Human Carboxylesterase Research Articles

Carboxylesterase 2a deletion provokes hepatic steatosis and insulin resistance in mice involving impaired diacylglycerol and lysophosphatidylcholine catabolism

ObjectiveHepatic triacylglycerol accumulation and insulin resistance are key features of NAFLD. However, NAFLD development and progression are rather triggered by the aberrant generation of lipid metabolites and signaling molecules including diacylglycerol (DAG) and lysophosphatidylcholine (lysoPC). Recent studies showed decreased expression of carboxylesterase 2 (CES2) in the liver of NASH patients and hepatic DAG accumulation was linked to low CES2 activity in obese individuals. The mouse genome encodes several Ces2 genes with Ces2a showing highest expression in the liver. Herein we investigated the role of mouse Ces2a and human CES2 in lipid metabolism in vivo and in vitro. MethodsLipid metabolism and insulin signaling were investigated in mice lacking Ces2a and in a human liver cell line upon pharmacological CES2 inhibition. Lipid hydrolytic activities were determined in vivo and from recombinant proteins. ResultsCes2a deficient mice (Ces2a-ko) are obese and feeding a high-fat diet (HFD) provokes severe hepatic steatosis and insulin resistance together with elevated inflammatory and fibrotic gene expression. Lipidomic analysis revealed a marked rise in DAG and lysoPC levels in the liver of Ces2a-ko mice fed HFD. Hepatic lipid accumulation in Ces2a deficiency is linked to lower DAG and lysoPC hydrolytic activities in liver microsomal preparations. Moreover, Ces2a deficiency significantly increases hepatic expression and activity of MGAT1, a PPAR gamma target gene, suggesting aberrant lipid signaling upon Ces2a deficiency. Mechanistically, we found that recombinant Ces2a and CES2 show significant hydrolytic activity towards lysoPC (and DAG) and pharmacological inhibition of CES2 in human HepG2 cells largely phenocopies the lipid metabolic changes present in Ces2a-ko mice including reduced lysoPC and DAG hydrolysis, DAG accumulation and impaired insulin signaling. ConclusionsCes2a and CES2 are critical players in hepatic lipid signaling likely via the hydrolysis of DAG and lysoPC at the ER.

The roles of serine hydrolases and serum albumin in alisol B 23-acetate hydrolysis in humans.

Introduction: Alisol B 23-acetate (AB23A), a major bioactive constituent in the Chinese herb Zexie (Rhizoma Alismatis), has been found with multiple pharmacological activities. AB23A can be readily hydrolyzed to alisol B in mammals, but the hydrolytic pathways of AB23A in humans and the key enzymes responsible for AB23A hydrolysis are still unrevealed. This study aims to reveal the metabolic organs and the crucial enzymes responsible for AB23A hydrolysis in human biological systems, as well as to decipher the impact of AB23A hydrolysis on its biological effects. Methods: The hydrolytic pathways of AB23A in human plasma and tissue preparations were carefully investigated by using Q-Exactive quadrupole-Orbitrap mass spectrometer and LC-UV, while the key enzymes responsible for AB23A hydrolysis were studied via performing a set of assays including reaction phenotyping assays, chemical inhibition assays, and enzyme kinetics analyses. Finally, the agonist effects of both AB23A and its hydrolytic metabolite(s) on FXR were tested at the cellular level. Results: AB23A could be readily hydrolyzed to form alisol B in human plasma, intestinal and hepatic preparations, while human butyrylcholinesterase (hBchE) and human carboxylesterases played key roles in AB23A hydrolysis in human plasma and tissue preparations, respectively. It was also found that human serum albumin (hSA) could catalyze AB23A hydrolysis, while multiple lysine residues of hSA were covalently modified by AB23A, suggesting that hSA catalyzed AB23A hydrolysis via its pseudo-esterase activity. Biological tests revealed that both AB23A and alisol B exhibited similar FXR agonist effects, indicating AB23A hydrolysis did not affect its FXR agonist effect. Discussion: This study deciphers the hydrolytic pathways of AB23A in human biological systems, which is very helpful for deep understanding of the metabolic rates of AB23A in humans, and useful for developing novel prodrugs of alisol B with desirable pharmacokinetic behaviors.

Modular Catalysis: Aptamer Enhancement of Enzyme Kinetics in a Nanoparticle Reactor.

