Human Carboxylesterase Research Articles

Enzymatically Activated Glyco-Prodrugs of Doxorubicin Synthesized by a Catalysis-Free Diels-Alder Reaction.

The severe side effects associated with the use of anthracycline anticancer agents continues to limit their use. Herein we describe the synthesis and preliminary biological evaluation of three enzymatically activatable doxorubicin-oligosaccharide prodrugs. The synthetic protocol allows late stage variation of the carbohydrate and is compatible with the use of disaccharides such as lactose as well as more complex oligosaccharides such as xyloglucan oligomers. The enzymatic release of doxorubicin from the prodrugs by both protease (plasmin) and human carboxylesterases (hCE1 and 2) was demonstrated in vitro and the cytotoxic effect of the prodrugs was assayed on MCF-7 breast cancer cells.

Asymmetric Construction of a Multi-Pharmacophore-Containing Dispirotriheterocyclic Scaffold and Identification of a Human Carboxylesterase 1 Inhibitor.

A catalytic asymmetric [3 + 2] cyclization of novel 4-isothiocyanato pyrazolones and isatin-derived ketimines was developed, delivering a wide range of intriguing dispirotriheterocyclic products in high yield with excellent diastereoselectivity and enantioselectivity. A chiral sulfoxide derivative of this dispirocyclic product was identified to be a promising hit of the human carboxylesterase 1 inhibitor, and the significant difference of the activity between two enantiomers emphasized the importance of this asymmetric process.

A holographic sensor based on a biomimetic affinity ligand for the detection of cocaine

The increasing consumption of illicit drugs has become a worldwide social and medical problem. Eradicating the supply and use of these drugs requires the development of devices that provide a fast, selective and simple response in seized powder samples. This paper describes a novel strategy for the design and development of a holographic sensor modified with a rationally designed receptor for the detection of cocaine. The receptor was designed by reviewing protein–cocaine interactions, particularly those of human carboxylesterase-1 and catalytic monoclonal antibody GNL7A1, and constructing a four-component Ugi library of putative cocaine-binding ligands. A hybrid hydrogel of poly (2-hydroxyethyl methacrylate–co-ethylene dimethacrylate-co-methacrolein) was used as the carbonyl source for the Ugi reaction. A Bragg diffraction grating comprising gold nanoparticles was fabricated in the polymer gel and the replay wavelength of the combinatorial library of Ugi-modified holographic sensors measured via a spectrophotometer in response to cocaine as a function of pH and ionic strength. A smartphone-based instrument and cloud-based algorithm was developed, optimised with training and test samples of cocaine and used to assess the presence of cocaine in seized field samples. These findings confirm the potential application of this analytical method in field testing for illicit drugs.

Genetic variation in human carboxylesterase CES1 confers resistance to hepatic steatosis

Obesity often leads non-alcoholic fatty liver disease, insulin resistance and hyperlipidemia. Expression of carboxylesterase CES1 is positively correlated with increased lipid storage and plasma lipid concentration. Here we investigated structural and metabolic consequences of a single nucleotide polymorphism in CES1 gene that results in p.Gly143Glu amino acid substitution. We generated a humanized mouse model expressing CES1WT (control), CES1G143E and catalytically dead CES1S221A (negative control) in the liver in the absence of endogenous expression of the mouse orthologous gene. We show that the CES1G143E variant exhibits only 20% of the wild-type lipolytic activity. High-fat diet fed mice expressing CES1G143E had reduced liver and plasma triacylglycerol levels. The mechanism by which decreased CES1 activity exerts this hypolipidemic phenotype was determined to include decreased very-low density lipoprotein secretion, decreased expression of hepatic lipogenic genes and increased fatty acid oxidation as determined by increased plasma ketone bodies and hepatic mitochondrial electron transport chain protein abundance. We conclude that attenuation of human CES1 activity provides a beneficial effect on hepatic lipid metabolism. These studies also suggest that CES1 is a potential therapeutic target for non-alcoholic fatty liver disease management.

