Impact of Genetic Polymorphisms and Drug-Drug Interactions Mediated by Carboxylesterase1 on Remimazolam Deactivation.

Remimazolam (Byfavo®), a recent FDA-approved ester-linked benzodiazepine, offers advantages in sedation, such as rapid onset and predictable duration, making it suitable for broad anesthesia applications. Its favorable pharmacological profile is primarily attributed to rapid hydrolysis, the primary metabolism pathway for its deactivation. Thus, understanding remimazolam hydrolysis determinants is essential for optimizing its clinical use. This study aimed to identify the enzyme(s) and tissue(s) responsible for remimazolam hydrolysis and to evaluate the influence of genetic polymorphisms and drug-drug interactions (DDIs) on its hydrolysis in the human liver. An initial incubation study with remimazolam and phosphate buffer saline (PBS), human serum, and the S9 fractions of human liver and intestine demonstrated that remimazolam was exclusively hydrolyzed by human liver S9 fractions. Subsequent incubation studies utilizing a Carboxylesterase inhibitor (Bis-para-nitrophenylphosphate, BNPP), recombinant human Carboxylesterase1 (CES1) and Carboxylesterase 2 (CES2) confirmed that remimazolam is specifically hydrolyzed by CES1 in human liver. Furthermore, in vitro studies with wild-type CES1 and CES1 variants transfected cells revealed that certain genetic polymorphisms significantly impair remimazolam deactivation. Notably, the impact of CES1 G143E was verified using individual human liver samples. Moreover, our evaluation of the DDIs between remimazolam and several other substrates/inhibitors of CES1-including simvastatin, enalapril, clopidogrel and sacubitril- found that clopidogrel significantly inhibited remimazolam hydrolysis at clinically relevant concentrations, with CES1 genetic variants potentially influencing the interactions. In summary, CES1 genetic variants and its interacting drugs are crucial factors contributing to interindividual variability in remimazolam hepatic hydrolysis, holding the potential to serve as biomarkers for optimizing remimazolam use. Significance Statement This investigation demonstrates that remimazolam is deactivated by CES1 in the human liver, with CES1 genetic variants and DDIs significantly influencing its metabolism. These findings emphasize the need to consider CES1 genetic variability and potential DDIs in remimazolam use, especially in personalized pharmacotherapy to achieve optimal anesthetic outcomes.

Inhibitory effects of phthalate esters (PAEs) and phthalate monoesters towards human carboxylesterases (CESs).

Phthalate esters (PAEs), accompanied by phthalate monoesters as hydrolysis metabolites in humans, have been widely used as plasticizers and exhibited disruptive effects on the endocrine and metabolic systems. The present study aims to investigate the inhibition behavior of PAEs and phthalate monoesters on the activity of the important hydrolytic enzymes, carboxylesterases (CESs), to elucidate the toxicity mechanism from a new perspective. The results showed significant inhibition on CES1 and CES2 by most PAEs, but not by phthalate monoesters, above which the activity of CES1 was strongly inhibited by DCHP, DEHP, DiOP, DiPP, DNP, DPP and BBZP, with inhibition ratios exceeding 80%. Kinetic analyses and in vitro-in vivo extrapolation were conducted, revealing that PAEs have the potential to disrupt the metabolism of endogenous substances catalyzed by CES1 in vivo. Molecular docking results revealed that hydrogen bonds and hydrophobic contacts formed by ester bonds contributed to the interaction of PAEs towards CES1. These findings will be beneficial for understanding the adverse effect of PAEs and phthalate monoesters.

An Innovative Aggregation-Induced Emission-Based NIR Fluorescent Probe for Visualizing Carboxylesterases in Living Cells, Zebrafish, and Tumor-Bearing Mice.

In the human body, carboxylesterases (CEs) play crucial roles in xenobiotic metabolism and lipid homeostasis. But abnormal expression of CEs is highly associated with some diseases, such as hyperlipidemia, diabetes, and liver cancer. Therefore, it is of great importance to develop an efficient tool for the accurate detection of CEs in living organisms. Herein, an innovative near-infrared (NIR) fluorescent probe, TTAP-AB, was designed for CE detection based on the aggregation-induced emission (AIE) mechanism. This probe exhibits rapid response (2 min), excellent sensitivity (limit of detection = 8.14 × 10-6 U/mL), and high selectivity to CEs. Additionally, owing to its good biocompatibility, the TTAP-AB probe enables the monitoring of dynamic changes in CE levels under drug-induced modulation in living cells and zebrafish. More importantly, the TTAP-AB probe was successfully employed to image liver tumors and assist in tumor resection through the real-time monitoring of CEs, indicating that TTAP-AB is promising to guide liver cancer surgery. Therefore, the TTAP-AB probe can not only enrich the strategies for CE detection in biological systems but also has great potential for some clinical imaging applications, including medical diagnosis, preclinical research, and imaging-guided surgery.