Enzyme activity requires sequential binding and chemical transformation of substrates. While directed evolution and random mutagenesis are common methods for improving catalytic activity, these methods do not allow for independent control of KM and kcat. To achieve such control, we envisioned that the colocalization of aptamers and enzymes that act on the same molecule could increase catalytic efficiency through preconcentration of substrate. We explored this concept with cocaine esterase and anticocaine aptamers having varying KD values, both encapsulated in MS2 virus-like particles. Rate enhancements were observed with magnitudes dependent on both aptamer:enzyme stoichiometry and aptamer KD, peaking when aptamer KD and enzyme KM were roughly equivalent. This beneficial effect was lost when either aptamer binding was too tight or the aptamers were not constrained to be close to the catalyst. This work demonstrates a modular way to enhance catalysis by independently controlling substrate capture and release to the processing enzyme.

A novel genetically modified mouse seizure model for evaluating anticonvulsive and neuroprotective efficacy of an A1 adenosine receptor agonist following soman intoxication

Recently a novel humanized mouse strain has been successfully generated, in which serum carboxylesterase (CES) knock out (KO) mice (Es1−/−) were further genetically modified by knocking in (KI), or adding, the gene that encodes the human form of acetylcholinesterase (AChE). The resulting human AChE KI and serum CES KO (or KIKO) mouse strain should not only exhibit organophosphorus nerve agent (NA) intoxication in a manner more similar to humans, but also display AChE-specific treatment responses more closely mimicking those of humans to facilitate data translation to pre-clinic trials. In this study, we utilized the KIKO mouse to develop a seizure model for NA medical countermeasure investigation, and then applied it to evaluate the anticonvulsant and neuroprotectant (A/N) efficacy of a specific A1 adenosine receptor (A1AR) agonist, N-bicyclo-(2.2.1)hept-2-yl-5′-chloro-5′-deoxyadenosine (ENBA), which has been shown in a rat seizure model to be a potent A/N compound. Male mice surgically implanted with cortical electroencephalographic (EEG) electrodes a week earlier were pretreated with HI-6 and challenged with various doses (26 to 47 μg/kg, SC) of soman (GD) to determine a minimum effective dose (MED) that induced sustained status epilepticus (SSE) activity in 100% of animals while causing minimum lethality at 24 h. The GD dose selected was then used to investigate the MED doses of ENBA when given either immediately following SSE initiation (similar to wartime military first aid application) or at 15 min after ongoing SSE seizure activity (applicable to civilian chemical attack emergency triage). The selected GD dose of 33 μg/kg (1.4 x LD50) generated SSE in 100% of KIKO mice and produced only 30% mortality. ENBA at a dose as little as 10 mg/kg, IP, caused isoelectric EEG activity within minutes after administration in naïve un-exposed KIKO mice. The MED doses of ENBA to terminate GD-induced SSE activity were determined to be 10 and 15 mg/kg when treatment was given at the time of SSE onset and when seizure activity was ongoing for 15 min, respectively. These doses were much lower than in the non-genetically modified rat model, which required an ENBA dose of 60 mg/kg to terminate SSE in 100% GD-exposed rats. At MED doses, all mice survived for 24 h, and no neuropathology was observed when the SSE was stopped. The findings confirmed that ENBA is a potent A/N for both immediate and delayed (i.e., dual purposed) therapy to victims of NA exposure and serves as a promising neuroprotective antidotal and adjunctive medical countermeasure candidate for pre-clinical research and development for human application.

Insights into the Degradation of Polymer-Drug Conjugates by an Overexpressed Enzyme in Cancer Cells.

Intensive efforts have been made to provide better treatments to cancer patients. Currently, nanoparticle-based drug delivery systems have gained propulsion, as they can overcome the drawbacks of free drugs. However, drug stability inside the nanocapsule must be ensured to prevent burst release. To overcome this, drugs conjugated to amphiphilic copolymers, assembled into nanoparticles, can provide a sustained release if endogenously degraded. Thus, we have designed and assessed the drug release viability of polymer-drug conjugates by the human Carboxylesterase 2, for a targeted drug activation. We performed molecular dynamics simulations applying a quantum mechanics/molecular mechanics potential to study the degradation profiles of 30 designed conjugates, where six were predicted to be hydrolyzed by this enzyme. We further analyzed the enzyme-substrate environment to delve into what structural features may lead to successful hydrolysis. These findings contribute to the development of new medicines ensuring effective cancer treatments with fewer side effects.

Unlocking the strength of inducible promoters in Gram-negative bacteria.

