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 Situ Visualizing Carboxylesterase Activity in Type 2 Diabetes Mellitus Using an Activatable Endoplasmic Reticulum Targetable Proximity Labeling Far-Red Fluorescent Probe.

Carboxylesterase (CE), an enzyme widely present in organisms, is involved in various physiological and pathological processes. Changes in the levels of CEs in the liver may predict the presence of type 2 diabetes mellitus (T2DM). Here, a novel dicyanoisophorone (DCI)-based proximity-labeled far-red fluorescent probe DCI2F-Ac with endoplasmic reticulum targeting was proposed for real-time monitoring and imaging of the CEs activity. DCI2F-Ac featured very low cytotoxicity and biotoxicity and was highly selective and sensitive for CEs. Compared with traditional CEs probes, DCI2F-Ac was covalently anchored directly to CEs, thus effectively reducing the loss of in situ fluorescent signals due to diffusion. Through the "on-off" fluorescence signal readout, DCI2F-Ac was able to distinguish cell lines and screen for CEs inhibitors. In terms of endoplasmic reticulum (ER) stress, it was found that thapsigargin (Tg) induced upregulation of CEs levels but not tunicamycin (Tm), which was related to the calcium homeostasis of the ER. DCI2F-Ac could efficiently detect downregulated CEs in the livers of T2DM, and the therapeutic efficacy of metformin, acarbose, and a combination of these two drugs was assessed by tracking the fluctuation of CEs levels. The results showed that combining metformin and acarbose could restore CEs levels to near-normal levels with the best antidiabetic effect. Thus, the DCI2F-Ac probe provides a great opportunity to explore the untapped potential of CEs in liver metabolic disorders and drug efficacy assessment.

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.

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.

Synthesis and evaluation of indomethacin prodrugs with a diester structure that are metabolically activated by human carboxylesterases.

1. Carboxylesterase (CES) has been studied extensively, mostly with substrates in the monoester structures. We investigated the relationship between indomethacin diester prodrugs and metabolic activation by microsomes and recombinant human CES.2. Eight indomethacin diester prodrugs were synthesised in two steps. They were used as substrates and hydrolysis rates were calculated.3. As a result, the major hydrolysis enzyme was CES. The hydrolysis rate of recombinant CES2A1 was comparable to that of recombinant CES1A1.4. In this study, by changing the structure of the prodrug to a diester structure, it was found that CES2 activity was equivalent to CES1 activity.5. It should be noted that the use of diester prodrugs in prodrug discovery, where organ-specific hydrolysis reactions are expected, may not yield the expected results.

SlCarE054 in Spodoptera litura (Lepidoptera: Noctuidae) showed direct metabolic activity to ß-cypermethrin with stereoselectivity.

Carboxylesterases (CarEs) is an important detoxification enzyme system in phase Ⅰ participating in insecticides resistance. In our previous study, SlCarE054, a CarEs gene from lepidoptera class, was screened out to be upregulated in a pyrethroids and organophosphates resistant population. Its overexpression was verified in two field-collected populations of Spodoptera litura (Lepidoptera: Noctuidae) resistant to pyrethroids and organophosphates by qRT-PCR. Spatiotemporal expression results showed that SlCarE054 was highly expressed in the pupae stage and the digestive tissue midgut. To further explore its role in pyrethroids and organophosphates resistance, its metabolism activity to insecticides was determined by UPLC. Its recombinant protein showed significant metabolism activity to cyhalothrin and fenvalerate, but not to phoxim or chlorpyrifos. The metabolic activity of SlCarE054 to ß-cypermethrin showed stereoselectivity, with higher metabolic activity to θ-cypermethrin than the enantiomer α-cypermethrin. The metabolite of ß-cypermethrin was identified as 3-phenoxybenzaldehyde. Further modelling and docking analysis indicated that ß-cypermethrin, cyhalothrin and fenvalerate could bind with the catalytic triad of the 3D structure of SlCarE054. The interaction of ß-cypermethrin with SlCarE054 also showed the lowest binding energy. Our work provides evidence that SlCarE054 play roles in ß-cypermethrin resistance in S. litura.

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.

Determination of carboxylesterase by fluorescence probe to guide detection of carbamate pesticide.

A carboxylesterase fluorescent probe (Probe 1) was developed for determination of carboxylesterase to guide detection of carbamate pesticide. The probe uses benzothiazole as fluorescence group and phenyldimethyl carbamate as recognition group. The solution of the fluorescent probe gradually changes from light blue to dark blue as the concentration of carbamate pesticides increases. The concentration of carbamate pesticides can be quickly calculated according to the colour of the probe solution through Get Color software on a smartphone. It showed that Probe 1 can be used as a rapid detection tool to achieve rapid detection of carbamate pesticides in juice samples without professional personnel and equipment. Furthermore, the probe has been successfully used to detect carbamate pesticides in fruit juice and vegetable juice.

