ABCB1 attenuates brain exposure to the KRASG12C inhibitor opnurasib whereas binding to mouse carboxylesterase 1c influences its plasma exposure.

Opnurasib (JDQ443) is a newly developed oral KRASG12C inhibitor, with a binding mechanism distinct from the registered KRASG12C inhibitors sotorasib and adagrasib. Phase I and II clinical trials for opnurasib in NSCLC are ongoing. We evaluated the pharmacokinetic roles of the ABCB1 (P-gp/MDR1) and ABCG2 (BCRP) efflux and OATP1 influx transporters, and of the metabolizing enzymes CYP3A and CES1 in plasma and tissue disposition of oral opnurasib, using genetically modified cell lines and mouse models. In vitro, opnurasib was potently transported by human (h)ABCB1 and slightly by mouse (m)Abcg2. In Abcb1a/b- and Abcb1a/b;Abcg2-deficient mice, a significant ∼100-fold increase in brain-to-plasma ratios was observed. Brain penetration was unchanged in Abcg2-/- mice. ABCB1 activity in the blood-brain barrier may therefore potentially limit the efficacy of opnurasib against brain metastases. The Abcb1a/b transporter activity could be almost completely reversed by co-administration of elacridar, a dual ABCB1/ABCG2 inhibitor, increasing the brain penetration without any behavioral or postural signs of acute CNS-related toxicity. No significant pharmacokinetic roles of the OATP1 transporters were observed. Transgenic human CYP3A4 did not substantially affect the plasma exposure of opnurasib, indicating that opnurasib is likely not a sensitive CYP3A4 substrate. Interestingly, Ces1-/- mice showed a 4-fold lower opnurasib plasma exposure compared to wild-type mice, whereas no strong effect was seen on the tissue distribution. Plasma Ces1c therefore likely binds opnurasib, increasing its retention in plasma. The obtained pharmacokinetic insights may be useful for further optimization of the clinical efficacy and safety of opnurasib, and might reveal potential drug-drug interaction risks.

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.

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.

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.

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.

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.

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.

Evaluation on the Metabolic Activity of Two Carboxylesterase Isozymes in Mouse Liver Microsomes by a LC-MS/MS Method.

An applicable method for the precise measurement of major carboxylesterase (CESs) activity in liver still limited. Clopidogrel and irinotecan are specific substrates for CES1 and CES2, respectively. Clopidogrel is metabolized to the inactive metabolite clopidogrel carboxylate (CCAM) by CES1. Irinotecan is metabolized to the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) by CES2. In the present study, the LC-MS/MS method for the determination of CCAM and SN-38 were separately developed to characterize the metabolic activities of CES1 and CES2 in mouse liver microsomal. CCAM was separated on a Ecosil ODS column with an isocratic mobile phase consisted of 5 mmol/L ammonium formate and 0.1% formic acid in water and acetonitrile (15:85, V:V) at a flow rate of 0.4mL/min. SN-38 was separated on a Waters symmetry C18 column with an gradient mobile phase consisted of 5 mmol/L ammonium formate and 0.1% formic acid in water and acetonitrile at a flow rate of 0.3 mL/min. Calibration curves were linear within the concentration range of 100-20,000 ng/mL for CCAM and 1-200 ng/mL for SN-38. The results of method showed excellent accuracy and precision. The recovery rate, matrix effect and stability inspection results were within the acceptance criteria. The optimized incubation conditions were as follows: protein concentration of microsomes were all 0.1 mg/mL, incubation time was 60 min for clopidogrel and 30 min for irinotecan, respectively. This method was sensitive and applicable for the determination of the activity of CESs in the mouse liver microsomes.

Biodegradation of Free Gossypol by Helicoverpa armigera Carboxylesterase Expressed in Pichia pastoris.

