Cloning, Expression, Characterization and Immobilization of a Recombinant Carboxylesterase from the Halophilic Archaeon, Halobacterium salinarum NCR-1.

Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from Halobacterium salinarum NRC-1 was cloned and successfully expressed in Haloferax volcanii. The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using p-nitrophenyl valerate as substrate (KM = 78 µM, kcat = 0.67 s-1). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, n-hexane, n-heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications.

Preliminary Study on the Pathogenic Mechanism of Jujube Flower Disease in Honeybees (Apis mellifera ligustica) Based on Midgut Transcriptomics.

Honeybees are prone to poisoning, also known as jujube flower disease, after collecting nectar from jujube flowers, resulting in the tumultuous demise of foragers. The prevalence of jujube flower disease has become one of the main factors affecting the development of the jujube and beekeeping industries in Northern China. However, the pathogenic mechanisms underlying jujube flower disease in honeybees are poorly understood. Herein, we first conducted morphological observations of the midgut using HE-staining and found that jujube flower disease-affected honeybees displayed midgut damage with peritrophic membrane detachment. Jujube flower disease was found to increase the activity of chitinase and carboxylesterase (CarE) and decrease the activity of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and the content of CYP450 in the honeybee midgut. Transcriptomic data identified 119 differentially expressed genes in the midgut of diseased and healthy honeybees, including CYP6a13, CYP6a17, CYP304a1, CYP6a14, AADC, and AGXT2, which are associated with oxidoreductase activity and vitamin binding. In summary, collecting jujube flower nectar could reduce antioxidant and detoxification capacities of the honeybee midgut and, in more severe cases, damage the intestinal structure, suggesting that intestinal damage might be the main cause of honeybee death due to jujube nectar. This study provides new insights into the pathogenesis of jujube flower disease in honeybees.

Regulation of carboxylesterases and its impact on pharmacokinetics and pharmacodynamics: an up-to-date review.

INTRODUCTION: Carboxylesterase 1 (CES1) and carboxylesterase 2 (CES2) are among the most abundant hydrolases in humans, catalyzing the metabolism of numerous clinically important medications, such as methylphenidate and clopidogrel. The large interindividual variability in the expression and activity of CES1 and CES2 affects the pharmacokinetics (PK) and pharmacodynamics (PD) of substrate drugs. AREAS COVERED: This review provides an up-to-date overview of CES expression and activity regulations and examines their impact on the PK and PD of CES substrate drugs. The literature search was conducted on PubMed from inception to January 2024. EXPERT OPINION: Current research revealed modest associations of CES genetic polymorphisms with drug exposure and response. Beyond genomic polymorphisms, transcriptional and posttranslational regulations can also significantly affect CES expression and activity and consequently alter PK and PD. Recent advances in plasma biomarkers of drug-metabolizing enzymes encourage the research of plasma protein and metabolite biomarkers for CES1 and CES2, which could lead to the establishment of precision pharmacotherapy regimens for drugs metabolized by CESs. Moreover, our understanding of tissue-specific expression and substrate selectivity of CES1 and CES2 has shed light on improving the design of CES1- and CES2-activated prodrugs.

Assessing the impact of antiviral drugs commonly utilized during the COVID-19 pandemic on the embryonic development of Xenopus laevis.

The antiviral drugs favipiravir and oseltamivir are widely used to treat viral infections, including coronavirus 2019 (COVID-19), and their levels are expected to increase in the aquatic environment. In this study, the potential toxic and teratogenic effects of these drugs were evaluated using the frog embryo teratogenesis assay Xenopus (FETAX). In addition, glutathione S-transferase (GST), glutathione reductase (GR), catalase, carboxylesterase (CaE), and acetylcholinesterase (AChE) enzyme activities and malondialdehyde levels were measured as biochemical markers in embryos and tadpoles for comparative assessment of the sublethal effects of the test compounds. Prior to embryo exposure, drug concentrations in the exposure medium were measured with high-performance liquid chromatography. The 96-h median lethal concentration (LC50) was 137.9 and 32.3 mg/L for favipiravir and oseltamivir, respectively. The teratogenic index for favipiravir was 4.67. Both favipiravir and oseltamivir inhibited GR, CaE, and AChE activities in embryos, while favipiravir increased the GST and CaE activities in tadpoles. In conclusion, favipiravir, for which teratogenicity data are available in mammalian test organisms and human teratogenicity is controversial, inhibited Xenopus laevis embryo development and was teratogenic. In addition, sublethal concentrations of both drugs altered the biochemical responses in embryos and tadpoles, with differences between the developmental stages.