Inducible bacterial promoters are ubiquitous biotechnology tools that have a consistent architecture including two key elements: the operator region recognized by the transcriptional regulatory proteins, and the -10 and -35 consensus sequences required to recruit the sigma (σ) 70 subunits of RNA polymerase to initiate transcription. Despite their widespread use, leaky transcription in the OFF state remains a challenge. We have updated the architecture of the lac and tet promoters to improve their strength, control and portability by the adaptation of the consensus -10 and -35 sequence boxes strongly targeted by σ70 , incorporation of a strong ribosome binding site recognized broadly by Gram-negative bacteria, and independent control of the transcriptional regulators by constitutive promoters. To test the promoters, we use the far-red fluorescent protein mCardinal, which significantly improves the signal-to-background ratio of promoter measurements over widely utilized green fluorescent proteins. We validate the improvement in OFF state control and inducibility by demonstrating production of the toxic and aggregate-prone cocaine esterase enzyme CocE. We further demonstrate portability of the promoters to additional Gram-negative species Pseudomonas putida and Vibrio natriegens. Our results represent a significant improvement over existing protein expression systems that will enable advances in protein production for various biotechnology applications.

A Space-Dependent 'Enzyme-Substrate' Type Probe based on 'Carboxylesterase-Amide Group' for Ultrafast Fluorescent Imaging Orthotopic Hepatocellular Carcinoma.

Fast and selective fluorescence imaging for a biomarker to related-disease diagnosis remains a significant challenge due to complex physical environment. Human carboxylesterase (CE) is expected to be a potential biomarker of hepatocellular carcinoma (HCC) to improve the accuracy of diagnosis. However, existing probes for CE has slow response rate and low selectivity. Herein, the amide group is selected as CE-responsive sites based on the "substrate-hydrolysis enzymatic reaction" approach. From a series of off-on probes with leave groups in the amide unit, probe JFast is screened with the optimal combination of rapid response rate and high selectivity toward CE. JFast requires only 150s to reach the maximum fluorescence at 676nm in the presence of CE and free from the interference of other esterase. Computational docking simulations indicate the shortest distance between the CE and active site of JFast . Cell and in vivo imaging present that the probe can turn on the liver cancer cells and tumor region precisely. Importantly, JFast is allowed to specifically image orthotopic liver tumor rather than metastatic tumor and distinguish human primary liver cancer tissue from adjacent ones. This study provides a new tool for CE detection and promotes advancements in accurate HCC diagnosis.

Structural insights into catalytical capability for CPT11 hydrolysis and substrate specificity of a novel marine microbial carboxylesterase, E93.

CPT11 (Irinotecan; 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is an important camptothecin-based broad-spectrum anticancer prodrug. The activation of its warhead, SN38 (7-ethyl-10-hydroxycamptothecin), requires hydrolysis by carboxylesterases. NPC (7-ethyl-10-[4-(1-piperidino)-1-amino] carbonyloxycamptothecin) is a metabolic derivative of CPT11 and is difficult to be hydrolyzed by human carboxylesterase. Microbial carboxylesterase with capability on both CPT11 and NPC hydrolysis is rarely reported. A marine microbial carboxylesterase, E93, was identified to hydrolyze both substrates in this study. This enzyme was an appropriate subject for uncovering the catalytic mechanism of carboxylesterases to CPT11 and NPC hydrolysis. X-ray diffraction method was applied to obtain high-resolution structure of E93. Molecular docking was adopted to analyze the interaction of E93 with p-NP (p-nitrophenyl), CPT11, and NPC substrates. Mutagenesis and enzymatic assay were adopted to verify the binding pattern of substrates. Three core regions (Region A, B, and C) of the catalytic pocket were identified and their functions on substrates specificity were validated via mutagenesis assays. The Region A was involved in the binding with the alcohol group of all tested substrates. The size and hydrophobicity of the region determined the binding affinity. The Region B accommodated the acyl group of p-NP and CPT11 substrates. The polarity of this region determined the catalytic preference to both substrates. The Region C specifically accommodated the acyl group of NPC. The interaction from the acidic residue, E428, contributed to the binding of E93 with NPC. The study analyzed both unique and conserved structures of the pocket in E93, for the first time demonstrating the discrepancy of substrate-enzyme interaction between CPT11 and NPC. It also expanded the knowledge about the substrate specificity and potential application of microbial Family VII carboxylesterases.