Gossypol is a polyphenolic toxic secondary metabolite derived from cotton. Free gossypol in cotton meal is remarkably harmful to animals. Furthermore, microbial degradation of gossypol produces metabolites that reduce feed quality. We adopted an enzymatic method to degrade free gossypol safely and effectively. We cloned the gene cce001a encoding carboxylesterase (CarE) into pPICZαA and transformed it into Pichia pastoris GS115. The target protein was successfully obtained, and CarE CCE001a could effectively degrade free gossypol with a degradation rate of 89%. When esterase was added, the exposed toxic groups of gossypol reacted with different amino acids and amines to form bound gossypol, generating substances with (M + H) m/z ratios of 560.15, 600.25, and 713.46. The molecular formula was C27H28O13, C34H36N2O6, and C47H59N3O3. The observed instability of the hydroxyl groups caused the substitution and shedding of the group, forming a substance with m/z of 488.26 and molecular formula C31H36O5. These properties render the CarE CCE001a a valid candidate for the detoxification of cotton meal. Furthermore, the findings help elucidate the degradation process of gossypol in vitro.

Carboxylesterase 2 induces mitochondrial dysfunction via disrupting lipid homeostasis in oral squamous cell carcinoma.

OBJECTIVE: Oral squamous cell carcinoma (OSCC) is characterized by high recurrence and metastasis and places a heavy burden on societies worldwide. Cancer cells thrive in a changing microenvironment by reprogramming lipidomic metabolic processes to provide nutrients and energy, activate oncogenic signaling pathways, and manage redox homeostasis to avoid lipotoxicity. The mechanism by which OSCC cells maintain lipid homeostasis during malignant progression is unclear. METHODS: The altered expression of fatty acid (FA) metabolism genes in OSCC, compared with that in normal tissues, and in OSCC patients with or without recurrence or metastasis were determined using public data from the TCGA and GEO databases. Immunohistochemistry was performed to examine the carboxylesterase 2 (CES2) protein level in our own cohort. CCK-8 and Transwell assays and an in vivo xenograft model were used to evaluate the biological functions of CES2. Mass spectrometry and RNA sequencing were performed to determine the lipidome and transcriptome alterations induced by CES2. Mitochondrial mass, mtDNA content, mitochondrial membrane potential, ROS levels, and oxygen consumption and apoptosis rates were evaluated to determine the effects of CES2 on mitochondrial function in OSCC. RESULTS: CES2 was downregulated in OSCC patients, especially those with recurrence or metastasis. CES2high OSCC patients showed better overall survival than CES2low OSCC patients. Restoring CES2 expression reduced OSCC cell viability and suppressed their migration and invasion in vitro, and it inhibited OSCC tumor growth in vivo. CES2 reprogrammed lipid metabolism in OSCC cells by hydrolyzing neutral lipid diacylglycerols (DGs) to release free fatty acids and reduce the membrane structure lipid phospholipids (PLs) synthesis. Free FAs were converted to acyl-carnitines (CARs) and transferred to mitochondria for oxidation, which induced reactive oxygen species (ROS) accumulation, mitochondrial damage, and apoptosis activation. Furthermore, the reduction in signaling lipids, e.g., DGs, PLs and substrates, suppressed PI3K/AKT/MYC signaling pathways. Restoring MYC rescued the diminished cell viability, suppressed migratory and invasive abilities, damaged mitochondria and reduced apoptosis rate induced by CES2. CONCLUSIONS: We demonstrated that CES2 downregulation plays an important role in OSCC by maintaining lipid homeostasis and reducing lipotoxicity during tumor progression and may provide a potential therapeutic target for OSCC.

Selective detection of carboxylesterase 2 activity in cancer cells using an activity-based chemiluminescent probe.

Carboxylesterase 2 (CES2) has crucial roles in both xenobiotic metabolism and formation of pathogenic states including cancer. Thus, it is highly critical to monitor intracellular CES2 activity in living cancer cells. Here, we report a CES2 activatable phenoxy 1,2-dioxetane based chemiluminescent agent (CL-CES2). The probe exhibited a selective turn-on response in the presence of CES2 enzyme and enabled detection of CES2 activity in three different cancer cells that possess varying enzyme concentrations with high signal to noise ratios. In contrast no signal was obtained with CES1, an isoform of CES2 enzyme. CL-CES2 marks the first ever example of a CES2-responsive chemiluminescent luminophore and holds a great potential in further understanding the roles of CES2 activity in tumorogenesis.

Carbon nanomaterial functionalization with pesticide-detoxifying carboxylesterase.