Inhibition of cocaine self‐administration by a novel mutant cocaine esterase (ET CocE) in rats

Cocaine abuse is a serious public health problem for which there are currently no approved therapies. Cocaine esterase (CocE) is a bacterially‐derived esterase capable of rapidly metabolizing cocaine to the inactive metabolites ecgonine methyl ester, and benzoylecgonine; however, CocE is not stable at body temperature resulting in a very short in vivo duration of action. Previous mutagenesis studies have improved the thermostability of CocE, resulting in longer acting mutant forms of CocE (e.g., RQ CocE) capable of inhibiting the reinforcing, discriminative stimulus, cardiovascular, convulsant, and lethal effects of cocaine in both rodents and rhesus monkeys. Despite a ~500‐fold improvement in the in vivo duration of action, incorporation of the T172R/G173Q mutations did not enhance the catalytic efficiency (Kcat/Km) of CocE (~100 uM/min). Recent studies aimed to improve the catalytic efficiency of CocE by mutating both the binding site (A51E) and active site (S288T), resulting a CocE (ET CocE) with a catalytic efficiency (157 uM/min) ~1.6‐fold greater than that of RQ CocE. This study aimed to characterize the potency and effectiveness of ET CocE to decrease cocaine self‐administration. Male Sprague‐Dawley (n=12) rats were trained to self‐administer 0.32 mg/kg/inf cocaine under a fixed ratio (FR) 5 schedule of reinforcement. Half of these rats transitioned to a multiple component FR5 schedule capable of evaluating the reinforcing effects of multiple doses of cocaine (0.032–1 mg/kg/inf) in a single session, and the following tests were performed: saline was substituted for cocaine, pretreatment with ET CocE (0.1–3.2 mg/kg; IV) and RQ CocE (3.2 mg/kg; IV) prior to cocaine self‐administration sessions. The other 6 rats were used to test the effects of ET CocE (1 mg/kg; IV) during 90 min sessions in which either cocaine (0.1 and 1.0 mg/kg/inf) or MDPV (0.01 and 0.1 mg/kg/inf) were available for infusion. Pretreatment with 3.2 mg/kg RQ CocE resulted in a ~3‐fold rightward shift in the cocaine dose‐response curve. Pretreatment with ET CocE produced dose‐dependent rightward shifts in the cocaine dose‐response curve, with a doses of 1 and 3.2 mg/kg ET CocE producing ~3‐fold and ~10‐fold rightward shifts, respectively; low rates of responding were observed when saline was substituted for cocaine. When ET CocE (1 mg/kg) was evaluated in sessions in which rats could respond for either cocaine or MDPV, ET CocE decreased responding for the small dose of cocaine and increased responding for the large dose of cocaine, but failed to alter responding for MDPV. Together, these data show that the incorporation of novel mutations to the binding site (A51E) and active site (S288T) of CocE results in an enzyme (ET CocE) that is ~1.6‐fold more efficient at metabolizing cocaine and ~3‐fold more potent at inhibiting the self‐administration of cocaine, and provide additional strong support for the continued development of cocaine metabolizing enzymes, such as ET CocE, for treatment of cocaine abuse in humans.Support or Funding InformationSupported by a NIDA research grant (R01DA039146; GTC) and the ASPET SURF program (KJ).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A high-throughput pH-based colorimetric assay: application focus on alpha/beta hydrolases

Research involving α/β hydrolases, including α-amino acid ester hydrolase and cocaine esterase, has been limited by the lack of an online high throughput screening assay. The development of a high throughput screening assay capable of detecting α/β hydrolase activity toward specific substrates and/or chemical reactions (e.g., hydrolysis in lieu of amidase activity and/or synthesis instead of thioesterase activity) is of interest in a broad set of scientific questions and applications. Here we present a general framework for pH-based colorimetric assays, as well as the mathematical considerations necessary to estimate de novo the experimental response required to assign a ‘hit’ or a ‘miss,’ in the absence of experimental standard curves. This combination is valuable for screening the hydrolysis and synthesis activity of α/β hydrolases on a variety of substrates, and produces data comparable to the current standard technique involving High Performance Liquid Chromatography (HPLC). In contrast to HPLC, this assay enables screening experiments to be performed with greater efficiency.

Evidence for Shikonin acting as an active inhibitor of human carboxylesterases 2: Implications for herb-drug combination.

