Biophysical and functional properties of purified glucose-6-phosphatase catalytic subunit 1.

Glucose-6-phosphatase catalytic subunit 1 (G6PC1) plays a critical role in hepatic glucose production during fasting by mediating the terminal step of the gluconeogenesis and glycogenolysis pathways. In concert with accessory transport proteins, this membrane-integrated enzyme catalyzes glucose production from glucose-6-phosphate (G6P) to support blood glucose homeostasis. Consistent with its metabolic function, dysregulation of G6PC1 gene expression contributes to diabetes, and mutations that impair phosphohydrolase activity form the clinical basis of glycogen storage disease type 1a. Despite its relevance to health and disease, a comprehensive view of G6PC1 structure and mechanism has been limited by the absence of expression and purification strategies that isolate the enzyme in a functional form. In this report, we apply a suite of biophysical and biochemical tools to fingerprint the in vitro attributes of catalytically active G6PC1 solubilized in lauryl maltose neopentyl glycol (LMNG) detergent micelles. When purified from Sf9 insect cell membranes, the glycosylated mouse ortholog (mG6PC1) recapitulated functional properties observed previously in intact hepatic microsomes and displayed the highest specific activity reported to date. Additionally, our results establish a direct correlation between the catalytic and structural stability of mG6PC1, which is underscored by the enhanced thermostability conferred by phosphatidylcholine and the cholesterol analog cholesteryl hemisuccinate. In contrast, the N96A variant, which blocks N-linked glycosylation, reduced thermostability. The methodologies described here overcome long-standing obstacles in the field and lay the necessary groundwork for a detailed analysis of the mechanistic structural biology of G6PC1 and its role in complex metabolic disorders.

Label-Free but Still Constrained: Assessment of Global Proteomic Strategies for the Quantification of Hepatic Enzymes and Transporters.

Building and refining pharmacology models require "system" data derived from tissues and in vitro systems analyzed by quantitative proteomics. Label-free global proteomics offers a wide scope of analysis, allowing simultaneous quantification of thousands of proteins per sample. The data generated from such analysis offer comprehensive protein expression profiles that can address existing gaps in models. In this study, we assessed the performance of three widely used label-free proteomic methods, "high N" ion intensity approach (HiN), intensity-based absolute quantification (iBAQ) and total protein approach (TPA), in relation to the quantification of enzymes and transporters in 27 human liver microsomal samples. Global correlations between the three methods were highly significant (R2 > 0.70, P < 0.001, n = 2232 proteins). Absolute abundances of 57 pharmacokinetic targets measured by standard-based label-free methods (HiN and iBAQ) showed good agreement, whereas the TPA overestimated abundances by two- to threefold. Relative abundance distribution of enzymes was similar for the three methods, while differences were observed with TPA in the case of transporters. Variability (CV) was similar across methods, with consistent between-sample relative quantification. The back-calculated amount of protein in the samples based on each method was compared with the nominal protein amount analyzed in the proteomic workflow, revealing overall agreement with data from the HiN method with bovine serum albumin as standard. The findings herein present a critique of label-free proteomic data relevant to pharmacokinetics and evaluate the possibility of retrospective analysis of historic datasets. SIGNIFICANCE STATEMENT: This study provides useful insights for using label-free methods to generate abundance data applicable for populating pharmacokinetic models. The data demonstrated overall correlation between intensity-based label-free proteomic methods (HiN, iBAQ and TPA), whereas iBAQ and TPA overestimated the total amount of protein in the samples. The extent of overestimation can provide a means of normalization to support absolute quantification. Importantly, between-sample relative quantification was consistent (similar variability) across methods.

Identifying the Dominant Contribution of Human Cytochrome P450 2J2 to the Metabolism of Rivaroxaban, an Oral Anticoagulant.