Four carbon materials, spent coffee-ground biochar, carbon black, short CNTs, and nitrogen-doped few-layer graphene (N-graphene) were tested for their functionalization with a commercial carboxylesterase. Their robustness to variations in time and key physicochemical parameters (temperature and pH) was analysed. In general, carbon nanomaterials showed better performance than biochar, both in terms of binding capacity and resilience in harsh conditions, at statistically significant levels. Among the tested materials, functionalized N-graphene also showed the highest level of inhibition of carboxylesterase by pesticide exposure. Therefore, N-graphene was selected for biotechnological application of pesticide scavenging toxicity in T. thermophila, a ciliate bioindicator of water quality. While immobilization of the enzyme was not effective in the case of carbaryl, a methyl carbamate, in the case of the organophosphorus dichlorvos, a 1- or 30-min contact time with a water solution containing 5 times the LC100 - 0.5 mM - allowed 50% and 100% rescue of ciliate survival, respectively. These results suggest that functionalization with carboxylesterase may be of additional benefit compared to bare carbon in water clean-up procedures, especially for highly hydrophilic pesticides such as dichlorvos.

Endoplasmic stress-inducing variants in carboxyl ester lipase and pancreatic cancer risk.

BACKGROUND: Endoplasmic reticulum (ER) stress-inducing variants in several pancreatic secretory enzymes have been associated with pancreatic disease. Multiple variants in CEL, encoding carboxyl ester lipase, are known to cause maturity-onset diabetes of the young (MODY8) but have not been implicated in pancreatic cancer risk. METHODS: The prevalence of ER stress-inducing variants in the CEL gene was compared among pancreatic cancer cases vs. controls. Variants were identified by next-generation sequencing and confirmed by Sanger sequencing. Variants of uncertain significance (VUS) were assessed for their effect on the secretion of CEL protein and variants with reduced protein secretion were evaluated to determine if they induced endoplasmic reticulum stress. RESULTS: ER stress-inducing CEL variants were found in 34 of 986 cases with sporadic pancreatic ductal adenocarcinoma, and 21 of 1045 controls (P = 0.055). Most of the variants were either the CEL-HYB1 variant, the I488T variant, or the combined CEL-HYB1/I488T variant; one case had a MODY8 variant. CONCLUSION: This case/control analysis finds ER stress-inducing CEL variants are not associated with an increased likelihood of having pancreatic cancer.

Activation of Esterase D by FPD5 Inhibits Growth of A549 Lung Cancer Cells via JAB1/p53 Pathway.

Esterase D (ESD) is widely distributed in mammals, and it plays an important role in drug metabolism, detoxification, and biomarkers and is closely related to the development of tumors. In our previous work, we found that a chemical small-molecule fluorescent pyrazoline derivative, FPD5, an ESD activator, could inhibit tumor growth by activating ESD, but its molecular mechanism is still unclear. Here, by using RNA interference (RNAi), andco-immunoprecipitation techniques, we found that ESD suppressed the nucleus exportation of p53 through reducing the interaction between p53 and JAB1. The protein level of p53 in the nucleus was upregulated and the downstream targets of p53 were found by Human Gene Expression Array. p53 inhibited the expression of CDCA8 and CDC20. Lastly, the cell cycle of A549 cells was arrested at the G0/G1 phase. Together, our data suggest that ESD inhibited the cancer cell growth by arresting the cell cycle of A549 cells via the JAB1/p53 signaling pathway. Our findings provide a new insight into how to inhibit the growth of lung cancer with the activation of ESD by FPD5.

Human carboxylesterase 1A plays a predominant role in the hydrolytic activation of remdesivir in humans.