Biochemical and molecular-level effects of co-exposure to chlorpyrifos and lambda-cyhalothrin on the earthworm (Eisenia fetida).

Farmland soil organisms frequently encounter pesticide mixtures presented in their living environment. However, the underlying toxic mechanisms employed by soil animals to cope with such combined pollution have yet to be explored. This investigation aimed to reveal the changes in cellular and mRNA levels under chlorpyrifos (CPF) and lambda-cyhalothrin (LCT) co-exposures in earthworms (Eisenia fetida). Results exhibited that the combination of CPF and LCT triggered an acute synergistic influence on the animals. Most exposures resulted in significant alterations in the activities of total superoxide dismutase (T-SOD), copper/zinc superoxide dismutase (Cu/Zn-SOD), caspase 3, and carboxylesterase (CarE) compared to the basal level. Moreover, when exposed to chemical mixtures, the transcription levels of four genes [heat shock protein 70 (hsp70), gst, sod, and calreticulin (crt)] also displayed more pronounced changes compared with their individual exposures. These changes in determined parameters indicated the occurrence of oxidative stress, cell death, detoxification dysfunction, and endoplasmic reticulum damage after co-exposure to CPF and LCT in E. fetida. The comprehensive examination of mixture toxicities of CPF and LCT at different endpoints would help to understand the overall toxicity they cause to soil invertebrates. The augmented deleterious effect of these pesticides in a mixture suggested that mixture toxicity assessment was necessary for the safety evaluation and application of pesticide mixtures.

Design, synthesis, and evaluation of a carboxylesterase detection probe with therapeutic effects.

In this study, a lysosomal targeting fluorescent probe recognition on CEs was designed and synthesized. The obtained probe BF2-cur-Mor demonstrated excellent selectivity, sensitivity, pH-independence, and enzyme affinity towards CEs within 5 min. BF2-cur-Mor could enable recognition of intracellular CEs and elucidate that the CEs content of different cancer cells follows the rule of HepG2 > HCT-116 > A549 > HeLa, and the CEs expression level of hepatoma cancer cells far exceeds that of normal hepatic cells, being in good agreement with the previous reports. The ability of BF2-cur-Mor to monitor CEs in vivo was confirmed by zebrafish experiment. BF2-cur-Mor exhibits some pharmacological activity in that it can induce apoptosis in hepatocellular carcinoma cells but is weaker in normal hepatocyte cells, being expected to be a potential "diagnostic and therapeutic integration" tool for the clinical diagnosis of CEs-related diseases.

Carboxylesterase and Cytochrome P450 Confer Metabolic Resistance Simultaneously to Azoxystrobin and Some Other Fungicides in Botrytis cinerea.

Plant pathogens have frequently shown multidrug resistance (MDR) in the field, often linked to efflux and sometimes metabolism of fungicides. To investigate the potential role of metabolic resistance in B. cinerea strains showing MDR, the azoxystrobin-sensitive strain B05.10 and -resistant strain Bc242 were treated with azoxystrobin. The degradation half-life of azoxystrobin in Bc242 (9.63 days) was shorter than that in B05.10 (28.88 days). Azoxystrobin acid, identified as a metabolite, exhibited significantly lower inhibition rates on colony and conidia (9.34 and 11.98%, respectively) than azoxystrobin. Bc242 exhibited higher expression levels of 34 cytochrome P450s (P450s) and 11 carboxylesterase genes (CarEs) compared to B05.10 according to RNA-seq analysis. The expression of P450 genes Bcin_02g01260 and Bcin_12g06380, along with the CarEs Bcin_12g06360 in Saccharomyces cerevisiae, resulted in reduced sensitivity to various fungicides, including azoxystrobin, kresoxim-methyl, pyraclostrobin, trifloxystrobin, iprodione, and carbendazim. Thus, the mechanism of B. cinerea MDR is linked to metabolism mediated by the CarE and P450 genes.

Thermodynamics of a hyperthermostable carboxylesterase from Anoxybacillus geothermalis D9.