Shikonin, a natural naphthoquinone compound derived from the herb Lithospermum erythrorhizon, is widely used for its various pharmacological activities. However, its potential interactions with other medications by inhibiting human carboxylesterases 2 (hCE2) remain unknown. In this study, the inhibitory effects of shikonin on the activity of hCE2 in human liver microsomes are investigated by using fluorescein diacetate (FD), N-(2-butyl-1,3-dioxo-2,3-dihydro-1H-phenalen-6-yl)-2-chloroacetamide (NCEN), and CPT-11 as substrates of hCE2. The results demonstrate that shikonin significantly inhibits the activity of hCE2 when FD and NCEN are used as substrates, whereas the half inhibition concentration value of shikonin increased by 5-30 times when CPT-11 was used as the substrate. The inhibition types of shikonin against hCE2 activity reflected by 3 substrates were all best fit to noncompetitive manners. In addition, shikonin was found to distinctly suppress endogenous hCE2 activity, characterized with attenuated fluorescence. Furthermore, for drugs metabolized by hCE2 with the similar binding sites with FD or NCEN, the estimated magnitudes of area under the curve variation were approximately 9-357% in the presence of shikonin. Also, the area under the curve of CPT-11 could be increased by 1-14% following administration of shikonin. These findings have clear clinical implications for the combination of shikonin and hCE2-metabolizing prodrugs.

Chemical synthesis of an indomethacin ester prodrug and its metabolic activation by human carboxylesterase 1

It is necessary to consider the affinity of prodrugs for metabolic enzymes for efficient activation of the prodrugs in the body. Although many prodrugs have been synthesized with consideration of these chemical properties, there has been little study on the design of a structure with consideration of biological properties such as substrate recognition ability of metabolic enzymes. In this report, chemical synthesis and evaluation of indomethacin prodrugs metabolically activated by human carboxylesterase 1 (hCES1) are described. The synthesized prodrugs were subjected to hydrolysis reactions in solutions of human liver microsomes (HLM), human intestine microsomes (HIM) and hCES1, and the hydrolytic parameters were investigated to evaluate the hydrolytic rates of these prodrugs and to elucidate the substrate recognition ability of hCES1. It was found that the hydrolytic rates greatly change depending on the steric hindrance and stereochemistry of the ester in HLM, HIM and hCES1 solutions. Furthermore, in a hydrolysis reaction catalyzed by hCES1, the Vmax value of n-butyl thioester with chemically high reactivity was significantly lower than that of n-butyl ester.

Facile synthesis of 1,2-dione-containing abietane analogues for the generation of human carboxylesterase inhibitors

Recently, a series of selective human carboxylesterase inhibitors have been identified based upon the tanshinones, with biologically active molecules containing a 1,2-dione group as part of a naphthoquinone core. Unfortunately, the synthesis of such compounds is complex. Here we describe a novel method for the generation of 1,2-dione containing diterpenoids using a unified approach, by which boronic acids are joined to vinyl bromo-cyclohexene derivatives via Suzuki coupling, followed by electrocyclization and oxidation to the o-phenanthroquinones. This has allowed the construction of a panel of miltirone analogues containing an array of substituents (methyl, isopropyl, fluorine, methoxy) which have been used to develop preliminary SAR with the two human carboxylesterase isoforms. As a consequence, we have synthesized highly potent inhibitors of these enzymes (Ki < 15 nM), that maintain the core tanshinone scaffold. Hence, we have developed a facile and reproducible method for the synthesis of abietane analogues that have resulted in a panel of miltirone derivatives that will be useful tool compounds to assess carboxylesterase biology.

Inhibitory Influence of Panax notoginseng Saponins on Aspirin Hydrolysis in Human Intestinal Caco-2 Cells.

Herb-drug interactions are important safety concerns in clinical practice. The interactions occur firstly in the intestinal absorption for orally administered drugs. Aspirin and Panax notoginseng saponins (PNS)-based drugs are often combined in China to prevent larger-artery atherosclerosis. Here, we aimed to characterize the aspirin transport across Caco-2 cell monolayers, a model of the intestinal absorption, and further to evaluate the influence of PNS on aspirin hydrolysis and the relating mechanisms. Transcellular transport of aspirin and the influence of PNS were explored using Caco-2 cell monolayers. The protein expression of human carboxylesterase 1 (hCE1) and hCE2 in Caco-2 cells after PNS treatment was analyzed by ELISA, and the mRNA level were determined by qRT-PCR. In the study, Caco-2 cells showed high level of hydrolase activity, and most aspirin was hydrolyzed inside the cells during the transport process. Interestingly, PNS were demonstrated to inhibit the esterase activities responsible for aspirin hydrolysis in Caco-2 cells. PNS could also decrease the protein expression of hCE1 and hCE2, whereas exhibited minor effect on the mRNA expression. These results indicated that oral administration of PNS-based drugs might inhibit the hydrolysis of aspirin during intestinal absorption thus promoting its bioavailability.