Quantitative evaluation of human carboxylesterase 1 by SERS-ELISA using a synergistic enhancement strategy based on gold nanoparticles and metal–organic framework

In this study, we synthesized gold nanoparticles (AuNPs) in situ on the surface of two-dimensional Cu-TCPP (meso-tetra(4-carboxyphenyl)porphine) metal– organic framework (MOF) nanosheets to produce AuNP@Cu-TCPP nanocomposites as surface-enhanced Raman scattering (SERS) substrates. The SERS signal was enhanced in two ways: aggregation of the AuNPs@Cu-TCPP near the SERS hotspot through the strong adsorption capacity of the MOF, and the synergistic effect between the charge-transfer enhancement of the Cu-TCPP MOF and the electromagnetic enhancement of plasmonic metal AuNPs. The SERS activity of AuNPs@Cu-TCPP was evaluated by the analysis of multiple model analytes; Rhodamine 6G (R6G) and benzidine were detected with detection limits below 10–13 and 10–12 mol·L–1, respectively. We used AuNP@Cu-TCPP as a SERS substrate combined with an enzyme-linked immuno- sorbent assay (ELISA) to detect the tumor marker carboxylesterase 1 (hCE1) in human serum. The SERS signal of benzidine at 1609 cm−1 is linearly correlated with hCE1 in the concentration range of 0.1–120 ng·mL−1. Owing to their high sensitivity and excellent SERS sensing capability, AuNPs@Cu-TCPP nanocomposites have potential applicability in the analysis of clinical samples.

Characterisation of AMB-FUBINACA metabolism and CB1-mediated activity of its acid metabolite

PurposeAMB-FUBINACA is a synthetic cannabinoid receptor agonist (SCRA) which is primarily metabolised by hepatic enzymes producing AMB-FUBINACA carboxylic acid. The metabolising enzymes associated with this biotransformation remain unknown. This study aimed to determine if AMB-FUBINACA metabolism could be reduced in the presence of carboxylesterase (CES) inhibitors and recreational drugs commonly consumed with it. The affinity and activity of the AMB-FUBINACA acid metabolite at the cannabinoid type-1 receptor (CB1) was investigated to determine the activity of the metabolite.MethodsThe effect of CES1 and CES2 inhibitors, and delta-9-tetrahydrocannabinol (Δ9-THC) on AMB-FUBINACA metabolism were determined using both human liver microsomes (HLM) and recombinant carboxylesterases. Radioligand binding and cAMP assays comparing AMB-FUBINACA and AMB-FUBINACA acid were carried out in HEK293 cells expressing human CB1.ResultsAMB-FUBINACA was rapidly metabolised by HLM in the presence and absence of NADPH. Additionally, CES1 and CES2 inhibitors both significantly reduced AMB-FUBINACA metabolism. Furthermore, digitonin (100 µM) significantly inhibited CES1-mediated metabolism of AMB-FUBINACA by ~ 56%, while the effects elicited by Δ9-THC were not statistically significant. AMB-FUBINACA acid produced only 26% radioligand displacement consistent with low affinity binding. In cAMP assays, the potency of AMB-FUBINACA was ~ 3000-fold greater at CB1 as compared to the acid metabolite.ConclusionsCES1A1 was identified as the main hepatic enzyme responsible for the metabolism of AMB-FUBINACA to its less potent carboxylic acid metabolite. This biotransformation was significantly inhibited by digitonin. Since other xenobiotics may also inhibit similar SCRA metabolic pathways, understanding these interactions may elucidate why some users experience high levels of harm following SCRA use.

In vitro metabolic fate of the synthetic cannabinoid receptor agonists (quinolin-8-yl 4-methyl-3-(morpholine-4-sulfonyl)benzoate [QMMSB]) and (quinolin-8-yl 4-methyl-3-((propan-2-yl)sulfamoyl)benzoate [QMiPSB]) including isozyme mapping and carboxylesterases activity testing.