Hyperthermostable enzymes are highly desirable biocatalysts due to their exceptional stability at extreme temperatures. Recently, a hyperthermostable carboxylesterase EstD9 from Anoxybacillus geothermalis D9 was biochemically characterized. The enzyme exhibited remarkable stability at high temperature. In this study, we attempted to probe the conformational adaptability of EstD9 under extreme conditions via in silico approaches. Circular dichroism revealed that EstD9 generated new ß-sheets at 80 °C, making the core of the hydrolase fold more stable. Interestingly, the profiles of molecular dynamics simulation showed the lowest scores of radius of gyration and solvent accessible surface area (SASA) at 80 °C. Three loops were responsible for protecting the catalytic site, which resided at the interface between the large and cap domains. To further investigate the structural adaptation in extreme conditions, the intramolecular interactions of the native structure were investigated. EstD9 revealed 18 hydrogen bond networks, 7 salt bridges, and 9 hydrophobic clusters, which is higher than the previously reported thermostable Est30. Collectively, the analysis indicates that intramolecular interactions and structural dynamics play distinct roles in preserving the overall EstD9 structure at elevated temperatures. This work is relevant to both fundamental and applied research involving protein engineering of industrial thermostable enzymes.

Heterologous expression, biochemical characterization and prospects for insecticide biosensing potential of carboxylesterase Ha006a from Helicoverpa armigera.

Enzymes have attracted considerable scientific attention for their crucial role in detoxifying a wide range of harmful compounds. In today's global context, the extensive use of insecticides has emerged as a significant threat to the environment, sparking substantial concern. Insects, including economically important pests like Helicoverpa armigera, have developed resistance to conventional pest control methods through enzymes like carboxyl/cholinesterases. This study specifically focuses on a notable carboxyl/cholinesterase enzyme from Helicoverpa armigera (Ha006a), with the goal of harnessing its potential to combat environmental toxins. A total of six insecticides belonging to two different classes displayed varying inhibitory responses towards Ha006a, thereby rendering it effective in detoxifying a broader spectrum of insecticides. The significance of this research lies in discovering the bioremediation property of Ha006a, as it hydrolyzes synthetic pyrethroids (fenvalerate, λ-cyhalothrin and deltamethrin) and sequesters organophosphate (paraoxon ethyl, profenofos, and chlorpyrifos) insecticides. Additionally, the interaction studies between organophosphate insecticides and Ha006a helped in the fabrication of a novel electroanalytical sensor using a modified carbon paste electrode (MCPE). This sensor boasts impressive sensitivity, with detection limits of 0.019 µM, 0.15 µM, and 0.025 µM for paraoxon ethyl, profenofos, and chlorpyrifos, respectively. This study provides a comprehensive biochemical and biophysical characterization of the purified esterase Ha006a, showcasing its potential to remediate different classes of insecticides.

Identification and biochemical characterization of a carboxylesterase gene associated with ß-cypermethrin resistance in Dermanyssus gallinae.

Dermanyssus gallinae is a major hematophagous ectoparasite in layer hens. Although the acaricide ß-cypermethrin has been used to control mites worldwide, D. gallinae has developed resistance to this compound. Carboxylesterases (CarEs) are important detoxification enzymes that confer resistance to ß-cypermethrin in arthropods. However, CarEs associated with ß-cypermethrin resistance in D. gallinae have not yet been functionally characterized. Here, we isolated a CarE gene (Deg-CarE) from D. gallinae and assayed its activity. The results revealed significantly higher expression of Deg-CarE in the ß-cypermethrin-resistant strain (RS) than in the susceptible strain (SS) toward α-naphthyl acetate (α-NA) and ß-naphthyl acetate (ß-NA). These findings suggest that enhanced esterase activities might have contributed to ß-cypermethrin resistance in D. gallinae. Quantitative real-time PCR analysis revealed that Deg-CarE expression levels were significantly higher in adults than in other life stages. Although Deg-CarE was upregulated in the RS, significant differences in gene copy numbers were not observed. Additionally, Deg-CarE expression was significantly induced by ß-cypermethrin in both the SS and RS. Moreover, silencing Deg-CarE via RNA interference decreased the enzyme activity and increased the susceptibility of the RS to ß-cypermethrin, confirming that Deg-CarE is crucial for ß-cypermethrin detoxification. Finally, recombinant Deg-CarE (rDeg-CarE) expressed in Escherichia coli displayed high enzymatic activity toward α/ß-NA. However, metabolic analysis indicated that rDeg-CarE did not directly metabolize ß-cypermethrin. The collective findings indicate that D. gallinae resistance to ß-cypermethrin is associated with elevated CarEs protein activity and increased Deg-CarE expression levels. These findings provide insights into the metabolic resistance of D. gallinae and offer scientific guidance for the management and control of D. gallinae.