Metabolic Hydrolysis of Aromatic Amides in Selected Rat, Minipig, and Human In Vitro Systems

The release of aromatic amines from drugs and other xenobiotics resulting from the hydrolysis of metabolically labile amide bonds presents a safety risk through several mechanisms, including geno-, hepato- and nephrotoxicity. Whilst multiple in vitro systems used for studying metabolic stability display serine hydrolase activity, responsible for the hydrolysis of amide bonds, they vary in their efficiency and selectivity. Using a range of amide-containing probe compounds (0.5–10 µM), we have investigated the hydrolytic activity of several rat, minipig and human-derived in vitro systems - including Supersomes, microsomes, S9 fractions and hepatocytes - with respect to their previously observed human in vivo metabolism. In our hands, human carboxylesterase Supersomes and rat S9 fractions systems showed relatively poor prediction of human in vivo metabolism. Rat S9 fractions, which are commonly utilised in the Ames test to assess mutagenicity, may be limited in the detection of genotoxic metabolites from aromatic amides due to their poor concordance with human in vivo amide hydrolysis. In this study, human liver microsomes and minipig subcellular fractions provided more representative models of human in vivo hydrolytic metabolism of the aromatic amide compounds tested.

Bysspectin A, an unusual octaketide dimer and the precursor derivatives from the endophytic fungus Byssochlamys spectabilis IMM0002 and their biological activities

Bysspectin A (1), a polyketide-derived octaketide dimer with a novel carbon skeleton, and two new precursor derivatives, bysspectins B and C (2 and 3), were obtained from an organic extract of the endophytic fungus Byssochlamys spectabilis that had been isolated from a leaf tissue of the traditional Chinese medicinal plant Edgeworthia chrysantha, together with a known octaketide, paecilocin A (4). Their structures were determined by HRMS, 1D and 2D NMR spectroscopic analysis. A plausible route for their biosynthetic pathway is proposed. Compounds 1–3 were tested for their antimicrobial activities. Only compound 3 was weakly active against Escherichia coli and Staphyloccocus aureus with MIC values of 32 and 64 μg/mL, respectively. Further, the inhibitory effects on human carboxylesterases (hCE1, hCE2) of compounds 1 and 4 were evaluated. The results demonstrated that bysspectin A (1) was a novel and highly selective inhibitor against hCE2 with the IC50 value of 2.01 μM. Docking simulation also demonstrated that active compound 1 created interaction with the Ser-288 (the catalytic amino-acid in the catalytic cavity) of hCE2 via hydrogen bonding, revealing its highly selective inhibition toward hCE2.

Characterization and structure-activity relationship studies of flavonoids as inhibitors against human carboxylesterase 2

Human carboxylesterases (hCEs) are key enzymes from the serine hydrolase superfamily. Among all identified hCEs, human carboxylesterase 2 (hCE2) plays crucial roles in the metabolic activation of ester drugs including irinotecan and flutamide. Selective and potent hCE2 inhibitors could be used to alleviate the toxicity induced by hCE2-substrate drugs. In this study, more than fifty flavonoids were collected to assay their inhibitory effects against hCE2 using a fluorescence-based method. The results demonstrated that C3 and C6 hydroxy groups were essential for hCE2 inhibition, while O-glycosylation or C-glycosylation would lead to the loss of hCE2 inhibition. Among all tested flavonoids, 5,6-dihydroxyflavone displayed the most potent inhibitory effect against hCE2 with the IC50 value of 3.50 μM. The inhibition mechanism of 5,6-dihydroxyflavone was further investigated by both experimental and docking simulations. All these findings are very helpful for the medicinal chemists to design and develop more potent and highly selective flavonoid-type hCE2 inhibitors.