PBPK Modelling for Drugs Cleared by Non-CYP Enzymes: State-of-the-Art and Future Perspectives.

Physiologically-based pharmacokinetic (PBPK) modeling has become the established method for predicting human pharmacokinetics (PK) and drug-drug interactions (DDI). The number of drugs cleared by non-CYP enzyme metabolism has increased steadily and to date, there is no consolidated overview of PBPK modeling for drugs cleared by non-CYP enzymes. This review aims to describe the state-of-the-art for PBPK modeling for drugs cleared via non-CYP enzymes, to identify successful strategies, to describe gaps and to provide suggestion to overcome them. To this end, we conducted a detailed literature search and found 58 articles published before the 1st of January 2023 containing 95 examples of clinical PBPK models for 62 non-CYP enzyme substrates. Reviewed articles covered the drug clearance by uridine 5'-diphospho-glucuronosyltransferases (UGTs), aldehyde oxidase (AO), flavin-containing monooxygenases (FMOs), sulfotransferases (SULTs) and carboxylesterases (CES), with UGT2B7, UGT1A9, CES1, FMO3 and AO being the enzymes most frequently involved. In vitro-in vivo extrapolation (IVIVE) of intrinsic clearance and the bottom-up PBPK modeling involving non-CYP enzymes remains challenging. We observed that the middle-out modeling approach was applied in 80% of the cases, with metabolism parameters optimized in 73% of the models. Our review could not identify a standardized approach used for model optimization based on clinical data, with manual optimization employed most frequently. Successful development of models for UGT2B7, UGT1A9, CES1, and FMO3 substrates provides a foundation for other drugs metabolized by these enzymes and guides the way forward in creating PBPK models for other enzymes in these families. Significance Statement Our review charts the rise of PBPK modeling for drugs cleared by non-CYP enzymes. Analyzing 58 articles and 62 non-CYP enzyme substrates, we found that UGTs, AO, FMOs, SULTs, and CES were the main enzyme families involved and that UGT2B7, UGT1A9, CES1, FMO3 and AO are the individual enzymes with the strongest PBPK modeling precedents. Approaches established for these enzymes can now be extended to additional substrates and to drugs metabolized by enzymes that are similarly well characterized.

Effects of dietary fluoranthene on tissue-specific responses of carboxylesterases, acetylcholinesterase and heat shock protein 70 in two forest lepidopteran species.

In this study, responses of carboxylesterases, acetylcholinesterase, and stress protein Hsp70 were examined in the midgut and midgut tissue, and brain of fifth instar larvae of Lymantria dispar L. and Euproctis chrysorrhoea L. following chronic exposure to dietary fluoranthene. Specific carboxylesterase activity increased significantly in the midgut tissue of E. chrysorrhoea larvae treated with a lower fluoranthene concentration. The specific patterns of isoforms expression, recorded in larvae of both species, enable efficient carboxylesterase activity as a significant part of defense mechanisms. Increased Hsp70 concentration in the brain of L. dispar larvae points to a response to the proteotoxic effects of a lower fluoranthene concentration. Decreased Hsp70 in the brain of E. chrysorrhoea larvae in both treated groups can suggest induction of other mechanisms of defense. The results indicate the importance of the examined parameters in larvae of both species exposed to the pollutant, as well as their potential as biomarkers.

Identification, expression profiles and involvement in insecticides tolerance and detoxification of carboxylesterase genes in Bactrocera dorsalis.