PURPOSE: Rivaroxaban, an oral anticoagulant, undergoes the metabolism mediated by human cytochrome P450 (CYP). The present study is to quantitatively analyze and compare the contributions of multiple CYPs in the metabolism of rivaroxaban to provide new information for medication safety. METHODS: The metabolic stability of rivaroxaban in the presence of human liver microsomes (HLMs) and recombinant CYPs was systematically evaluated to estimate the participation of various CYP isoforms. Furthermore, the catalytic efficiency of CYP isoforms was compared via metabolic kinetic studies of rivaroxaban with recombinant CYP isoenzymes, as well as via CYP-specific inhibitory studies. Additionally, docking simulations were used to illustrate molecular interactions. RESULTS: Multiple CYP isoforms were involved in the hydroxylation of rivaroxaban, with decreasing catalytic rates as follows: CYP2J2 > 3A4 > 2D6 > 4F3 > 1A1 > 3A5 > 3A7 > 2A6 > 2E1 > 2C9 > 2C19. Among the CYPs, 2J2, 3A4, 2D6, and 4F3 were the four major isoforms responsible for rivaroxaban metabolism. Notably, the intrinsic clearance of rivaroxaban catalyzed by CYP2J2 was nearly 39-, 64-, and 100-fold that catalyzed by CYP3A4, 2D6, and 4F3, respectively. In addition, rivaroxaban hydroxylation was inhibited by 41.1% in the presence of the CYP2J2-specific inhibitor danazol, which was comparable to the inhibition rate of 43.3% by the CYP3A-specific inhibitor ketoconazole in mixed HLMs. Furthermore, molecular simulations showed that rivaroxaban is principally bound to CYP2J2 by π-alkyl bonds, carbon-hydrogen bonds, and alkyl interactions. CONCLUSION: CYP2J2 dominated the hydroxylation of rivaroxaban, which may provide new insight into clinical drug interactions involving rivaroxaban.

Construction and Characterization of CRISPR/Cas9 Knockout Rat Model of Carboxylesterase 2a Gene.

Carboxylesterase (CES) 2, an important metabolic enzyme, plays a critical role in drug biotransformation and lipid metabolism. Although CES2 is very important, few animal models have been generated to study its properties and functions. Rat Ces2 is similar to human CES2A-CES3A-CES4A gene cluster, with highly similar gene structure, function, and substrate. In this report, CRISPR-associated protein-9 (CRISPR/Cas9) technology was first used to knock out rat Ces2a, which is a main subtype of Ces2 mostly distributed in the liver and intestine. This model showed the absence of CES2A protein expression in the liver. Further pharmacokinetic studies of diltiazem, a typical substrate of CES2A, confirmed the loss of function of CES2A both in vivo and in vitro. At the same time, the expression of CES2C and CES2J protein in the liver decreased significantly. The body and liver weight of Ces2a knockout rats also increased, but the food intake did not change. Moreover, the deficiency of Ces2a led to obesity, insulin resistance, and liver fat accumulation, which are consistent with the symptoms of nonalcoholic fatty liver disease (NAFLD). Therefore, this rat model is not only a powerful tool to study drug metabolism mediated by CES2 but also a good disease model to study NAFLD. SIGNIFICANCE STATEMENT: Human carboxylesterase (CES) 2 plays a key role in the first-pass hydrolysis metabolism of most oral prodrugs as well as lipid metabolism. In this study, CRISPR/Cas9 technology was used to knock out Ces2a gene in rats for the first time. This model can be used not only in the study of drug metabolism and pharmacokinetics but also as a disease model of nonalcoholic fatty liver disease (NAFLD) and other metabolic disorders.

Proteomic Quantification of Changes in Abundance of Drug-Metabolizing Enzymes and Drug Transporters in Human Liver Cirrhosis: Different Methods, Similar Outcomes.