Remdesivir, an intravenous nucleotide prodrug, has been approved for treating COVID-19 in hospitalized adults and pediatric patients. Upon administration, remdesivir can be readily hydrolyzed to form its active form GS-441524, while the cleavage of the carboxylic ester into GS-704277 is the first step for remdesivir activation. This study aims to assign the key enzymes responsible for remdesivir hydrolysis in humans, as well as to investigate the kinetics of remdesivir hydrolysis in various enzyme sources. The results showed that remdesivir could be hydrolyzed to form GS-704277 in human plasma and the microsomes from human liver (HLMs), lung (HLuMs) and kidney (HKMs), while the hydrolytic rate of remdesivir in HLMs was the fastest. Chemical inhibition and reaction phenotyping assays suggested that human carboxylesterase 1 (hCES1A) played a predominant role in remdesivir hydrolysis, while cathepsin A (CTSA), acetylcholinesterase (AchE) and butyrylcholinesterase (BchE) contributed to a lesser extent. Enzymatic kinetic analyses demonstrated that remdesivir hydrolysis in hCES1A (SHUTCM) and HLMs showed similar kinetic plots and much closed Km values to each other. Meanwhile, GS-704277 formation rates were strongly correlated with the CES1A activities in HLM samples from different individual donors. Further investigation revealed that simvastatin (a therapeutic agent for adjuvant treating COVID-19) strongly inhibited remdesivir hydrolysis in both recombinant hCES1A and HLMs. Collectively, our findings reveal that hCES1A plays a predominant role in remdesivir hydrolysis in humans, which are very helpful for predicting inter-individual variability in response to remdesivir and for guiding the rational use of this anti-COVID-19 agent in clinical settings.

Enzyme Control Over Ferric Iron Magnetostructural Properties.

Fe3+ complexes in aqueous solution can exist as discrete mononuclear species or multinuclear magnetically coupled species. Stimuli-driven change to Fe3+ speciation represents a powerful mechanistic basis for magnetic resonance sensor technology, but ligand design strategies to exert precision control of aqueous Fe3+ magnetostructural properties are entirely underexplored. In pursuit of this objective, we rationally designed a ligand to strongly favor a dinuclear µ-oxo-bridged and antiferromagnetically coupled complex, but which undergoes carboxylesterase mediated transformation to a mononuclear high-spin Fe3+ chelate resulting in substantial T1 -relaxivity increase. The data communicated demonstrate proof of concept for a novel and effective strategy to exert biochemical control over aqueous Fe3+ magnetic, structural, and relaxometric properties.

Acute Toxicity of Insecticide Thiamethoxam to Crayfish (Astacus leptodactylus): Alterations in Oxidative Stress Markers, ATPases and Cholinesterase.

Thiamethoxam (Thmx) is a globally used neonicotinoid pesticide contaminated in freshwater ecosystems with residues detected in fishery products. Astacus leptodactylus is a popular freshwater crustacean that is cultivated and exported in many countries. In this study, we investigated the acute toxic effects of Thmx on A. leptodactylus using various biomarkers (acetylcholinesterase, carboxylesterase, glutathione S-transferase, glutathione, superoxide dismutase, glutathione peroxidase, glutathione reductase, and adenosinetriphosphatases). The 96-h LC50 value of Thmx was calculated as 8.95 mg active ingredient L-1. As the dose of Thmx increased, oxidative stress was induced by the inhibition/activation of antioxidant enzymes, while the activities of acetylcholinesterase, carboxylesterase and adenosinetriphosphatases were inhibited. As a result, it can be said that Thmx has highly toxic effects on crayfish, therefore they are under threat in the areas where this pesticide is used.

m6A modification impacts hepatic drug and lipid metabolism properties by regulating carboxylesterase 2.

Methylation of adenosine at the N6 position to form N6-methyladenosine (m6A) is the most prevalent epitranscriptomic modification of mammalian mRNA. This modification is catalyzed by a methyltransferase-like 3 (METTL3)-METTL14 complex and is erased by demethylases such as fat mass and obesity-associated protein (FTO) or AlkB homolog 5 (ALKBH5). m6A modification regulates mRNA stability, nuclear export, splicing, and/or protein translation via recognition by reader proteins such as members of YT521-B homology (YTH) family. Carboxylesterase 2 (CES2) is a serine esterase responsible for the hydrolysis of drugs and endogenous substrates, such as triglycerides and diacylglycerides. Here, we examined the potential regulation of human CES2 expression by m6A modification. CES2 mRNA level was significantly increased by double knockdown of METTL3 and METTL14 but was decreased by knockdown of FTO or ALKBH5 in HepaRG and HepG2 cells, leading to changes in its protein level and hydrolase activity for 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11), suggesting that m6A modification negatively regulates CES2 expression. Consistent with the changes in CES2 expression, lipid accumulation in the cells was decreased by double knockdown of METTL3 and METTL14 but was increased by knockdown of FTO or ALKBH5. RNA immunoprecipitation assays using an anti-m6A antibody showed that adenosines in the 5'-untranslated region (UTR) and the last exon of CES2 are methylated. Luciferase assays revealed that YTHDC2, which degrades m6A-containing mRNA, downregulates CES2 expression by recognition of m6A in the 5'-UTR of CES2. Collectively, we demonstrated that m6A modification has a great impact on the regulation of CES2, affecting pharmacokinetics, drug response and lipid metabolism.