Carboxylesterases (CarEs) are a multifunctional superfamily of enzymes and play an important role in detoxification of various insecticides in insects. The oriental fruit fly, Bactrocera dorsalis, is one of the most destructive agricultural pests and has developed different degrees of resistance to organophosphates in field. However, the involvement of BdCarEs in tolerance or resistance to other alternative insecticides are still unclear. In the present study, 33 BdCarEs genes were identified based on the genome database of B. dorsalis. Phylogenetic analysis demonstrated that they were classified into nine clades, with abundance of α-esterases. Meanwhile, the sequence characterization and the chromosome distribution were also analyzed. The spatiotemporal expression analysis of BdCarEs genes suggested that the diversity of potential function in different physiological processes. With the exception of BdCarE21, all BdCarEs genes responded to at least one insecticide exposure, and BdCarE20 was found to be up-regulated after exposure to all five tested insecticides individually. Eight BdCarEs genes were overexpressed in MR strain when compared to that in SS strain. Subsequently, knockdown the expression of representative BdCarEs genes significantly increased the susceptibility of the oriental fruit fly to corresponding insecticides, which indicated that the tested BdCarEs genes contributed to one or multiple insecticide detoxification. These findings provide valuable insights into the potential role in respond to tolerance or resistance to insecticides with different mode of action, and will facilitate development of efficiency management strategy for B. dorsalis.

Structural and Biochemical Insights into Bis(2-hydroxyethyl) Terephthalate Degrading Carboxylesterase Isolated from Psychrotrophic Bacterium Exiguobacterium antarcticum.

This study aimed to elucidate the crystal structure and biochemically characterize the carboxylesterase EaEst2, a thermotolerant biocatalyst derived from Exiguobacterium antarcticum, a psychrotrophic bacterium. Sequence and phylogenetic analyses showed that EaEst2 belongs to the Family XIII group of carboxylesterases. EaEst2 has a broad range of substrate specificities for short-chain p-nitrophenyl (pNP) esters, 1-naphthyl acetate (1-NA), and 1-naphthyl butyrate (1-NB). Its optimal pH is 7.0, losing its enzymatic activity at temperatures above 50 °C. EaEst2 showed degradation activity toward bis(2-hydroxyethyl) terephthalate (BHET), a polyethylene terephthalate degradation intermediate. We determined the crystal structure of EaEst2 at a 1.74 Å resolution in the ligand-free form to investigate BHET degradation at a molecular level. Finally, the biochemical stability and immobilization of a crosslinked enzyme aggregate (CLEA) were assessed to examine its potential for industrial application. Overall, the structural and biochemical characterization of EaEst2 demonstrates its industrial potency as a biocatalyst.

A Turn-On Lipid Droplet-Targeted Near-Infrared Fluorescent Probe with a Large Stokes Shift for Detection of Intracellular Carboxylesterases and Cell Viability Imaging.

Carboxylesterases (CEs) play important physiological roles in the human body and are involved in numerous cellular processes. Monitoring CEs activity has great potential for the rapid diagnosis of malignant tumors and multiple diseases. Herein, we developed a new phenazine-based "turn-on" fluorescent probe DBPpys by introducing 4-bromomethyl-phenyl acetate to DBPpy, which can selectively detect CEs with a low detection limit (9.38 × 10-5 U/mL) and a large Stokes shift (more than 250 nm) in vitro. In addition, DBPpys can also be converted into DBPpy by carboxylesterase in HeLa cells and localized in lipid droplets (LDs), emitting bright near-infrared fluorescence under the irradiation of white light. Moreover, we achieved the detection of cell health status by measuring the intensity of NIR fluorescence after co-incubation of DBPpys with H2O2-pretreated HeLa cells, indicating that DBPpys has great potential applications for assessing CEs activity and cellular health.

Carbonate-Based Fluorescent Chemical Tool for Uncovering Carboxylesterase 1 (CES1) Activity Variations in Live Cells.

Carboxylesterase 1 (CES1) plays a key role in the metabolism of endogenous biomolecules and xenobiotics including a variety of pharmaceuticals. Despite the established importance of CES1 in drug metabolism, methods to study factors that can vary CES1 activity are limited with only a few suitable for use in live cells. Herein, we report the development of FCP1, a new CES1 specific fluorescent probe with a unique carbonate substrate constructed from commercially available reagents. We show that FCP-1 can specifically report on endogenous CES1 activity with a robust fluorescence response in live HepG2 cells through studies with inhibitors and genetic knockdowns. Subsequently, we deployed FCP-1 to develop a live cell fluorescence microscopy-based approach to identify activity differences between CES1 isoforms. To the best of our knowledge, this is the first application of a fluorescent probe to measure the activity of CES1 sequence variants in live cells.

Plasmatic B-esterases as potential biomarkers of exposure to marine plastics in loggerhead turtles.