Model-based assessment of the effects of liver disease on drug pharmacokinetics requires quantification of changes in enzymes and transporters responsible for drug metabolism and disposition. Different proteomic methods are currently used for protein quantification in tissues and in vitro systems, each with specific procedures and requirements. The outcome of quantitative proteomic assays using four different methods (one targeted and three label-free) applied to the same sample set was compared in this study. Three pooled cirrhotic liver microsomal samples corresponding to cirrhosis with nonalcoholic fatty liver disease, biliary disease, or cancer and a control microsomal pool were analyzed using quantification concatemer-based targeted proteomics, the total protein approach (TPA), high three ion intensity (Hi3) approach, and intensity-based absolute quantification (iBAQ) to determine the absolute and relative abundance in disease compared with control. The relative abundance data provided a "disease perturbation factor" (DPF) for each target protein. Absolute and relative abundances generated by standard-based label-free methods (iBAQ and Hi3) showed good agreement with targeted proteomics (limited bias and scatter), but TPA (standard-free method) overestimated absolute abundances by approximately 2-fold. The DPF was consistent between different proteomic methods but varied between enzymes and transporters, indicating discordance of effects of cirrhosis on various metabolism-related proteins. The DPF ranged from no change (e.g., for glucuronosyltransferase-1A6 in nonalcoholic fatty liver disease group) to less than 0.3 (e.g., carboxylesterases-1 in cirrhosis of biliary origin). SIGNIFICANCE STATEMENT: This study demonstrated that relative changes in enzymes and transporters (DPF) are independent of the quantitative proteomic methods used. Standard-based label-free methods, such as high three ion intensity (Hi3) and intensity-based absolute quantification (iBAQ) methods, were less biased and more precise than the total protein approach (TPA) when compared with targeted data. The DPF reconciled differences across proteomic methods observed with absolute levels. Using this approach, differences were revealed in the expression of enzymes/transporters in cirrhosis associated with different etiologies.

Exposure to an Environmental Mixture of Polycyclic Aromatic Hydrocarbons Induces Hepatic Cytochrome P450 Enzymes in Mice.

Cytochrome P450 enzymes (CYPs) play an important role in bioactivating or detoxifying polycyclic aromatic hydrocarbons (PAHs), common environmental contaminants. While it is widely accepted that exposure to PAHs induces CYPs, effectively increasing rates of xenobiotic metabolism, dose- and time-response patterns of CYP induction are not well-known. In order to better understand dose- and time-response relationships of individual CYPs following induction, we exposed B6129SF1/J mice to single or repeated doses (2-180 µmol/kg/d) of benzo[a]pyrene (BaP) or Supermix-10, a mixture of the top 10 most abundant PAHs found at the Portland Harbor Superfund Site. In hepatic microsomes from exposed mice, we measured amounts of active CYPs using activity-based protein profiling and total CYP expression using global proteomics. We observed rapid Cyp1a1 induction after 6 h at the lowest PAH exposures and broad induction of many CYPs after 3 daily PAH doses at 72 h following the first dose. Using samples displaying Cyp1a1 induction, we observed significantly higher metabolic affinity for BaP metabolism (Km reduced 3-fold), 3-fold higher intrinsic clearance, but no changes to the Vmax. Mice dosed with the highest PAH exposures exhibited 1.7-5-fold higher intrinsic clearance rates for BaP compared to controls and higher Vmax values indicating greater amounts of enzymes capable of metabolizing BaP. This study demonstrates exposure to PAHs found at superfund sites induces enzymes in dose- and time-dependent patterns in mice. Accounting for specific changes in enzyme profiles, relative rates of PAH bioactivation and detoxification, and resulting risk will help translate internal dosimetry of animal models to humans and improve risk assessments of PAHs at superfund sites.

In Vitro and In Vivo Studies of Metabolic Activation of Marrubiin, a Bioactive Constituent from Marrubium Vulgare.