Homozygosity of short VNTR lengths in the CEL gene may confer susceptibility to idiopathic chronic pancreatitis.

OBJECTIVE: The carboxyl-ester lipase (CEL) gene contains a variable number of tandem repeats (VNTR) region. It remains unclear whether the number of repeats in the CEL VNTR is related to the risk of pancreatic diseases. The aim of this study was to investigate whether CEL VNTR length is associated with idiopathic chronic pancreatitis (ICP), alcoholic chronic pancreatitis (ACP), or pancreatic cancer in a cohort of Chinese patients. METHODS: CEL VNTRs were genotyped in patients diagnosed with ICP (n = 771), ACP (n = 222), or pancreatic cancer (n = 263), and in healthy controls (n = 927). CEL VNTR lengths were determined using a screening method combining PCR and DNA fragment analysis. RESULTS: Overall, the CEL VNTR lengths ranged from 5 to 22 repeats, with the 16-repeat allele ('normal' size, N) accounting for 73.82% of all observed alleles. The VNTR allele frequencies and genotype distributions were not significantly different between healthy controls and patients with ACP or pancreatic cancer. For the ICP group, allele frequencies did not differ significantly from the controls, while the frequency of the SS genotype (homozygosity for 5-15 repeats) was significantly higher in the patients (4.67%) than in the controls (1.94%) (p = 0.0014; OR = 2.47; 95% CI = 1.39-4.39). CONCLUSIONS: There were no associations between the CEL VNTR length and ACP or pancreatic cancer. However, homozygosity for short VNTR lengths may confer susceptibility to ICP.

Arylacetamide deacetylase as a determinant of the hydrolysis and activation of abiraterone acetate in mice and humans.

AIM: Abiraterone acetate for metastatic castration-resistant prostate cancer is an acetylated prodrug to be hydrolyzed to abiraterone. Abiraterone acetate is known to be hydrolyzed by pancreatic cholesterol esterase secreted into the intestinal lumen. This study aimed to investigate the possibility that arylacetamide deacetylase (AADAC) expressed in enterocytes contributes to the hydrolysis of abiraterone acetate based on its substrate preference. MATERIALS AND METHODS: Abiraterone acetate hydrolase activity was measured using human intestinal (HIM) and liver microsomes (HLM) as well as recombinant AADAC. Correlation analysis between activity and AADAC expression was performed in 14 individual HIMs. The in vivo pharmacokinetics of abiraterone acetate was examined using wild-type and Aadac knockout mice administered abiraterone acetate with or without orlistat, a pancreatic cholesterol esterase inhibitor. KEY FINDINGS: Recombinant AADAC showed abiraterone acetate hydrolase activity with similar Km value to HIM and HLM. The positive correlation between activity and AADAC levels in individual HIMs supported the responsibility of AADAC for abiraterone acetate hydrolysis. The area under the plasma concentration-time curve (AUC) of abiraterone after oral administration of abiraterone acetate in Aadac knockout mice was 38% lower than that in wild-type mice. The involvement of pancreatic cholesterol esterase in abiraterone formation was revealed by the decreased AUC of abiraterone by coadministration of orlistat. Orlistat potently inhibited AADAC, implying its potential as a perpetrator of drug-drug interactions. SIGNIFICANCE: AADAC is responsible for the hydrolysis of abiraterone acetate in the intestine and liver, suggesting that concomitant use of abiraterone acetate and drugs potently inhibiting AADAC should be avoided.