Sea turtles are particularly vulnerable to plastic exposures, and the associated chemical additives, due to their feeding strategies. The species Caretta caretta is a proposed sentinel of plastic pollution worldwide. Thus, there is a need to find adequate biomarkers of plastic exposure through non-invasive protocols for this IUCN protected species. Plasmatic acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and carboxylesterase (CE) which participate in xenobiotic and endogenous metabolic reactions could all serve as biomarkers, as they are responsive to plasticizers and have already proved adequate for identifying organophosphorus esters exposures. Here we measured plasmatic B-esterases in wild specimens captured as accidental by-catch. Measurements were taken in each individual either at entry into the rehabilitation program or immediately before release after a recovery period. For CE measurements, 4 commercial substrates were used as potentially indicative of distinct enzyme isoforms. Increased activity was seen with the butyrate-derived substrates. Plasmatic CE activities were over one order of magnitude higher than AChE and BuChE substrates. Moreover, an in vitro protocol with the inclusion of plastic additives such as tetrabromobisphenol A (TBBPA), bisphenol A and some of its analogues was considered a proxy of enzymatic interactions. A clear inhibition by TBBPA was found when using commercially purified AChE and recombinant CE proteins. Overall, from in vitro and in vivo evidences, CEs in plasma are sensitive and easily measurable and have been shown to significantly increase after turtles have been rehabilitated in rescue centres. Nevertheless, the inclusion of plastic (or plasticizers) characterisation would help to confirm its association with plasmatic enzyme modifications before they can be adopted as biomarkers of plastic contamination.

B-esterases characterisation in the digestive tract of the common octopus and the European cuttlefish and their in vitro responses to contaminants of environmental concern.

Cephalopods are a group of marine invertebrates that have received little attention as sentinel species in comparison to other molluscs, such as bivalves. Consequently, their physiological and biochemical xenobiotic metabolism responses are poorly understood. Here we undertake a comparative analysis of the enzymatic activities involved in detoxification reactions and neural transmission in the digestive tract of two commercial cephalopods: the Common octopus, Octopus vulgaris, and the European cuttlefish, Sepia officinalis. For methodological purposes, several common B-esterases (five carboxylesterase (CE) substrates and three cholinesterase (ChE) determinations) were assayed as a proxy of metabolic and neuronal activities, respectively. Four components of the digestive tract in each species were considered: salivary glands, the stomach, the digestive gland and the caecum. The in vitro responses of digestive gland homogenates to model chemicals and contaminants of environmental concern were contrasted between both cephalopod species. The baseline biochemical activities in the four digestive tract components were also determined. Moreover, in order to validate the protocol, purified proteins, recombinant human CE (CE1 and CE2) and purified eel acetylcholinesterase (AChE) were included in the analysis. Overall, carboxylesterase activities were higher in octopus than in cuttlefish, with the activity quantified in the digestive tract components in the following order: digestive gland ≈ caecum > stomach ≈ salivary glands, with higher hydrolysis rates reached with naphthyl-derived substrates. In contrast, cuttlefish hydrolysis rates with ChE substrates were higher than in octopus. This trend was also reflected in a higher sensitivity to CE inhibitors in octopus and to AChE inhibitors in cuttlefish. Given the detoxification character of CEs and its protective role preventing AChE inhibition, octopus could be regarded as more efficiently protected than cuttlefish from neurotoxic exposures. A full characterisation of B-esterases in the digestive tract of the two common cephalopods is also provided.

Cross effects of heat stress and three insecticides on the survival of the codling moth Cydia pomonella (L.): Investigating the molecular and biochemical mechanisms.

As temperature is expected to strongly increase in the future, understanding temperature-mediated toxicity of insecticides is determinant to assess pest management efficiency in a warming world. Investigating molecular and biochemical mechanisms associated with cross mechanisms of temperature and insecticides on pests' tolerance would also be useful in this context. This study aimed to investigate cross effects between temperature and insecticides on the survival of a major pest, the codling moth Cydia pomonella, and their underlying mechanisms. The effect of three insecticidal active ingredients, i.e. chlorantraniliprole, emamectin and spinosad, was assessed at different temperatures on: (i) C. pomonella larval survival; (ii) detoxification enzymes activities (cytochrome P450 multi-function oxygenases, carboxylesterases and glutathione S-transferases) and (iii) genes expression of some detoxification enzymes, heat shock proteins and receptors targeted by the insecticides. We observed a decreased efficiency of emamectin and spinosad at high temperature to control the codling moth while no influence of temperature on chlorantraniliprole efficacy was observed. Detoxification enzymes activities were improved by heat stress alone but not by double stress (temperature + insecticides). Moreover, two detoxification genes (Cyp9A61 and Gst1) were over-expressed by a single stress but not by two stresses while Hsp70 and Cyp6B2 genes may be involved in tolerance to two stresses in C. pomonella. These results confirmed the cross effects of temperature and insecticides on C. pomonella for emamectin and spinosad and provided clues to understand how temperature affects the susceptibility of C. pomonella to insecticides. They illustrate however the complexity of molecular and biochemical responses of individuals facing multiple stresses.