Role of carboxylesterase and arylacetamide deacetylase in drug metabolism, physiology, and pathology.

Carboxylesterases (CES1 and CES2) and arylacetamide deacetylase (AADAC), which are expressed primarily in the liver and/or gastrointestinal tract, hydrolyze drugs containing ester and amide bonds in their chemical structure. These enzymes often catalyze the conversion of prodrugs, including the COVID-19 drugs remdesivir and molnupiravir, to their pharmacologically active forms. Information on the substrate specificity and inhibitory properties of these enzymes, which would be useful for drug development and toxicity avoidance, has accumulated. Recently,in vitroandin vivostudies have shown that these enzymes are involved not only in drug hydrolysis but also in lipid metabolism. CES1 and CES2 are capable of hydrolyzing triacylglycerol, and the deletion of their orthologous genes in mice has been associated with impaired lipid metabolism and hepatic steatosis. Adeno-associated virus-mediated human CES overexpression decreases hepatic triacylglycerol levels and increases fatty acid oxidation in mice. It has also been shown that overexpression of CES enzymes or AADAC in cultured cells suppresses the intracellular accumulation of triacylglycerol. Recent reports indicate that AADAC can be up- or downregulated in tumors of various organs, and its varied expression is associated with poor prognosis in patients with cancer. Thus, CES and AADAC not only determine drug efficacy and toxicity but are also involved in pathophysiology. This review summarizes recent findings on the roles of CES and AADAC in drug metabolism, physiology, and pathology.

Ganodermanontriol Suppresses the Progression of Lung Adenocarcinoma by Activating CES2 to Enhance the Metabolism of Mycophenolate Mofetil.

New anti-lung cancer therapies are urgently required to improve clinical outcomes. Since ganodermanontriol (GDNT) has been identified as a potential antineoplastic agent, its role in lung adenocarcinoma (LUAD) is investigated in this study. Concretely, lung cancer cells were treated with GDNT and/or mycophenolate mofetil (MMF), after which MTT assay, flow cytometry and Western blot were conducted. Following bioinformatics analysis, carboxylesterase 2 (CES2) was knocked down and rescue assays were carried out in vitro. Xenograft experiment was performed on mice, followed by drug administration, measurement of tumor growth and determination of CES2, IMPDH1 and IMPDH2 expressions. As a result, the viability of lung cancer cells was reduced by GDNT or MMF. GDNT enhanced the effects of MMF on suppressing viability, promoting apoptosis and inducing cell cycle arrest in lung cancer cells. GDNT up-regulated CES2 level, and strengthened the effects of MMF on down-regulating IMPDH1 and IMPDH2 levels in the cells. IMPDH1 and IMPDH2 were highly expressed in LUAD samples. CES2 was a potential target for GDNT. CES2 knockdown reversed the synergistic effect of GDNT and MMF against lung cancer in vitro. GDNT potentiated the role of MMF in inhibiting tumor growth and expressions of CES2 and IMPDH1/2 in lung cancer in vivo. Collectively, GDNT suppresses the progression of LUAD by activating CES2 to enhance the metabolism of MMF.

Variability in plasma rifampicin concentrations and role of SLCO1B1, ABCB1, AADAC2 and CES2 genotypes in Ethiopian patients with tuberculosis.