Marrubiin, a furanoid compound, is a well-known diterpenoid lactone isolated from Marrubium vulgare, which displays a wide spectrum of pharmacological effects and potential hepatotoxicity. Considering that marrubiin contains a structural alert, furan ring, metabolic activation may be one of the major metabolic pathways, and the reactive metabolite may be involved in the hepatotoxicity. The present study was carried out to investigate the bioactivation mechanism of marrubiin in rats and humans. Marrubiin was initially metabolized into cis-butene-1,4-dial intermediate, which was readily trapped by glutathione (GSH) and N-acetyl-lysine (NAL) in the microsomal incubations supplemented with NADPH. A total of nine conjugates were detected and identified by high-resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. M1-M3 and M6 and M7 were characterized as mono-GSH conjugates, and M4 and M5 were identified as bis-GSH conjugates. M8 and M9 were identified as NAL conjugates. In rat bile, five GSH conjugates (M1-M3; M6 and M7) were detected. M1, M8, and M9 were chemically synthesized, and their structures were characterized by 13C NMR. Sulfaphenazole, ticlopidine, and ketoconazole displayed significant inhibitory effect on the bioactivation of marrubiin. Further phenotyping revealed that CYP2C9, CYP2C19, and CYP3A4 were the primary enzymes catalyzing the bioactivation of marrubiin. The current study provides evidence for the CYP-dominated bioactivation of marrubiin to the corresponding cis-butene-1,4-dial intermediate, which enables us to better understand the potential side effects caused by marrubiin.

Influence of Proteome Profiles and Intracellular Drug Exposure on Differences in CYP Activity in Donor-Matched Human Liver Microsomes and Hepatocytes.

Human liver microsomes (HLM) and human hepatocytes (HH) are important in vitro systems for studies of intrinsic drug clearance (CLint) in the liver. However, the CLint values are often in disagreement for these two systems. Here, we investigated these differences in a side-by-side comparison of drug metabolism in HLM and HH prepared from 15 matched donors. Protein expression and intracellular unbound drug concentration (Kpuu) effects on the CLint were investigated for five prototypical probe substrates (bupropion-CYP2B6, diclofenac-CYP2C9, omeprazole-CYP2C19, bufuralol-CYP2D6, and midazolam-CYP3A4). The samples were donor-matched to compensate for inter-individual variability but still showed systematic differences in CLint. Global proteomics analysis outlined differences in HLM from HH and homogenates of human liver (HL), indicating variable enrichment of ER-localized cytochrome P450 (CYP) enzymes in the HLM preparation. This suggests that the HLM may not equally and accurately capture metabolic capacity for all CYPs. Scaling CLint with CYP amounts and Kpuu could only partly explain the discordance in absolute values of CLint for the five substrates. Nevertheless, scaling with CYP amounts improved the agreement in rank order for the majority of the substrates. Other factors, such as contribution of additional enzymes and variability in the proportions of active and inactive CYP enzymes in HLM and HH, may have to be considered to avoid the use of empirical scaling factors for prediction of drug metabolism.

Transfer learning enables prediction of CYP2D6 haplotype function.

Cytochrome P450 2D6 (CYP2D6) is a highly polymorphic gene whose protein product metabolizes more than 20% of clinically used drugs. Genetic variations in CYP2D6 are responsible for interindividual heterogeneity in drug response that can lead to drug toxicity and ineffective treatment, making CYP2D6 one of the most important pharmacogenes. Prediction of CYP2D6 phenotype relies on curation of literature-derived functional studies to assign a functional status to CYP2D6 haplotypes. As the number of large-scale sequencing efforts grows, new haplotypes continue to be discovered, and assignment of function is challenging to maintain. To address this challenge, we have trained a convolutional neural network to predict functional status of CYP2D6 haplotypes, called Hubble.2D6. Hubble.2D6 predicts haplotype function from sequence data and was trained using two pre-training steps with a combination of real and simulated data. We find that Hubble.2D6 predicts CYP2D6 haplotype functional status with 88% accuracy in a held-out test set and explains 47.5% of the variance in in vitro functional data among star alleles with unknown function. Hubble.2D6 may be a useful tool for assigning function to haplotypes with uncurated function, and used for screening individuals who are at risk of being poor metabolizers.

Clopidogrel Resistance in a Murine Model of Diet-Induced Obesity Is Mediated by the Interleukin-1 Receptor and Overcome With DT-678.