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.

Various functions of detoxification enzymes against insecticides in Nilaparvata lugens selected by toxicity assays and RNAi methods.

The brown planthopper (BPH), Nilaparvata lugens is a devastating agricultural pest of rice, and they have developed resistance to many pesticides. In this study, we assessed the response of BPH nymphs to nitenpyram, imidacloprid, and etofenprox using contact and dietary bioassays, and investigated the underlying functional diversities of BPH glutathione-S-transferase (GST), carboxylesterase (CarE) and cytochrome P450 monooxygenase (P450) against these insecticides. Both contact and ingestion toxicity of nitenpyram to BPH were significantly higher than either imidacloprid or etofenprox. Under the LC50 concentration of each insecticide, they triggered a distinct response for GST, CarE, and P450 activities, and each insecticide induced at least one detoxification enzyme activity. These insecticides almost inhibited the expression of all tested GST, CarE, and P450 genes in contact bioassays but induced the transcriptional levels of these genes in dietary bioassays. Silencing of NlGSTD2 expression had the greatest effect on BPH sensitivity to nitenpyram in contact test and imidacloprid in dietary test. The sensitivities of BPH to insecticide increased the most in the contact test was etofenprox after silencing of NlCE, while the dietary test was nitenpyram. Knockdown of NlCYP408A1 resulted in BPH sensitivities to insecticide increasing the most in the contact test was nitenpyram, while the dietary test was imidacloprid. Taken together, these findings reveal that NlGSTD2, NlCE, and NlCYP408A1 play an indispensable role in the detoxification of the contact and ingestion toxicities of different types of insecticides to BPH, which is of great significance for the development of new strategies for the sucking pest control.

Sensitivity of cholinesterases and carboxylesterases to pharmaceutical products in Tinca tinca.

Discharges to the aquatic environment of pharmaceuticals represent a hazard to the aquatic organisms. Subchronic assay with 17-alpha-ethinylestradiol (EE2) and in vitro essays with pharmaceuticals of environmental concern were conducted to examine the sensitivity of tissue acetylcholinesterase (AChE) and carboxylesterase (CbE) activities of Tinca tinca to them. Subchronic exposure to 17-alpha-EE2 caused significant effects on brain, liver, and muscle CbE, but no on AChE activities. Most of the pharmaceuticals tested in vitro were considered as weak inhibitors of tissular AChE activity. Depending on the tissues, some compounds were classified as moderate inhibitors of CbE activity while other were categorized as weak enzymatic inhibitors. An opposite trend was observed depending on the tissue, while brain and liver CbE activities were inhibited, the muscle CbE activity was induced. Changes experienced on enzymatic activities after exposure to pharmaceuticals might affect the physiological functions in which these enzymes are involved. In vitro exposure to 17-alpha-EE2 in tench could be an informative, but not a surrogate model to know the effect of this synthetic estrogen on AChE and CbE activities.

The ability of selected fungal strains to produce carboxylesterase enzymes for biodegradation and use of bifenthrin insecticide as carbon source: in vitro and in silico approaches.

Bifenthrin (BF) is a broad-spectrum type I pyrethroid insecticide that acts on insects by impairing the nervous system and inhibiting ATPase activity, and it has toxic effects on non-target organisms and high persistence in the environment. This study aimed to determine the potential of six different fungi, including Pseudozyma hubeiensis PA, Trichoderma reesei PF, Trichoderma koningiopsis PD, Purpureocillium lilacinum ACE3, Talaromyces pinophilus ACE4, and Aspergillus niger AJ-F3, to degrade BF. Three different concentrations of BF, including 0.1%, 0.2%, and 0.3% w/v, were used in the sensitivity testing that revealed a significant (p ≤ 0.01) impact of BF on fungal growth. Enzymatic assays demonstrated that both intracellular and extracellular carboxylesterases hydrolyzed BF with the enzymatic activity of up to 175 ± 3 U (µmol/min) and 45 ± 1 U, respectively. All tested fungi were capable of utilizing BF as a sole carbon source producing 0.06 ± 0.01 to 0.45 ± 0.01 mg dry biomass per mg BF. Moreover, the presence of PytH was determined in the fungi using bioinformatics tools and was found in A. niger, T. pinophilus, T. reesei, and P. lilacinum. 3D structures of the PytH homologs were predicted using AlphaFold2, and their intermolecular interactions with pyrethroids were determined using MOE. All the homologs interacted with different pyrethroids with a binding energy of lesser than - 10 kcal/mol. Based on the study, it was concluded that the investigated fungi have a greater potential for the biodegradation of BF.