The Functional Characterization of Carboxylesterases Involved in the Degradation of Volatile Esters Produced in Strawberry Fruits.

Volatile ester compounds are important contributors to the flavor of strawberry, which affect consumer preference. Here, the GC-MS results showed that volatile esters are the basic aroma components of strawberry, banana, apple, pear, and peach, and the volatile esters were significantly accumulated with the maturation of strawberry fruits. The main purpose of this study is to discuss the relationship between carboxylesterases (CXEs) and the accumulation of volatile ester components in strawberries. FaCXE2 and FaCXE3 were found to have the activity of hydrolyzing hexyl acetate, Z-3-hexenyl acetate, and E-2-hexenyl acetate to the corresponding alcohols. The enzyme kinetics results showed that FaCXE3 had the higher affinity for hexyl acetate, E-2-hexenyl acetate, and Z-3-hexenyl acetate compared with FaCXE2. The volatile esters were mainly accumulated at the maturity stages in strawberry fruits, less at the early stages, and the least during the following maturation stages. The expression of FaCXE2 gradually increased with fruit ripening and the expression level of FaCXE3 showed a decreasing trend, which suggested the complexity of the true function of CXEs. The transient expression of FaCXE2 and FaCXE3 genes in strawberry fruits resulted in a significantly decreased content of volatile esters, such as Z-3-hexenyl acetate, methyl hexanoate, methyl butyrate, and other volatile esters. Taken together, FaCXE2 and FaCXE3 are indeed involved in the regulation of the synthesis and degradation of strawberry volatile esters.

Effect of microplastics on the activity of carboxylesterase and phosphatase enzymes in Scinax squalirostris tadpoles.

Microplastics (MPs) are critical emerging pollutants around the world. There is a growing interest in the effects of MP ingestion, non-digestion, and toxicity on aquatic organisms. Amphibian tadpoles are the vertebrate group that has received the least attention regarding this issue. The aim of the present study was to determine the ingestion of polyethylene MPs by Scinax squalirostris tadpoles by atomic force microscopy (AFM) and to evaluate the activities of carboxylesterase (CbE, using 4-naphthyl butyrate-NB-, and 1-naphthyl acetate -NA- as substrates) and alkaline phosphatase (ALP) under MP exposure. Enzyme activities were analyzed spectrophotometrically at 2 and 10 days of exposure. Tadpoles were exposed to two different treatments during 10 days: a negative control (CO, dechlorinated water) and MP (60 mg L-1). AFM images of the digestive contents of tadpoles revealed the presence of MPs. After 10 days of MP exposure, CbE (NB) activity was significantly higher and CbE (NA) activity was significantly lower in MP treatments than in controls. ALP activity decreased in MP treatments after 2 and 10 days of exposure. The detection of MP particles in the intestinal contents and the effects on metabolic enzymes in a common frog species evidenced the potential health risk of MP to aquatic vertebrates. Thus, the differential response in enzymes and substrates demonstrate the need for considering the complex effects of contaminants and nutrients on ecosystems for ecotoxicological risk characterization.

Carboxylesterases for the hydrolysis of acetoacetate esters and their applications in terpenoid production using Escherichia coli.