OBJECTIVE: Clopidogrel is a commonly used P2Y12 inhibitor to treat and prevent arterial thrombotic events. Clopidogrel is a prodrug that requires bioactivation by CYP (cytochrome P450) enzymes to exert antiplatelet activity. Diabetes mellitus is associated with an increased risk of ischemic events, and impaired ability to generate the active metabolite (AM) from clopidogrel. The objective of this study is to identify the mechanism of clopidogrel resistance in a murine model of diet-induced obesity (DIO). Approach and Results: C57BL/6J mice and IL-1R-/- mice were given high-fat diet for 10 weeks to generate a murine model of diet-induced obesity. Platelet aggregation and carotid arterial thrombosis were assessed in response to clopidogrel treatment. Wild-type DIO mice exhibited resistance to antiplatelet and antithrombotic effects of clopidogrel that was associated with reduced hepatic expression of CYP genes and reduced generation of the AM. IL (Interleukin)-1 receptor-deficient DIO (IL1R-/- DIO) mice showed no resistance to clopidogrel. Lack of resistance was accompanied by increased exposure of the clopidogrel AM. This resistance was also absent when wild-type DIO mice were treated with the conjugate of the clopidogrel AM, DT-678. CONCLUSIONS: These findings indicate that antiplatelet effects of clopidogrel may be impaired in the setting of diabetes mellitus due to reduced prodrug bioactivation related to IL-1 receptor signaling. Therapeutic targeting of P2Y12 in patients with diabetes mellitus using the conjugate of clopidogrel AM may lead to improved outcomes.

Metabolic study of ginsenoside Rg3 and glimepiride in type 2 diabetic rats by liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry.

RATIONALE: Ginsenoside Rg3 and glimepiride have been applied to treat type 2 diabetes (T2DM) because of their good hypoglycemic effects. In this study, the effects of ginsenoside Rg3 acting synergistically with glimepiride were investigated in liver microsomes from rats with type 2 diabetes. METHODS: An in vitro incubation system with normal rat liver microsomes (RLM) and type 2 diabetic rat liver microsomes (TRLM) was developed. The system also included two experimental groups consisting of RLM and TRLM pretreated with ginsenoside Rg3 and glimepiride (named the RLMR and TRLMR groups, respectively). The metabolism in the different groups was analyzed by ultra-performance liquid chromatography coupled with quadrupole-orbitrap mass spectrometry (UPLC/Q-Orbitrap MS). RESULTS: The results showed that the concentration of glimepiride increased in RLM and TRLM after treatment with ginsenoside Rg3. Five metabolites (M1-M5) of glimepiride were found, and they were named 3N-hydroxyglimepiride, hydroxyglimepiride, 1,2-epoxy ether-3-hydroxyglimepiride, 1N-hydroxyglimepiride and 1N,2C,S,O,O-epoxy ether-3-hydroxyglimepiride. The metabolite of ginsenoside Rg3 was ginsenoside Rh2. CONCLUSIONS: An in vitro incubation system with RLM and TRLM was developed. The system revealed pathways that produce glimepiride metabolites. Ginsenoside Rg3 may inhibit the activity of cytochrome P450 enzymes in vitro. The present study showed that ginsenoside Rg3 and glimepiride may be combined for the treatment of T2DM.

Comparison on Metabolism of Estradiol and Its 17-Sulfate by Recombinant Human CYP Isoforms.

To identify the CYP isoforms involved in the production of 2-hydroxyestradiol 17-sulfate (2-OH-ES), which we assume to be an antioxidant in vivo, the 2-hydroxylation reaction of estradiol 17-sulfate (ES) by human liver microsome was investigated. As a result, it was estimated that CYP2C8 and 2C9 were largely involved in the production of 2-OH-ES. Therefore, the 2-hydroxylation kinetic analysis of ES was performed for both CYPs, and the metabolic clearance Vmax/Km (µL/nmol CYP/min) was determined. On comparing the results of ES with those of estradiol (E2), it was found that CYP2C8 was about 2.5 times higher and CYP2C9 was about 3 times higher, and ES was more likely to be a substrate for the 2-hydroxylation reaction by both CYPs. The CYP isoforms involved in A-ring hydroxylation of E2 and ES differed. From this, it was speculated that 2-OH-ES plays a different role to 2-hydroxyestradiol (2-OH-E2), which is recognized as an antioxidant in the body.