BACKGROUND: Rifampicin, a key drug against tuberculosis (TB), displays wide between-patient pharmacokinetics variability and concentration-dependent antimicrobial effect. We investigated variability in plasma rifampicin concentrations and the role of SLCO1B1, ABCB1, arylacetamide deacetylase (AADAC) and carboxylesterase 2 (CES-2) genotypes in Ethiopian patients with TB. METHODS: We enrolled adult patients with newly diagnosed TB (n = 119) who had received 2 weeks of rifampicin-based anti-TB therapy. Venous blood samples were obtained at three time points post-dose. Genotypes for SLCO1B1 (c.388A > G, c.521T > C), ABCB1 (c.3435C > T, c.4036A > G), AADACc.841G > A and CES-2 (c.269-965A > G) were determined. Rifampicin plasma concentration was quantified using LC-MS/MS. Predictors of rifampicin Cmax and AUC0-7 h were analysed. RESULTS: The median rifampicin Cmax and AUC0-7 were 6.76 µg/mL (IQR 5.37-8.48) and 17.05 µg·h/mL (IQR 13.87-22.26), respectively. Only 30.3% of patients achieved the therapeutic efficacy threshold (Cmax>8 µg/mL). The allele frequency for SLCO1B1*1B (c.388A > G), SLCO1B1*5 (c.521T > C), ABCB1 c.3435C > T, ABCB1c.4036A > G, AADAC c.841G > A and CES-2 c.269-965A > G were 2.2%, 20.2%, 24.4%, 14.6%, 86.1% and 30.6%, respectively. Sex, rifampicin dose and ABCB1c.4036A > G, genotypes were significant predictors of rifampicin Cmax and AUC0-7. AADACc.841G > A genotypes were significant predictors of rifampicin Cmax. There was no significant influence of SLCO1B1 (c.388A > G, c.521T > C), ABCB1c.3435C > T and CES-2 c.269-965A > G on rifampicin plasma exposure variability. CONCLUSIONS: Subtherapeutic rifampicin plasma concentrations occurred in two-thirds of Ethiopian TB patients. Rifampicin exposure varied with sex, dose and genotypes. AADACc.841G/G and ABCB1c.4036A/A genotypes and male patients are at higher risk of lower rifampicin plasma exposure. The impact on TB treatment outcomes and whether high-dose rifampicin is required to improve therapeutic efficacy requires further investigation.

Evolution and diversification of carboxylesterase-like [4+2] cyclases in aspidosperma and iboga alkaloid biosynthesis.

Monoterpene indole alkaloids (MIAs) are a large and diverse class of plant natural products, and their biosynthetic construction has been a subject of intensive study for many years. The enzymatic basis for the production of aspidosperma and iboga alkaloids, which are produced exclusively by members of the Apocynaceae plant family, has recently been discovered. Three carboxylesterase (CXE)-like enzymes from Catharanthus roseus and Tabernanthe iboga catalyze regio- and enantiodivergent [4+2] cycloaddition reactions to generate the aspidosperma (tabersonine synthase, TS) and iboga (coronaridine synthase, CorS; catharanthine synthase, CS) scaffolds from a common biosynthetic intermediate. Here, we use a combined phylogenetic and biochemical approach to investigate the evolution and functional diversification of these cyclase enzymes. Through ancestral sequence reconstruction, we provide evidence for initial evolution of TS from an ancestral CXE followed by emergence of CorS in two separate lineages, leading in turn to CS exclusively in the Catharanthus genus. This progression from aspidosperma to iboga alkaloid biosynthesis is consistent with the chemotaxonomic distribution of these MIAs. We subsequently generate and test a panel of chimeras based on the ancestral cyclases to probe the molecular basis for differential cyclization activity. Finally, we show through partial heterologous reconstitution of tabersonine biosynthesis using non-pathway enzymes how aspidosperma alkaloids could have first appeared as "underground metabolites" via recruitment of promiscuous enzymes from common protein families. Our results provide insight into the evolution of biosynthetic enzymes and how new secondary metabolic pathways can emerge through small but important sequence changes following co-option of preexisting enzymatic functions.

In Silico Study of Camptothecin-Based Pro-Drugs Binding to Human Carboxylesterase 2.

