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.

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.

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.

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.

Characteristics of CXE family of Salvia miltiorrhiza and identification of interactions between SmGID1s and SmDELLAs.

Carboxylesterase (CXE) is a class of hydrolases that contain an α/ß folding domain, which plays critical roles in plant growth, development, and stress responses. Based on the genomic and transcriptomic data of Salvia miltiorrhiza, the SmCXE family was systematically analyzed using bioinformatics. The results revealed 34 SmCXE family members in S. miltiorrhiza, and the SmCXE family could be divided into five groups (Group I, Group II, Group III, Group IV, and Group V). Cis-regulatory elements indicated that the SmCXE promoter region contained tissue-specific and development-related, hormone-related, stress-related, and photoresponsive elements. Transcriptome analysis revealed that the expression levels of SmCXE2 were highest in roots and flowers (SmCXE8 was highest in stems and SmCXE19 was highest in leaves). Further, two GA receptors SmCXE1 (SmGID1A) and SmCXE2 (SmGID1B) were isolated from the SmCXE family, which are homologous to other plants. SmGID1A and SmGID1B have conserved HGGSF motifs and active amino acid sites (Ser-Asp-Val/IIe), which are required to maintain their GA-binding activities. SmGID1A and SmGID1B were significantly responsive to gibberellic acid (GA3) and methyl jasmonate (MeJA) treatment. A subcellular assay revealed that SmCXE1 and SmCXE2 resided within the nucleus. SmGID1B can interact with SmDELLAs regardless of whether GA3 exists, whereas SmGID1A can only interact with SmDELLAs in the presence of GA3. A Further assay showed that the GRAS domain mediated the interactions between SmGID1s and SmDELLAs. This study lays a foundation for further elucidating the role of SmCXE in the growth and development of S. miltiorrhiza.

Computational analysis of carboxylesterase genes and proteins in non-pathogenic food bacterium Alicyclobacillus acidocaldarius: insights from proteogenomics.

Over recent years, Alicyclobacillus acidocaldarius, a Gram-positive nonpathogenic rod-shaped thermo-acid-tolerant bacterium, has posed numerous challenges for the fruit juice industry. However, the bacterium's unique characteristics, particularly its nonpathogenic and thermophilic capabilities, offer significant opportunities for genetic exploration by biotechnologists. This study presents the computational proteogenomics report on the carboxylesterase (CE) enzyme in A. acidocaldarius, shedding light on structural and evolutional of CEs from this bacterium. Our analysis revealed that the average molecular weight of CEs in A. acidocaldarius was 41 kDa, with an isoelectric point around 5. The amino acid composition favored negative amino acids over positive ones. The aliphatic index and hydropathicity were approximately 88 and - 0.15, respectively. While the protein sequence showed no disulfide bonds in the CEs' structure, the presence of Cys amino acids was observed in the structure of CEs. Phylogenetic analysis presented more than 99% similarity between CEs, indicating their close evolutionary relationship. By applying homology modeling, the 3-dimensional structural models of the carboxylesterase were constructed, which with the help of structural conservation and solvent accessibility analysis highlighted key residues and regions responsible for enzyme stability and conformation. The specific patterns presented the total solvent accessibility of less than 25 (Å2) was in considerable position as well as Gly residues were noticeably have high accessibility to solvent in all structures. Ala was the more frequent amino acids in the conserved-SASA of carboxylesterases. Furthermore, unsupervised agglomerative hierarchical clustering based on solvent accessibility feature successfully clustered and even distinguished this enzyme from proteases from the same genome. These findings contribute to a deeper understanding of the nonpathogenic A. acidocaldarius carboxylesterase and its potential applications in biotechnology. Additionally, structural analysis of CEs would help to address potential solutions in fruit juice industry with utilization of computational structural biology.

Discovery of novel carboxylesterase 2 inhibitors for the treatment of delayed diarrhea and ulcerative colitis.

Human carboxylesterase 2 (hCES2) is an enzyme that metabolizes irinotecan to SN-38, a toxic metabolite considered a significant source of side effects (lethal delayed diarrhea). The hCES2 inhibitors could block the hydrolysis of irinotecan in the intestine and thus reduce the exposure of intestinal SN-38, which may alleviate irinotecan-associated diarrhea. However, existing hCES2 inhibitors (except loperamide) are not used in clinical applications due to lack of validity or acceptable safety. Therefore, developing more effective and safer drugs for treating delayed diarrhea is urgently needed. This study identified a lead compound 1 with a novel scaffold by high-throughput screening in our in-house library. After a comprehensive structure-activity relationship study, the optimal compound 24 was discovered as an efficient and highly selective hCES2 inhibitor (hCES2: IC50 = 6.72 µM; hCES1: IC50 > 100 µM). Further enzyme kinetics study indicated that compound 24 is a reversible inhibitor of hCES2 with competitive inhibition mode (Ki = 6.28 µM). The cell experiments showed that compound 24 could reduce the level of hCES2 in living cells (IC50 = 6.54 µM). The modeling study suggested that compound 24 fitted very well with the binding pocket of hCES2 by forming multiple interactions. Notably, compound 24 can effectively treat irinotecan-induced delayed diarrhea and DSS-induced ulcerative colitis, and its safety has also been verified in subtoxic studies. Based on the overall pharmacological and preliminary safety profiles, compound 24 is worthy of further evaluation as a novel agent for irinotecan-induced delayed diarrhea.