In vitro study on the effect of ophiopogonin D on the activity of cytochrome P450 enzymes.

Ophiopogonin D is a commonly used herb in cardiology and pediatrics for its variuos pharmacological effects. It is necessary to investigate the effect of ophiopogonin D on the activity of cytochrome P450 enzymes (CYP450s) to provide more guidance for the clinical application of ophiopogonin D. Eight isoforms of CYP450s, including CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 were incubated with 100 µM ophiopogonin D in pooled human liver microsomes. The inhibition model and corresponding parameters were also investigated. Ophiopogonin D exerted a significant inhibitory effect on the activity of CYP3A4, 2C9, and 2E1 in a dose-dependent manner with the IC50 values of 8.08, 12.92, and 22.72 µM, respectively (p < 0.05). The inhibition of CYP3A4 by ophiopogonin D was performed non-competitively and time-dependently with the Ki value of 4.08 µM and the KI/Kinact value of 5.02/0.050 min-1·µM-1. Whereas, ophiopogonin D acts as a competitive inhibitor of CYP2E1 and 2C9 with the Ki value of 6.69 and 11.07 µM, respectively. The inhibitory effect of ophiopogonin D on the activity of CYP3A4, 2C9, and 2E1 indicated the potential drug-drug interaction between ophiopogonin D and drugs metabolized by these CYP450s, which needs further in vivo investigation and validation.

Assessment of the in vitro cytochrome P450 (CYP) inhibition potential of nafithromycin, a next generation lactone ketolide antibiotic.

Nafithromycin is a next generation lactone ketolide antibiotic slated to enter phase III clinical development in India for the treatment of CABP as a shorter 800 mg-OD X3 day therapeutic regimen. Nafithromycin exhibits potent activity against MDR Streptococcus pneumoniae including erythromycin and telithromycin-resistant resistant strains. Older macrolides/ketolides are reported to be potent inhibitors of CYP3A4/5. To facilitate comparative assessment of drug-drug interaction potential, CYP inhibitory activities of nafithromycin was evaluated in comparison with clarithromycin, telithromycin, cethromycin and solithromycin. CYP inhibitory activities were assessed against key CYP isoforms (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and CYP3A4/5) using human liver microsomes. Additionally, time-dependent inhibition (TDI), metabolism-based inhibition (MBI) and k inact /K I activities were also investigated for CYP3A4/5. Nafithromycin did not inhibit key CYP enzymes and was found to be a weak inhibitor of CYP3A4/5. Comparator antibiotics were found to be potent inhibitors with 2- to 50-fold leftward shifts in CYP3A4/5 IC50 values, while such shift was not noted for nafithromycin. k inact /K I ratio of nafithromycin was 3- to 153-fold lower than comparator drugs, further substantiating its lower affinity for CYP3A4/5. In sum, weaker inhibition and lower k inact /K I ratio for CYP3A4/5, points towards nafithromycin's lower propensities towards clinical drug-drug interactions as compared to other macrolides/ketolides antibiotics.

Prediction of dose-dependent in vivo acetylcholinesterase inhibition by profenofos in rats and humans using physiologically based kinetic (PBK) modeling-facilitated reverse dosimetry.