The synthetic cannabinoid receptor agonists (SCRAs) (quinolin-8-yl 4-methyl-3-(morpholine-4-sulfonyl)benzoate [QMMSB]) and (quinolin-8-yl 4-methyl-3-((propan-2-yl)sulfamoyl)benzoate [QMiPSB], also known as SGT-46) are based on the structure of quinolin-8-yl 4-methyl-3-(piperidine-1-sulfonyl)benzoate (QMPSB) that has been identified on seized plant material in 2011. In clinical toxicology, knowledge of the metabolic fate is important for their identification in biosamples. Therefore, the aim of this study was the identification of in vitro Phase I and II metabolites of QMMSB and QMiPSB in pooled human liver S9 fraction (pHLS9) incubations for use as screening targets. In addition, the involvement of human monooxygenases and human carboxylesterases (hCES) was examined. Analyses were performed by liquid chromatography coupled with high-resolution tandem mass spectrometry. Ester hydrolysis was found to be an important step in the Phase I metabolism of both SCRAs, with the carboxylic acid product being found only in negative ionization mode. Monohydroxy and N-dealkyl metabolites of the ester hydrolysis products were detected as well as glucuronides. CYP2C8, CYP2C9, CYP3A4, and CYP3A5 were involved in hydroxylation. Whereas enzymatic ester hydrolysis of QMiPSB was mainly catalyzed by hCES1 isoforms, nonenzymatic ester hydrolysis was also observed. The results suggest that ester hydrolysis products of QMMSB and QMiPSB and their glucuronides are suitable targets for toxicological screenings. The additional use of the negative ionization mode is recommended to increase detectability of analytes. Different cytochrome P450 (CYP) isozymes were involved in the metabolism; thus, the probability of drug-drug interactions due to CYP inhibition can be assessed as low.

Involvement of esterases in the pulmonary metabolism of beclomethasone dipropionate and the potential influence of cannabis use

Beclomethasone dipropionate (BDP) is an inhaled glucocorticoid used for maintenance treatment of asthma in adults and children. BDP is a prodrug activated in lung when hydrolyzed to its major active metabolite beclomethasone-17-monopropionate (17-BMP), which can be further deactivated to beclomethasone (BOH). The specific hydrolases contributing to these processes have not been identified which warrants an investigation to enable a better assessment of the drug-drug interaction (DDI) liability and a better management of drug efficacy and systemic toxicity. In the present study, the pulmonary metabolism of BDP was investigated using both human lung S9 (HLuS9) and recombinant carboxylesterase 1 (CES1) S9. By employing the relative activity approach, we tested the hypothesis of CES1 being the major enzyme involved. Assessment of other hydrolases were conducted in an assay with selective esterase inhibitors. In addition, the DDI potentials between BDP and Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) were evaluated due to the increasing use of inhaled cannabis both recreationally and medically. The mechanism of DDI was conducted in an in vitro time-dependent inhibition assay, and further interpreted utilizing a proposed model. In HLuS9, BDP was efficiently metabolized almost completely to 17-BMP, which was then converted to BOH at a much lower rate. CES1 was found as a minor contributor accounting for only 1.4% of BDP metabolism in HLuS9, while arylacetamide deacetylase might be the main enzyme involved. Both THC and CBD inhibited the HLuS9 mediated BDP hydrolysis in a reversible manner, with reported IC50 values estimated as 8.98 and 36.8 μM, respectively. Our proposed model suggested a moderately decreased 17-BMP exposure in lung by concomitant THC from a cannabis cigarette, while the effects from orally administered CBD was expected to be of no clinical relevance.

Esterases Involved in the Rapid Bioconversion of Esmolol after Intravenous Injection in Humans.

Esmolol is indicated for the acute and temporary control of ventricular rate due to its rapid onset of action and elimination at a rate greater than cardiac output. This rapid elimination is achieved by the hydrolysis of esmolol to esmolol acid. It has previously been reported that esmolol is hydrolyzed in the cytosol of red blood cells (RBCs). In order to elucidate the metabolic tissues and enzymes involved in the rapid elimination of esmolol, a hydrolysis study was performed using different fractions of human blood and liver. Esmolol was slightly hydrolyzed by washed RBCs and plasma proteins while it was extensively hydrolyzed in plasma containing white blood cells and platelets. The negligible hydrolysis of esmolol in RBCs is supported by its poor hydrolysis by esterase D, the sole cytosolic esterase in RBCs. In human liver microsomes, esmolol was rapidly hydrolyzed according to Michaelis-Menten kinetics, and its hepatic clearance, calculated by the well-stirred model, was limited by hepatic blood flow. An inhibition study and a hydrolysis study using individual recombinant esterases showed that human carboxylesterase 1 isozyme (hCE1) is the main metabolic enzyme of esmolol in both white blood cells and human liver. These studies also showed that acyl protein thioesterase 1 (APT1) is involved in the cytosolic hydrolysis of esmolol in the liver. The hydrolysis of esmolol by hCE1 and APT1 also results in its pulmonary metabolism, which might be a reason for its high total clearance (170-285 mL/min/kg bodyweight), 3.5-fold greater than cardiac output (80.0 mL/min/kg bodyweight).