Pharmacokinetics of the KRASG12C inhibitor adagrasib is limited by CYP3A and ABCB1, and influenced by binding to mouse plasma carboxylesterase 1c.

Adagrasib (Krazati™) is the second FDA-approved specific KRASG12C inhibitor for non-small cell lung cancer (NSCLC) patients harboring this mutation. The impact of the drug efflux transporters ABCB1 and ABCG2, and the drug-metabolizing enzymes CYP3A and carboxylesterase 1 (CES1) on the pharmacokinetics of oral adagrasib were studied using genetically modified mouse models. Adagrasib was potently transported by human ABCB1 and modestly by mouse Abcg2 in vitro. In Abcb1a/b-/- and Abcb1a/b;Abcg2-/- mice, the brain-to-plasma ratios were enhanced by 33- and 55-fold, respectively, compared to wild-type mice, whereas ratios in Abcg2-/- mice remained unchanged. The influence of ABC transporters was completely reversed by coadministration of the dual ABCB1/ABCG2 inhibitor elacridar, increasing the brain penetration in wild-type mice by 41-fold while no signs of acute CNS toxicity were observed. Tumor ABCB1 overexpression may thus confer adagrasib resistance. Whereas the ABC transporters did not affect adagrasib plasma exposure, CYP3A and Ces1 strongly impacted its apparent oral availability. The plasma AUC0-8 h was significantly enhanced by 2.3-fold in Cyp3a-/- compared to wild-type mice, and subsequently 4.3-fold reduced in transgenic CYP3A4 mice, indicating substantial CYP3A-mediated metabolism. Adagrasib plasma exposure was strongly reduced in Ces1-/- compared to wild-type mice, but tissue exposure was slightly increased, suggesting that adagrasib binds to plasma Ces1c in mice and is perhaps metabolized by Ces1. This binding could complicate interpretation of mouse studies, especially since humans lack circulating CES1 enzyme(s). Our results may be useful to further optimize the clinical safety and efficacy of adagrasib, and give more insight into potential drug-drug interactions risks.

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.

Generation of Caco-2 cells with predictable metabolism by CYP3A4, UGT1A1 and CES using the PITCh system.

Caco-2 cells are widely used as an in vitro intestinal model. However, the expression levels of the drug-metabolizing enzymes CYP3A4 and UGT1A1 are lower in these cells than in intestinal cells. Furthermore, the majority of prodrugs in use today are ester-containing, and carboxylesterase (CES) 1 and CES2 are among the enzymes that process the prodrugs into drugs. In the human small intestine, CES1 is hardly expressed while CES2 is highly expressed, but the CES expression pattern in Caco-2 cells is the opposite. In this study, we generated CYP3A4-POR-UGT1A1-CES2 knock-in (KI) and CES1 knock-out (KO) Caco-2 (genome-edited Caco-2) cells using a PITCh system. Genome-edited Caco-2 cells were shown to express functional CYP3A4, POR, UGT1A1 and CES2 while the expression of the CES1 protein was completely knocked out. We performed transport assays using temocapril. The Papp value of temocapril in genome-edited Caco-2 cells was higher than that in WT Caco-2 cells. Interestingly, the amount of temocaprilat on the apical side in genome-edited Caco-2 cells was lower than that in WT Caco-2 cells. These results suggest that genome-edited Caco-2 cells are more suitable than WT Caco-2 cells as a model for predicting intestinal drug absorption and metabolism.

Discovery of seven-membered ring berberine analogues as highly potent and specific hCES2A inhibitors.

Human carboxylesterase 2A (hCES2A) is a key serine hydrolase responsible for the metabolic clearance of large number of compounds bearing the ester- or amide-bond(s). Inhibition of hCES2A can relieve the chemotherapy-induced toxicity and alter the pharmacokinetic bahaviors of some orally administrate esters-containing agents. However, most of the hCES2A inhibitors show poor cell-membrane permeability and poor specificity. Herein, guided by the structure activity relationships (SAR) of fifteen natural alkaloids against hCES2A, fifteen new seven-membered ring berberine analogues were designed and synthesized, and their anti-hCES2A activities were evaluated. Among all tested compounds, compound 28 showed potent anti-hCES2A effect (IC50 = 1.66 µM) and excellent selectivity over hCES1A (IC50 > 100 µM). The SAR analysis revealed that the seven-membered ring of these berberine analogues was a crucial moiety for hCES2A inhibition, while the secondary amine group of the ring-C is important for improving their specificity over other serine hydrolases. Inhibition kinetic analyses and molecular dynamic simulation demonstrated that 28 strongly inhibited hCES2A in a mixed-inhibition manner, with an estimated Ki value of 1.035 µM. Moreover, 28 could inhibit intracellular hCES2A in living HepG2 cells and exhibited suitable metabolic stability. Collectively, the SAR of seven-membered ring berberine analogues as hCES2A inhibitors were studied, while compound 28 acted as a promising candidate for developing highly selective hCES2A inhibitors.