Pro-drugs, which ideally release their active compound only at the site of action, i.e., in a cancer cell, are a promising approach towards an increased specificity and hence reduced side effects in chemotherapy. A popular form of pro-drugs is esters, which are activated upon their hydrolysis. Since carboxylesterases that catalyse such a hydrolysis reaction are also abundant in normal tissue, it is of great interest whether a putative pro-drug is a probable substrate of such an enzyme and hence bears the danger of being activated not just in the target environment, i.e., in cancer cells. In this work, we study the binding mode of carboxylesters of the drug molecule camptothecin, which is an inhibitor of topoisomerase I, of varying size to human carboxylesterase 2 (HCE2) by molecular docking and molecular dynamics simulations. A comparison to irinotecan, known to be a substrate of HCE2, shows that all three pro-drugs analysed in this work can bind to the HCE2 protein, but not in a pose that is well suited for subsequent hydrolysis. Our data suggest, moreover, that for the irinotecan substrate, a reactant-competent pose is stabilised once the initial proton transfer from the putative nucleophile Ser202 to the His431 of the catalytic triad has already occurred. Our simulation work also shows that it is important to go beyond the static models obtained from molecular docking and include the flexibility of enzyme-ligand complexes in solvents and at a finite temperature. Under such conditions, the pro-drugs studied in this work are unlikely to be hydrolysed by the HCE2 enzyme, indicating a low risk of undesired drug release in normal tissue.

Interplay of Ritonavir-Boosted Oral Cabazitaxel with the Organic Anion-Transporting Polypeptide (OATP) Uptake Transporters and Carboxylesterase 1 in Mice.

Intravenously administered chemotherapeutic cabazitaxel is used for palliative treatment of prostate cancer. An oral formulation would be more patient-friendly and reduce the need for hospitalization. We therefore study determinants of the oral pharmacokinetics of cabazitaxel in a ritonavir-boosted setting, which reduces the CYP3A-mediated first-pass metabolism of cabazitaxel. We here assessed the role of organic anion-transporting polypeptides (OATPs) in the disposition of orally boosted cabazitaxel and its active metabolites, using the Oatp1a/b-knockout and the OATP1B1/1B3-transgenic mice. These transporters may substantially affect plasma clearance and hepatic and intestinal drug disposition. The pharmacokinetics of cabazitaxel and DM2 were not significantly affected by Oatp1a/b and OATP1B1/1B3 activity. In contrast, the plasma AUC0-120 min of DM1 in Oatp1a/b-/- was 1.9-fold (p < 0.05) higher than that in wild-type mice, and that of docetaxel was 2.4-fold (p < 0.05) higher. We further observed impaired hepatic uptake and intestinal disposition for DM1 and docetaxel in the Oatp-ablated strains. None of these parameters showed rescue by the OATP1B1 or -1B3 transporters in the humanized mouse strains, suggesting a minimal role of OATP1B1/1B3. Ritonavir itself was also a potent substrate for mOatp1a/b, showing a 2.9-fold (p < 0.0001) increased plasma AUC0-120 min and 3.5-fold (p < 0.0001) decreased liver-to-plasma ratio in Oatp1a/b-/- compared to those in wild-type mice. Furthermore, we observed the tight binding of cabazitaxel and its active metabolites, including docetaxel, to plasma carboxylesterase (Ces1c) in mice, which may complicate the interpretation of pharmacokinetic and pharmacodynamic mouse studies. Collectively, these results will help to further optimize (pre)clinical research into the safety and efficacy of orally applied cabazitaxel.

Tight-Binding Small-Molecule Carboxylesterase 2 Inhibitors Reduce Intracellular Irinotecan Activation.

As the primary enzyme responsible for the activatable conversion of Irinotecan (CPT-11) to SN-38, carboxylesterase 2 (CES2) is a significant predictive biomarker toward CPT-11-based treatments for pancreatic ductal adenocarcinoma (PDAC). High SN-38 levels from high CES2 activity lead to harmful effects, including life-threatening diarrhea. While alternate strategies have been explored, CES2 inhibition presents an effective strategy to directly alter the pharmacokinetics of CPT-11 conversion, ultimately controlling the amount of SN-38 produced. To address this, we conducted a high-throughput screening to discover 18 small-molecule CES2 inhibitors. The inhibitors are validated by dose-response and counter-screening and 16 of these inhibitors demonstrate selectivity for CES2. These 16 inhibitors inhibit CES2 in cells, indicating cell permeability, and they show inhibition of CPT-11 conversion with the purified enzyme. The top five inhibitors prohibited cell death mediated by CPT-11 when preincubated in PDAC cells. Three of these inhibitors displayed a tight-binding mechanism of action with a strong binding affinity.