Defining the Chemical Additives Driving In Vitro Toxicities of Plastics.

Increases in the global use of plastics have caused concerns regarding potential adverse effects on human health. Plastic products contain hundreds of potentially toxic chemical additives, yet the exact chemicals which drive toxicity currently remain unknown. In this study, we employed nontargeted analysis and in vitro bioassays to identify the toxicity drivers in plastics. A total of 56 chemical additives were tentatively identified in five commonly used plastic polymer pellets (i.e., PP, LDPE, HDPE, PET, and PVC) by employing suspect screening and nontargeted analysis. Phthalates and organophosphates were found to be dominant in PVC pellets. Triphenyl phosphate and 2-ethylhexyl diphenyl phosphate accounted for a high amount (53.6%) of the inhibition effect of PVC pellet extract on human carboxylesterase 1 (hCES1) activity. Inspired by the high abundances of chemical additives in PVC pellets, six different end-user PVC-based products including three widely used PVC water pipes were further examined. Among them, extracts of PVC pipe exerted the strongest PPARγ activity and cell viability suppression. Organotins were identified as the primary drivers to these in vitro toxicities induced by the PVC pipe extracts. This study clearly delineates specific chemical additives responsible for hCES1 inhibition, PPARγ activity, and cell viability suppression associated with plastic.

Carboxylesterase 1d Inactivation Augments Lung Inflammation in Mice.

Carboxylesterases are members of the serine hydrolase superfamily and metabolize drugs, pesticides, and lipids. Previous research showed that inhibition of carboxylesterase 1 (CES1) in human macrophages altered the immunomodulatory effects of lipid mediators called prostaglandin glyceryl esters, which are produced by cyclooxygenase-catalyzed oxygenation of the endocannabinoid 2-arachidonoylglycerol (2-AG). Ces1d - the mouse ortholog of human CES1 - is the most abundant Ces isoform in murine lung tissues and alveolar macrophages and a major target of organophosphate poisons. Monoacylglycerol lipase (Magl) is also expressed in murine lung and is the main enzyme responsible for 2-AG catabolism. Several metabolic benefits are observed in Ces1d-/- mice fed a high-fat diet; thus, we wondered whether pharmacological and genetic inactivation of Ces1d in vivo might also ameliorate the acute inflammatory response to lipopolysaccharide (LPS). C57BL/6 mice were treated with WWL229 (Ces1d inhibitor) or JZL184 (Magl inhibitor), followed 30 min later by either LPS or saline. Wild-type (WT) and Ces1d-/- mice were also administered LPS to determine the effect of Ces1d knockout. Mice were sacrificed at 6 and 24 h, and cytokines were assessed in serum, lung, liver, and adipose tissues. Lipid mediators were quantified in lung tissues, while activity-based protein profiling and enzyme assays determined the extent of lung serine hydrolase inactivation by the inhibitors. WWL229 was shown to augment LPS-induced lung inflammation in a female-specific manner, as measured by enhanced neutrophil infiltration and Il1b mRNA. The marked Ces inhibition in female lung by 4 h after drug treatment might explain this sex difference, although the degree of Ces inhibition in female and male lungs was similar at 6 h. In addition, induction of lung Il6 mRNA and prostaglandin E2 by LPS was more pronounced in Ces1d-/- mice than in WT mice. Thus, WWL229 inhibited lung Ces1d activity and augmented the female lung innate immune response, an effect observed in part in Ces1d-/- mice and Ces1d/CES1-deficient murine and human macrophages. In contrast, JZL184 attenuated LPS-induced Il1b and Il6 mRNA levels in female lung, suggesting that Ces1d and Magl have opposing effects. Mapping the immunomodulatory molecules/pathways that are regulated by Ces1d in the context of lung inflammation will require further research.

In Vitro Metabolic Fate of the Synthetic Cannabinoid Receptor Agonists 2F-QMPSB and SGT-233 Including Isozyme Mapping and Carboxylesterases Activity Testing.