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.

Carboxylesterase Activatable Molecular Probe for Personalized Treatment Guidance by Analyte-Induced Molecular Transformation.

Accurate visualization of tumor microenvironment is of great significance for personalized medicine. Here, we develop a near-infrared (NIR) fluorescence/photoacoustic (FL/PA) dual-mode molecular probe (denoted as NIR-CE) for distinguishing tumors based on carboxylesterase (CE) level by an analyte-induced molecular transformation (AIMT) strategy. The recognition moiety for CE activity is the acetyl unit of NIR-CE, generating the pre-product, NIR-CE-OH, which undergoes spontaneous hydrogen atom exchange between the nitrogen atoms in the indole group and the phenol hydroxyl group, eventually transforming into NIR-CE-H. In cellular experiments and in vivo blind studies, the human hepatoma cells and tumors with high level of CE were successfully distinguished by both NIR FL and PA imaging. Our findings provide a new molecular imaging strategy for personalized treatment guidance.

Esterase D interacts with metallothionein 2A and inhibits the migration of A549 lung cancer cells in vitro.

Esterase D (ESD) is a nonspecific esterase widely distributed in various organisms. ESD plays an important role in regulating cholesterol efflux, inhibiting viral replication and lung cancer growth. MT2A (metallothionein 2A) is the most important isoform of metallothionein (MTs) in human and high expression of MT2A in tumors represents poor prognosis and metastatic behavior. However, there are no reports about the molecular mechanism of ESD in the regulation of tumor metastasis. In this study, we found for the first time that activation ESD promoted its interaction with MT2A and decreased the protein level of MT2A, which resulting in the concentration of free zinc ions up-regulated, and inhibited the migration of A549 lung cancer cells in vitro.

Enhanced Bioactivity of Enzyme/MOF Biocomposite via Host Framework Engineering.

This study reports the successful development of a sustainable synthesis protocol for a phase-pure metal azolate framework (MAF-6) and its application in enzyme immobilization. An esterase@MAF-6 biocomposite was synthesized, and its catalytic performance was compared with that of esterase@ZIF-8 and esterase@ZIF-90 in transesterification reactions. Esterase@MAF-6, with its large pore aperture, showed superior enzymatic performance compared to esterase@ZIF-8 and esterase@ZIF-90 in catalyzing transesterification reactions using both n-propanol and benzyl alcohol as reactants. The hydrophobic nature of the MAF-6 platform was shown to activate the immobilized esterase into its open-lid conformation, which exhibited a 1.5- and 4-times enzymatic activity as compared to free esterase in catalyzing transesterification reaction using n-propanol and benzyl alcohol, respectively. The present work offers insights into the potential of MAF-6 as a promising matrix for enzyme immobilization and highlights the need to explore MOF matrices with expanded pore apertures to broaden their practical applications in biocatalysis.

Rationally Engineered hCES2A Near-Infrared Fluorogenic Substrate for Functional Imaging and High-Throughput Inhibitor Screening.

Human carboxylesterase 2A (hCES2A) is an important endoplasmic reticulum (ER)-resident enzyme that is responsible for the hydrolytic metabolism or activation of numerous ester-bearing drugs and environmental toxins. The previously reported hCES2A fluorogenic substrates suffer from limited emission wavelength, low specificity, and poor localization accuracy, thereby greatly limiting the in situ functional imaging of hCES2A and drug discovery. Herein, a rational ligand design strategy was adopted to construct a highly specific near-infrared (NIR) substrate for hCES2A. Following scaffold screening and recognition group optimization, HTCF was identified as a desirable NIR fluorophore with excellent photophysical properties and high ER accumulation ability, while several HTCF esters held a high potential to be good hCES2A substrates. Further investigations revealed that TP-HTCF (the tert-pentyl ester of HTCF) was an ideal substrate with ultrahigh sensitivity, excellent specificity, and a substantial signal-to-noise ratio. Upon the addition of hCES2A, TP-HTCF could be rapidly hydrolyzed to release HTCF, a chemically stable product that emitted bright fluorescent signals at around 670 nm. A TP-HTCF-based biochemical assay was then established for the high-throughput screening of potent and cell-active hCES2A inhibitors from an in-house compound library. Furthermore, TP-HTCF displayed high imaging resolution for imaging hCES2A in living cells as well as mouse liver slices and tumor-xenograft mice. Collectively, this study demonstrates a rational strategy for developing highly specific fluorogenic substrates for an ER-resident target enzyme, while TP-HTCF can act as a practical tool for sensing hCES2A in living systems.