Carboxylesterase-activated near-infrared fluorescence probe for highly sensitive imaging of liver tumors.

Carboxylesterases (CESs) are critical for metabolizing ester-containing biomolecules and are specifically important in liver metabolic disorders. The modulation of CESs is also an important issue in pharmacology and clinical applications. Herein, we present a near-infrared (NIR) CES fluorescent probe (NCES) based on the protection-deprotection of the hydroxyl group for monitoring CES levels in living systems. The NCES probe has good selectivity and sensitivity for CESs with a limit of detection (LOD) of 5.24 mU mL-1, which allows for tracing the fluctuation of cellular CES after treatment with anticancer drugs and under inflammation and apoptosis states. Furthermore, NCES can be successfully applied for guiding liver cancer surgery with high-contrast in vivo imaging and detecting clinical serum samples from liver cancer patients. This work showed that the NCES probe has great potential in drug development, imaging applications for medical diagnosis, and early-stage detection for clinical liver diseases.

Histochemical research of enzymes involved in cellular digestion in the digestive tract of tub gurnard, Chelidonichthys lucerna.

The tub gurnard Chelidonichthys lucerna (Linnaeus, 1758), Triglidae, is an opportunistic, demersal carnivorous fish. Data on the digestive enzymes of tub gurnard have not been reported in the literature. Therefore, the aim of this research was to investigate the distribution and intensity of alkaline phosphatase, acid phosphatase, non-specific esterase, and aminopeptidase in the digestive tract of tub gurnard. To investigate data about those enzymes tissue samples of the esophagus, anterior and posterior part of the stomach, pyloric caeca, anterior, middle and posterior part of the intestine proper, and rectum were taken. Azo-coupling methods were used to detect the enzymatic reactions. The intensities of the reactions were measured using ImageJ software. Alkaline phosphatase, acid phosphatase, and non-specific esterase activities were found in all parts of the digestive tract. The brush border of the pyloric caeca and intestine proper were the main sites of alkaline phosphatase reaction, with intensity decreasing toward the posterior parts of the digestive tract. The high intensities of acid phosphatase were found in the epithelium of the anterior part of the stomach, pyloric caeca, anterior part of the intestine proper, and in the rectum. The intensity of non-specific esterase was mainly increased from the anterior to the posterior parts of the digestive tract. Aminopeptidase activity was found in the esophagus, pyloric caeca, and intestine proper. Our results suggest that the entire digestive tract of the tub gurnard is involved in the digestion and absorption of dietary components.

Enantioselective metabolism of fenpropathrin enantiomers by carboxyl/choline esterase 6 in Tetranychus cinnabarinus.

BACKGROUND: Tetranychus cinnabarinus is a polyphagous pest mite commonly found in agriculture. As an excellent acaricide, fenpropathrin (FEN) is frequently used to control T. cinnabarinus in agriculture. However, commercial FEN is a racemate with two enantiomers, R-FEN and S-FEN. Considering that investigations on the metabolism of FEN by T. cinnabarinus are based on racemate FEN, it is important to investigate the enantioselective metabolism of FEN in T. cinnabarinus. RESULTS: S-FEN was more toxic to T. cinnabarinus than R-FEN by more than 68.8-fold. Moreover, the synergist bioassay revealed that carboxylesterase and cytochrome P450 were the primary enzymes engaged in the detoxification of FEN in T. cinnabarinus, with carboxylesterase playing a leading role. Seven genes were substantially different after the induction of S-FEN and R-FEN. TcCCE06 was screened and selected as a key gene that related to FEN metabolism in T. cinnabarinus. The metabolic results showed that the recombinant TcCCE06 effectively metabolized 32.1% of the R-FEN and 13.8% of the S-FEN within 4 h of incubation. Moreover, R-FEN was demonstrated to have a higher affinity for the TcCCE06 protein than S-FEN based on molecular docking. CONCLUSION: Our results indicated that TcCCE06 mediates the enantioselective metabolism of FEN in T. cinnabarinus. Our findings will contribute to a more comprehensive understanding of the mechanisms underlying the differential toxicity of the FEN enantiomers against T. cinnabarinus. Furthermore, they also provide a new perspective for the development of enantiomer-enriched acaricides with higher activity and lower pesticide dosage and pollution risks. © 2023 Society of Chemical Industry.