Quinolin-8-yl 3-(4,4-difluoropiperidine-1-sulfonyl)-4-methylbenzoate (2F-QMPSB) and 3-(4,4-difluoropiperidine-1-sulfonyl)-4-methyl-N-(2-phenylpropan-2-yl)benzamide (SGT-233) belong to a new group of synthetic cannabinoid receptor agonists containing a sulfamoyl benzoate or sulfamoyl benzamide core structure. 2F-QMPSB was identified in herbal material seized in Europe in 2018. The aims of this study were the identification of in vitro Phase I and II metabolites of 2F-QMPSB and SGT-233 to find analytical targets for toxicological screenings. Furthermore, the contribution of different monooxygenases and human carboxylesterases to Phase I metabolism was investigated. Liquid chromatography coupled to high-resolution tandem mass spectrometry was used for analysis. Ester hydrolysis was found to be an important step in the metabolism of 2F-QMPSB, which was catalyzed mainly by human carboxylesterases (hCES)1 isoforms. Additionally, nonenzymatic ester hydrolysis was observed in case of 2F-QMPSB. Notably, the carboxylic acid product derived from ester hydrolysis and metabolites thereof were only detectable in negative ionization mode. In case of SGT-233, mono- and dihydroxy metabolites were identified, as well as glucuronides. The cytochrome P450 (CYP) isozymes CYP2C8, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 were found to be involved in the hydroxylation of both compounds. The results of these in vitro experiments suggest that the ester hydrolysis products of 2F-QMPSB and their glucuronides are suitable targets for toxicological screenings. In the case of SGT-233, the mono- and dihydroxy metabolites were identified as suitable screening targets. The involvement of various CYP isoforms in the metabolism of both substances reduces the likelihood of drug-drug interactions due to CYP inhibition.

In vitro evaluation of the impact of Covid-19 therapeutic agents on the hydrolysis of the antiviral prodrug remdesivir

Remdesivir (RDV, Veklury®) is an FDA-approved prodrug for the treatment of hospitalized patients with COVID-19. Recent in vitro studies have indicated that human carboxylesterase 1 (CES1) is the major metabolic enzyme catalyzing RDV activation. COVID-19 treatment for hospitalized patients typically also involves a number of antibiotics and anti-inflammatory drugs. Further, individuals who are carriers of a CES1 variant (polymorphism in exon 4 codon 143 [G143E]) may experience impairment in their ability to metabolize therapeutic agents which are CES1 substrates. The present study assessed the potential influence of nine therapeutic agents (hydroxychloroquine, ivermectin, erythromycin, clarithromycin, roxithromycin, trimethoprim, ciprofloxacin, vancomycin, and dexamethasone) commonly used in treating COVID-19 and 5 known CES1 inhibitors on the metabolism of RDV. Additionally, we further analyzed the mechanism of inhibition of cannabidiol (CBD), as well as the impact of the G143E polymorphism on RDV metabolism. An in vitro S9 fraction incubation method and in vitro to in vivo pharmacokinetic scaling were utilized. None of the nine therapeutic agents evaluated produced significant inhibition of RDV hydrolysis; CBD was found to inhibit RDV hydrolysis by a mixed type of competitive and noncompetitive partial inhibition mechanism. In vitro to in vivo modeling suggested a possible reduction of RDV clearance and increase of AUC when coadministration with CBD. The same scaling method also suggested a potentially lower clearance and higher AUC in the presence of the G143E variant. In conclusion, a potential CES1-mediated DDI between RDV and the nine assessed medications appears unlikely. However, a potential CES1-mediated DDI between RDV and CBD may be possible with sufficient exposure to the cannabinoid. Patients carrying the CES1 G143E variant may exhibit a slower biotransformation and clearance of RDV. Further clinical studies would be required to evaluate and characterize the clinical significance of a CBD-RDV interaction.

Fluorine impairs carboxylesterase 1-mediated hydrolysis of T-2 toxin and increases its chondrocyte toxicity.