An iridium complex-based probe for photoluminescence lifetime imaging of human carboxylesterase 2 in living cells.

A novel photoluminescence lifetime probe (Ir-TB) has been developed for the detection and imaging of hCE2 in living cells. A large lifetime increase by around 300 ns after the enzymatic reaction makes it an ideal tool to distinguish hCE2-hydrolyzed probes from those non-hydrolyzed ones via PLIM for the first time.

A novel quantification method for serine hydrolases in cellular expression system using fluorophosphonate-biotin probe

In the present study, we established a quantitative western blotting method to measure the expression level of recombinant serine hydrolases based on their catalytic mechanism. Fluorophosphonate (FP)-biotin was selected as a universal probe to quantify their expression levels, since FP moiety irreversibly inhibits serine hydrolases through strong stoichiometric binding to active serine residue. The linearity of detection using FP-biotin was assessed on three serine hydrolases; human carboxylesterase (CES) 1, butyrylcholinesterase and porcine liver esterases (PLE). Similar response signals were obtained from the equimolar concentrations of these enzymes and excellent linearity was observed at the range of 0.4–3.4pmol/lane (r2>0.99). Accuracy and precision of the proposed method were proved using PLE with recovery of 97.1–107.2% and relative standard deviation of 5.56%. PLE was selected as a calibration standard because of its high stability and commercial availability. As an application of the developed method, we measured the expression levels of four recombinant CES isozymes from human and cynomolgus macaque in S9 fraction of HEK293 cell homogenates. The expression levels of human CES1 and CES2, and cynomolgus macaque CES1 and CES2 were 2.51±0.1, 1.63±0.17, 0.79±0.09 and 1.37±0.13pmol/5μg S9 protein, respectively. Based on these determinations, their hydrolytic activities were accurately assessed. Cynomolgus CESs showed lower hydrolysis activities for p-nitrophenyl esters than human CESs. The hydrolase activities of CES2 isozymes were higher than CES1 in both species. Three to five folds faster hydrolysis for p-nitrophenyl butyrate than p-nitrophenyl acetate was observed in all CES isozymes except of cynomolgus CES1 that showed nearly same hydrolysis for both substrates. The provided method could be widely used for universal quantitative analysis of recombinant serine hydrolases.

Diterpenoids from the roots of Euphorbia ebracteolata and their inhibitory effects on human carboxylesterase 2

A chemical investigation of the roots of Euphorbia ebracteolata identified eighteen diterpenoids and glycosides. On the basis of spectroscopic data, they were determined to be ent-kauranes, ent-atisanes, tigliane derivatives, ingenane, and ent-abietanes, among which were eleven previously undescribed diterpenoids. The inhibitory effects of the isolated compounds against human carboxylesterase 2 (hCE-2) were evaluated in vitro, which revealed moderate inhibitory effects with IC50 values < 50 μM. Next, the inhibitory kinetics were evaluated for the putative hCE-2 inhibitor 4β,9α,16,20-tetrahydroxy-14(13 → 12)-abeo-12αH-1,6-tigliadiene-3,13-dione (IC50 3.88 μM), and results indicated competitive inhibition with Ki 4.94 μM. Additionally, none of the diterpenoids showed cytotoxic effects against five human tumor cell lines as determined by MTT assays.

A bioactive new protostane-type triterpenoid from Alisma plantago-aquatica subsp. orientale (Sam.) Sam.

A new protostane-type triterpenoid, 5β,29-dihydroxy alisol A (1) was isolated from Alisma plantago-aquatica subsp. orientale (Sam.) Sam. as well as 12-deoxyphorbol-13α-pentadecanoate (2). We first report the presence of compound 2 in the genus Alisma. Their structures were established on the basis of 1D and 2D NMR, and HRESIMS spectroscopic analyses. All the isolated compounds were assayed for their inhibitory effects against human carboxylesterase 2 (HCE-2). Compounds 1 and 2 displayed inhibitory activities against HCE-2 with IC50 values of 29.2 and 4.6 μM, respectively. The interaction mechanisms of HCE-2 with compounds 1 and 2 were investigated by molecular docking, respectively.