Evaluation of Drug-Drug Interactions via Inhibition of Hydrolases by Orlistat, an Anti-Obesity Drug.

Drug-drug interactions (DDI) have a significant impact on drug efficacy and safety. It has been reported that orlistat, an anti-obesity drug, inhibits the hydrolysis of p-nitrophenol acetate, a common substrate of the major drug-metabolizing hydrolases, carboxylesterase (CES) 1, CES2, and arylacetamide deacetylase (AADAC), in vitro. The aim of this study was to examine whether orlistat affects the pharmacokinetics of drug(s) metabolized by hydrolases in vivo after evaluating its inhibitory potencies against CES1, CES2, and AADAC in vitro. Orlistat potently inhibited the hydrolysis of acebutolol, a specific substrate of CES2, in a non-competitive manner (inhibition constant, K i = 2.95 ± 0.16 nM), whereas it slightly inhibited the hydrolysis of temocapril and eslicarbazepine acetate, specific substrates of CES1 and AADAC, respectively (IC50 >100 nM). The in vivo DDI potential was elucidated using mice, in which orlistat showed strong inhibition against acebutolol hydrolase activities in the liver and intestinal microsomes, similar to humans. The area under the curve (AUC) of acebutolol was increased by 43%, whereas the AUC of acetolol, a hydrolyzed metabolite of acebutolol, was decreased by 47% by co-administration of orlistat. The ratio of the K i value to the maximum unbound plasma concentration of orlistat (<0.012) is lower than the risk criteria for DDI in the liver defined by the US Food and Drug Administration guideline (>0.02), whereas the ratio of the K i value to the estimated intestinal luminal concentration (3.3 × 105) is considerably higher than the risk criteria in the intestine (>10). Therefore, this suggests that orlistat causes DDI by inhibiting hydrolases in the intestine. SIGNIFICANCE STATEMENT: This study demonstrated that orlistat, an anti-obesity drug, causes drug-drug interactions in vivo by potently inhibiting carboxylesterase 2 in the intestine. This is the first evidence that inhibition of hydrolases causes drug-drug interactions.

Molecular identification of carboxylesterase genes and their potential roles in the insecticides susceptibility of Grapholita molesta.

Grapholita molesta is one of the most damaging pests worldwide in stone and pome fruits. Application of chemical pesticides is still the main method to control this pest, which results in resistance to several types of insecticides. Carboxylesterase (CarE) is one of the important enzymes involved in the detoxification metabolism and tolerance of xenobiotics and insecticides. However, the roles of CarEs in insecticides susceptibility of G. molesta are still unclear. In the present study, the enzyme activity of CarEs and the mRNA expression of six CarE genes were consistently elevated after treatment with three insecticides (emamectin benzoate, lambda-cyhalothrin, and chlorantraniliprole). According to spatio-temporal expression profiles, six CarE genes expressed differently in different developmental stages, and highly expressed in some detoxification metabolic organs. RNAi-mediated knockdown of these six CarE genes indicated that the susceptibility of G. molesta to all these three insecticides were obviously raised after GmCarE9, GmCarE14, GmCarE16, and GmCarE22 knockdown, respectively. Overall, these results demonstrated that GmCarE9, GmCarE14, GmCarE16, and GmCarE22 play a role in the susceptibility of G. molesta to emamectin benzoate, lambda-cyhalothrin, and chlorantraniliprole treatment. This study expands our understanding of CarEs in insects, that the same CarE gene could participate in the susceptibility to different insecticides.

Enzyme-targeted near-infrared fluorescent probe for organophosphorus pesticide residue detection.

Pesticide residues significantly affect food safety and harm human health. In this work, a series of near-infrared fluorescent probes were designed and developed by acylating the hydroxyl group of the hemicyanine skeleton with a quenching moiety for monitoring the presence of organophosphorus pesticides in food and live cells. The carboxylic ester bond on the probe was hydrolyzed catalytically in the presence of carboxylesterase and thereby the fluorophore was released with near-infrared emission. Notably, the proposed probe 1 exhibited excellent sensitivity against organophosphorus based on the carboxylesterase inhibition mechanism and the detection limit for isocarbophos achieved 0.1734 µg/L in the fresh vegetable sample. More importantly, probe 1 allowed for situ visualization of organophosphorus in live cells and bacteria, meaning great potential for tracking the organophosphorus in biological systems. Consequently, this study presents a promising strategy for tracking pesticide residues in food and biological systems.