Pathway engineering is a useful technology for producing desired compounds on a large scale by modifying the biosynthetic pathways of host organisms using genetic engineering. We focused on acetoacetate esters as novel low-cost substrates and established an efficient terpenoid production system using pathway-engineered recombinant Escherichia coli. Functional analysis using recombinant E. coli proteins of 18 carboxylesterases identified from the microbial esterases and lipases database showed that the p-nitrobenzyl esterase (PnbA) from Bacillus subtilis specifically hydrolyzed two acetoacetate esters: methyl acetoacetate (MAA) and ethyl acetoacetate (EAA). We generated a plasmid (pAC-Mev/Scidi/Aacl/PnbA) co-expressing PnbA and six enzymes of the mevalonate pathway gene cluster from Streptomyces, isopentenyl diphosphate isomerase type I from Saccharomyces cerevisiae, and acetoacetyl-coenzyme A ligase from Rattus norvegicus. The plasmid pAC-Mev/Scidi/Aacl/PnbA was introduced into E. coli along with plasmid expressing carotenoid (lycopene) or sesquiterpene (ß-bisabolene) biosynthesis genes, and the terpenoid production was evaluated following the addition of acetoacetate esters as substrates. These recombinant E. coli strains used MAA and EAA as substrates for the biosynthesis of terpenoids and produced almost equivalent concentrations of target compounds compared with the previous production system that used mevalonolactone and lithium acetoacetate. The findings of this study will enable the production of useful terpenoids from low-cost substrates, which may facilitate their commercial production on an industrial scale in the future. KEY POINTS: • PnbA from Bacillus subtilis exhibits acetoacetate hydrolysis activity. • A plasmid enabling terpenoid synthesis from acetoacetate esters was constructed. • Acetoacetate esters as substrates enable a low-cost production of terpenoids.

Old pesticide, new use: Smart and safe enantiomer of isocarbophos in locust control.

Locust plagues are still worldwide problems. Selecting active enantiomers from current chiral insecticides is necessary for controlling locusts and mitigating the pesticide pollution in agricultural lands. Herein, two enantiomers of isocarbophos (ICP) were separated and the enantioselectivity in insecticidal activity against the pest Locusta migratoria manilensis (L. migratoria) and mechanisms were investigated. The significant difference of LD50 between (+)-ICP (0.609 mg/kg bw) and (-)-ICP (79.412 mg/kg bw) demonstrated that (+)-ICP was a more effective enantiomer. The enantioselectivity in insecticidal activity of ICP enantiomers could be attributed to the selective affinity to acetylcholinesterase (AChE). Results of in vivo and in vitro assays suggested that AChE was more sensitive to (+)-ICP. In addition, molecular docking showed that the -CDOKER energies of (+)-ICP and (-)-ICP were 25.6652 and 24.4169, respectively, which suggested a stronger affinity between (+)-ICP and AChE. Significant selectivity also occurred in detoxifying enzymes activities (carboxylesterases (CarEs) and glutathione S-transferases (GSTs)) and related gene expressions. Suppression of detoxifying enzymes activities with (+)-ICP treatment suggested that (-)-ICP may induce the detoxifying enzyme-mediated ICP resistance. A more comprehensive understanding of the enantioselectivity of ICP is necessary for improving regulation and risk assessment of ICP.

Chemoproteomics-Enabled De Novo Discovery of Photoswitchable Carboxylesterase Inhibitors for Optically Controlled Drug Metabolism.

Herein, we report arylazopyrazole ureas and sulfones as a novel class of photoswitchable serine hydrolase inhibitors and present a chemoproteomic platform for rapid discovery of optically controlled serine hydrolase targets in complex proteomes. Specifically, we identify highly potent and selective photoswitchable inhibitors of the drug-metabolizing enzymes carboxylesterases 1 and 2 and demonstrate their pharmacological application by optically controlling the metabolism of the immunosuppressant drug mycophenolate mofetil. Collectively, this proof-of-concept study provides a first example of photopharmacological tools to optically control drug metabolism by modulating the activity of a metabolizing enzyme. Our arylazopyrazole ureas and sulfones offer synthetically accessible scaffolds that can be expanded to identify specific photoswitchable inhibitors for other serine hydrolases, including lipases, peptidases, and proteases. Our chemoproteomic platform can be applied to other photoswitches and scaffolds to achieve optical control over diverse protein classes.

Detection techniques of carboxylesterase activity: An update review.

Mammalian carboxylesterases (CESs) are essential members of serine esterase hydrolase superfamily, which are widely distributed in many tissues including liver, intestine, lung and kidney. CESs play an important role in the metabolism of various xenobiotics including ester drugs and environmental toxicants, and also participate in lipid homeostasis, so the development of CESs activity detection techniques are of great significance for drug discovery and biomedical research. With the rapid development of separated and detection technologies such as chromatography, capillary electrophoresis, fluorescent probe-based detection technology, bioluminescent sensor and colorimetric sensor in recent decade, the research of physiological functions of CESs have make huge breakthrough. This review summarizes the development and application of CESs activity detection techniques, as well as comparatively analyzes the characteristics of various detection techniques. The information and knowledge represented here will help the researchers carry out various biochemical studies for understanding activation mechanism and role of CESs in drug metabolism.