Development of amide-based fluorescent probes for selective measurement of carboxylesterase 1 activity in tissue extracts

Carboxylesterases are well known for their role in the metabolism of xenobiotics. However, recent studies have also implicated carboxylesterases in regulating a number of physiological processes including metabolic homeostasis and macrophage development, underlying the need to quantify them individually. Unfortunately, current methods for selectively measuring the catalytic activity of individual carboxylesterases are not sufficiently sensitive to support many biological studies. In order to develop a more sensitive and selective method to measure the activity of human carboxylesterase 1 (hCE1), we generated and tested novel substrates with a fluorescent aminopyridine leaving group. hCE1 showed at least a 10-fold higher preference for the optimized substrate 4-MOMMP than the 13 other esterases tested. Because of the high stability of 4-MOMMP and its hydrolysis product, this substrate can be used to measure esterase activity over extended incubation periods yielding a low picogram (femtomol) limit of detection. This sensitivity is comparable to current ELISA methods; however, the new assay quantifies only the catalytically active enzyme facilitating direct correlation to biological processes. The method described herein may allow hCE1 activity to be used as a biomarker for predicting drug pharmacokinetics, early detection of hepatocellular carcinoma, and other disease states where the activity of hCE1 is altered.

Improvement of the self-consistent-charge density-functional-tight-binding theory by a modified Mulliken charge

Although extensive efforts had been carried out to improve the accuracy of the self-consistent-charge density-functional-tight-binding (SCC-DFTB), the application of SCC-DFTB on biological systems (e.g., enzymes) is still limited. Our benchmark calculations show that the original SCC-DFTB/MM is not able to properly descript the human carboxylesterase 1 (CES1) catalyzed hydrolysis of d-threo-methylphenidate (dMD). In contrast to the ab initio QM/MM results, SCC-DFTB/MM underestimates the activation free energy barrier and renders the acylation process as a single-step reaction without a tetrahedral intermediate. It seems like that SCC-DFTB/MM misestimates the developing negative QM Mulliken charge of the substrate oxygen atom in the oxyanion hole. To improve the SCC-DFTB energy and the electrostatic interaction between QM (SCC-DFTB) and MM atoms, we adopt an optimization strategy for QM Mulliken charge, in which an empirical parameter is trained to fit the SCC-DFTB Mulliken charge to high-level DFT method along the reaction coordinate. Herein, the optimized Mulliken charge-based SCC-DFTB method is denoted as SCC-DFTBMR. Finally, benchmark and free energy calculations were performed to prove the applicability of SCC-DFTBMR to CES1-catalyzed reaction.

Difference in substrate specificity of carboxylesterase and arylacetamide deacetylase between dogs and humans

Carboxylesterase (CES) and arylacetamide deacetylase (AADAC) are the major enzymes responsible for the hydrolysis of various clinical drugs. Our recent study demonstrated that the identity of the responsible hydrolase can be roughly surmised based on the chemical structures of compounds in humans. Dogs are used for preclinical studies in drug development, but the substrate specificities of dog CES and AADAC remain to be clarified. The purpose of this study is to characterize their substrate specificities. We prepared recombinant dog CES1, CES2, and AADAC. p-Nitrophenyl acetate, a general substrate for esterases, was hydrolyzed by dog CES1 and AADAC, while it was not hydrolyzed by CES2. CES2 protein was not substantially detected in the recombinant system or in the dog liver and intestinal microsomes by Western blot using anti-human CES2 antibodies. In silico analyses demonstrated slight differences in the three-dimensional structures of dog CES2 and human CES2, indicating that dog CES2 might be unstable or inactive. By evaluating the hydrolase activities of 22 compounds, which are known to be substrates of human CES and/or AADAC, we found that the activities of dog recombinant CES1 and AADAC as well as dog tissue preparations for nearly all compounds were lower than those of human enzymes. The dog enzymes that were responsible for the hydrolysis of most compounds corresponded to the human enzymes, but the following differences were observed: oseltamivir, irinotecan, and rifampicin were not hydrolyzed in the dog liver or by any of the recombinant esterases and procaine, a human CES2 substrate, was hydrolyzed by dog CES1. In conclusion, the present study could provide new finding to facilitate our understanding of species differences in drug hydrolysis, which can facilitate drug development and drug safety evaluation.