates (OP) was also lower. Investigation into the altered catalytic proficiency using pH-activity studies indicated that the catalytic triad of the mutant enzyme was preserved. Furthermore, viscosity variation and carbamate inhibition experiments indicated that rates of substrate association and acylation/deacylation were lower. Finally, recombinant CEs were found to possess lipolytic activity toward cholesteryl oleate and 2-arachidonylglycerol. In summary, the L362R mutant CE markedly slowed the rate of ester hydrolysis and was less sensitive to OP inhibition. The apparent causes of the diminished catalysis are discussed. [ABSTRACT FROM AUTHOR]

Doxazolidine (Doxaz), a formaldehyde−doxorubicin (Dox) conjugate, exhibits markedly increased tumor toxicity with respect to Dox without a concurrent increase in toxicity to cardiomyocytes. Pentyl PABC-Doxaz (PPD) is a Doxaz carbamate prodrug that is hydrolyzed by carboxylesterases. Here, we identify human intestinal carboxylesterase (hiCE) as the agent of activation for PPD. Upon prodrug treatment, cells that express higher levels of hiCE responded with lower IC 50values for growth inhibition. Exposing MCF-7 human breast cancer cells, which respond poorly and express little hiCE, to PPD together with hiCE resulted in a dramatic decrease in the IC 50, a decrease that was absent when human carboxylesterase 1 was added to prodrug treatment. Finally, U373MG glioblastoma cells overexpressing hiCE displayed ∼100-fold reduction in the IC 50for PPD compared to cells lacking the carboxylesterase. Overall, our studies indicate that PPD is selectively hydrolyzed to the active metabolite by hiCE. [ABSTRACT FROM AUTHOR]

Carboxylesterases (CE) are ubiquitous enzymes responsible for the detoxification of xenobiotics, including numerous clinically used drugs. Therefore, the selective inhibition of these proteins may prove useful in modulating drug half-life and bioavailability. Recently, we identified 1,2-diones as potent inhibitors of CEs, although little selectivity was observed in the inhibition of either human liver CE (hCE1) or human intestinal CE (hiCE). In this paper, we have further examined the inhibitory properties of ethane-1,2-diones toward these proteins and determined that, when the carbonyl oxygen atoms are cis-coplanar, the compounds demonstrate specificity for hCE1. Conversely, when the dione oxygen atoms are not planar (or are trans-coplanar), the compounds are more potent at hiCE inhibition. These properties have been validated in over 40 1,2-diones that demonstrate inhibitory activity toward at least one of these enzymes. Statistical analysis of the results confirms the correlation (P< 0.001) between the dione dihedral angle and the preferential inhibition of either hiCE or hCE1. Overall, the results presented here define the parameters necessary for small molecule inhibition of human CEs. [ABSTRACT FROM AUTHOR]

Carboxylesterases (CE) are ubiquitous enzymes thought to be responsible for the metabolism and detoxification of xenobiotics. Numerous clinically used drugs including Demerol, lidocaine, capecitabine, and CPT-11 are hydrolyzed by these enzymes. Hence, the identification and application of selective CE inhibitors may prove useful in modulating the metabolism of esterified drugs in vivo. Having recently identified benzil (diphenylethane-1,2-dione) as a potent selective inhibitor of CEs, we sought to evaluate the inhibitory activity of related 1,2-diones toward these enzymes. Biochemical assays and kinetic studies demonstrated that isatins (indole-2,3-diones), containing hydrophobic groups attached at a variety of positions within these molecules, could act as potent, specific CE inhibitors. Interestingly, the inhibitory potency of the isatin compounds was related to their hydrophobicity, such that compounds with clogP values of <1.25 were ineffective at enzyme inhibition. Conversely, analogs demonstrating clogP values >5 routinely yielded Kivalues in the nM range. Furthermore, excellent 3D QSAR correlates were obtained for two human CEs, hCE1 and hiCE. While the isatin analogues were generally less effective at CE inhibition than the benzils, the former may represent valid lead compounds for the development of inhibitors for use in modulating drug metabolism in vivo. [ABSTRACT FROM AUTHOR]