BackgroundT-2 toxin is recognized as one of the high-risk environmental factors for etiology and pathogenesis of Kashin-Beck disease (KBD). Previous evidence indicates decreased serum fluorine level in KBD patients. However, whether fluoride could regulate carboxylesterase 1 (CES1)-mediated T-2 toxin hydrolysis and alter its chondrocyte toxicity remains largely unknown.MethodsIn this study, in vitro hydrolytic kinetics were explored using recombinant human CES1. HPLC-MS/MS was used to quantitative determination of hydrolytic metabolites of T-2 toxin. HepG2 cells were treated with different concentration of sodium fluoride (NaF). qRT-PCR and western blot analysis were used to compare the mRNA and protein expression levels of CES1. C28/I2 cells were treated with T-2 toxin, HT-2 toxin, and neosolaniol (NEO), and then cell viability was determined by MTT assay, cell apoptosis was determined by Annexin V-FITC/PI, Hoechst 33258 staining, and cleaved caspase-3, and cell cycle was monitored by flow cytometry assay, CKD4 and CDK6.ResultsWe identified that recombinant human CES1 was involved in T-2 toxin hydrolysis to generate HT-2 toxin, but not NEO, and NaF repressed the formation of HT-2 toxin. Both mRNA and protein expression of CES1 were significantly down-regulated in a dose-dependent manner after NaF treatment in HepG2 cells. Moreover, we evaluated the chondrocyte toxicity of T-2 toxin and its hydrolytic metabolites. Results showed that T-2 toxin induced strongest cell apoptosis, followed by HT-2 toxin and NEO. The decreased the proportion of cells in G0/G1 phase was observed with the descending order of T-2 toxin, HT-2 toxin, and NEO.ConclusionsThis study reveals that CES1 is responsible for the hydrolysis of T-2 toxin, and that fluoride impairs CES1-mediated T-2 toxin detoxification to increase its chondrocyte toxicity. This study provides novel insight into understanding the relationship between fluoride and T-2 toxin in the etiology of KBD.

Going deeper into the toxicokinetics of synthetic cannabinoids: in vitro contribution of human carboxylesterases

Synthetic cannabinoids (SC) are new psychoactive substances known to cause intoxications and fatalities. One reason may be the limited data available concerning the toxicokinetics of SC, but toxicity mechanisms are insufficiently understood so far. Human carboxylesterases (hCES) are widely known to play a crucial role in the catalytic hydrolysis of drugs (of abuse). The aim of this study was to investigate the in vitro contribution of hCES to the metabolism of the 13 SC 3,5-AB-5F-FUPPYCA, AB-5F-P7AICA, A-CHMINACA, DMBA-CHMINACA, MBA-CHMINACA, MDMB-4F-BINACA, MDMB-4en-PINACA, MDMB-FUBICA, MDMB-5F-PICA, MMB-CHMICA, MMB-4en-PICA, MMB-FUBINACA, and MPhP-5F-PICA. The SC were incubated with recombinant hCES1b, hCES1c, or hCES2 and analyzed by liquid chromatography–ion trap mass spectrometry to assess amide or ester hydrolysis in an initial activity screening. Enzyme kinetic studies were performed if sufficient hydrolysis was observed. No hydrolysis of the amide linker was observed using those experimental conditions. Except for MDMB-5F-PICA, ester hydrolysis was always detected if an ester group was present in the head group. In general, SC with a terminal ester bearing a small alcohol part and a larger acyl part showed higher affinity to hCES1 isozymes. Due to the low hydrolysis rates, enzyme kinetics could not be modeled for the SC with a tert-leucine-derived moiety, but hydrolysis reactions of MPhP-5F-PICA and of those containing a valine-derived moiety followed classic Michaelis–Menten kinetics. In conclusion, drug–drug/drug–food interactions or hCES polymorphisms may prolong the half-life of SC and the current results help to estimate the risk of toxicity in the future after combining them with activity and clinical data.

Two-Photon Photodynamic Therapy Targeting Cancers with Low Carboxylesterase 2 Activity Guided by Ratiometric Fluorescence.

Human carboxylesterase 2 (hCES2) converts anticancer prodrugs, such as irinotecan, into their active metabolites via phase I drug metabolism. Owing to interindividual variability, hCES2 serves as a predictive marker of patient response to hCES2-activated prodrug-based therapy, whereby a low intratumoral hCES2 activity leads to therapeutic resistance. Despite the ability to identify nonresponders, effective treatments for resistant patients are needed. Clinically approved photodynamic therapy is an attractive alternative for irinotecan-resistant patients. Here, we describe the application of our hCES2-selective small-molecule ratiometric fluorescent chemosensor, Benz-AP, as a single theranostic agent given its discovered functionality as a photosensitizer. Benz-AP produces singlet oxygen and induces photocytotoxicity in cancer cells in a strong negative correlation with hCES2 activity. Two-photon excitation of Benz-AP produces fluorescence, singlet oxygen, and photocytotoxicity in tumor spheroids. Overall, Benz-AP serves as a novel theranostic agent with selective photocytotoxicity in hCES2-prodrug resistant cancer cells, making Benz-AP a promising agent for in vivo applications.