Synthesis of novel flexible tamoxifen analogues to overcome CYP2D6 polymorphism and their biological evaluation on MCF-7 cell line.

Tamoxifen (TAM) is currently the endocrine treatment of choice for all stages of breast cancer; it has proven success in ER positive and ER negative patients. TAM is activated by endogenous CYP450 enzymes to the more biologically active metabolites 4-hydroxytamoxifen and endoxifen mainly via CYP2D6 and CYP3A4/5. CYP2D6 has been investigated for polymorphism; there is a large interindividual variation in the enzyme activity, this drastically effects clinical outcomes of tamoxifen treatment. Here in we report the design and synthesis of 10 novel compounds bearing a modified tamoxifen skeleton, ring C is substituted with different ester groups to bypass the CYP2D6 enzyme metabolism and employ esterase enzymes for activation. All compounds endorse flexibility on ring A. Compounds (II-X) showed MCF-7% growth inhibition >50% at a screening dose of 10 µM. These results were validated by yeast estrogen screen (YES) and E-Screen assay combined with XTT assay. Compound II (E/Z 4-[1-4-(3-Dimethylamino-propoxy)-phenyl)-3-(4-methoxy-phenyl)-2-methyl-propenyl]-phenol) showed nanomolar antiestrogenic activity (IC50 = 510 nM in YES assay) and was five times more potent in inhibiting the growth of MCF-7 BUS (IC50 = 96 nM) compared to TAM (IC50 = 503 nM). Esterified analogues VI, VII were three times more active than TAM on MCF-7 BUS (IC50 = 167 nM). Novel analogues are prodrugs that can ensure equal clinical outcomes to all breast cancer patients.

Comparison of several non-specific skin mucus immune defences in three piscine species of aquaculture interest.

Fish skin mucus is a viscous and semipermeable barrier made mainly of water, glycoproteins and soluble proteins. It represents an important defence against the environment and previous studies have reported the presence of different substances involved in immune defence responses in it. The aim of the present work was to characterize skin mucus protease activity by zymography and esterase activity of the subfamily of carboxylesterases in three species of interest for aquaculture: gilthead sea bream, sea bass and meagre. Mucus antioxidant power was also determined by adapting ferric reducing antioxidant power (FRAP) analysis. As a result of these non-specific immune defence parameters, we compared the antibacterial capacity of skin mucus in these species via in vitro dual bacteria strains-skin mucus co-culture growths. We used Pseudomonas anguilliseptica and Vibrio anguillarum as marine pathogenic bacteria and Escherichia coli as non-pathogenic. For each fish species, in the respective zymograms, we determined a pattern of proteolytic digestion bands. A high-molecular-weight band (around 200 kDa; H-band) was evident in sea bream and sea bass, and showed chymotrypsin activity. One or two intermediate-molecular-weight bands (around 75 kDa; I-bands) with non-trypsin and non-chymotrypsin activity, and putatively with metalloprotease activity, were evident in all species. Finally, low-molecular-weight bands (between 14 and 30 kDa; L-bands) showed distinct patterns for each species and matched trypsin activity. Despite the conservative pattern of digestion bands, the levels of total proteolytic activity (TPA) were 5 and 10 times higher in meagre than in sea bass and sea bream, respectively. In parallel, three carboxylesterase activities were detected in the mucus of the three fish species, using myristate (pNPM-CE activity), butyrate (pNPB-CE activity) and acetate (pNPA-CE activity) as substrates. Both pNPB-CE and pNPA-CE were the most abundant in fish mucus, and meagre was again the species with the highest levels. In contrast, the antioxidant power of meagre skin mucus was the lowest. We established the capacity of skin mucus to block or limit bacterial growth (lytic activity) using 24 h growth curves. The log-growth phase of V. anguillarum was strongly blocked by sea bream and meagre mucus for a few hours; but not by sea bass mucus. However, if mucus was not renewed, log-growth was at the end of 24 h studied period. For its part, P. anguilliseptica growth curve was delayed by the three mucus types during the entire growth period. Only meagre achieved lytic activity against E. coli growth. All parameters studied here will be of a great interest as non-invasive bioindicators of non-specific immune defences in fish skin mucus.