Organophosphate pesticides (OPs) are known to inhibit acetylcholine esterase (AChE), a critical effect used to establish health-based guidance values. This study developed a combined in vitro-in silico approach to predict AChE inhibition by the OP profenofos in rats and humans. A physiologically based kinetic (PBK) model was developed for both species. Parameter values for profenofos conversion to 4-bromo-2-chlorophenol (BCP) were derived from in vitro incubations with liver microsomes, liver cytosol, and plasma from rats (catalytic efficiencies of 1.1, 2.8, and 0.19 ml/min/mg protein, respectively) and humans (catalytic efficiencies of 0.17, 0.79, and 0.063 ml/min/mg protein, respectively), whereas other chemical-related parameter values were derived using in silico calculations. The rat PBK model was evaluated against literature data on urinary excretion of conjugated BCP. Concentration-dependent inhibition of rat and human AChE was determined in vitro and these data were translated with the PBK models to predicted dose-dependent AChE inhibition in rats and humans in vivo. Comparing predicted dose-dependent AChE inhibition in rats to literature data on profenofos-induced AChE inhibition revealed an accurate prediction of in vivo effect levels. Comparison of rat predictions (BMDL10 of predicted dose-response data of 0.45 mg/kg bw) and human predictions (BMDL10 of predicted dose-response data of 0.01 mg/kg bw) suggests that humans are more sensitive than rats, being mainly due to differences in kinetics. Altogether, the results demonstrate that in vivo AChE inhibition upon acute exposure to profenofos was closely predicted in rats, indicating the potential of this novel approach method in chemical hazard assessment.

Anthraquinones inhibit cytochromes P450 enzyme activity in silico and in vitro.

Anthraquinones exhibit various pharmacological activities (e.g., antioxidant and laxative) and are commonly found in consumer products including foods, dietary supplements, drugs, and traditional medicines. Despite their widespread use, there are limited data available on their toxicokinetic properties. Cytochrome P450 enzymes (CYPs) in the liver play major roles in metabolizing exogenous chemicals (e.g., pharmaceuticals, food ingredients, and environmental pollutants) and endogenous biomolecules (e.g., steroid hormones and cholesterol). Inhibition of CYP activities may lead to serious interactions among these compounds. Here, in silico (quantitative structure-activity relationship modeling) and in vitro (human recombinant enzymes and liver microsomes) methods were used to identify inhibitors of five major CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) among 22 anthraquinones. First, in silico prediction and in vitro human recombinant enzyme assays were conducted for all compounds, and results showed that most of the anthraquinones were potent CYP1A2 inhibitors. Second, five selected anthraquinones (emodin, aloe-emodin, rhein, purpurin, and rubiadin) were further evaluated in human liver microsomes. Finally, plasma concentrations of the five anthraquinones in animal and humans were identified in the literature and compared to their in vitro inhibition potency (IC50 values) towards CYP activities. Emodin, rhein, and aloe-emodin inhibited activities of multiple CYPs in human liver microsomes and potential in vivo inhibition may occur due to their high plasma concentrations. These in silico and in vitro results enabled rapid identification of potential CYP inhibitors and prioritized future in-depth studies.

Selective inhibitory effects of HYIpro-3-1 on CYP1A2 in human liver microsomes.

CYP1A2 is one of the main Cytochrome P450 enzymes in the human liver associated with the metabolism of several xenobiotics. CYP1A2 is especially involved in the metabolic activation of different procarcinogens. Therefore, the development of cancer may be inhibited by inhibiting CYP1A2 activity. Here, the inhibitory effect of HYIpro-3-1 and its derivatives on CYP1A2 activity in human liver microsomes (HLM) was studied through LC-MS/MS using a cocktail assay. Among the four compounds, HYIpro-3-1 showed the most selective and strongest inhibitory effect on CYP1A2 at IC50 values of 0.1 µM in HLMs and inhibition was confirmed using purified human CYP1A2. It was determined that inhibition is reversible because the inhibitory effect of HYIpro-3-1 is not dependent on preincubation time. HYIpro-3-1 showed a typical pattern of competitive inhibition for CYP1A2-catalyzed phenacetin O-deethylation, based on the Lineweaver-Burk plot, with a Ki value of 0.05 µM in HLMs; the secondary plot also showed a linear pattern. In our study, HYIpro-3-1 was proposed as a novel inhibitor with the capacity to selectively inhibit CYP1A activity in HLMs.

Novel Inhibitors of Nicotinamide-N-Methyltransferase for the Treatment of Metabolic Disorders.

Nicotinamide-N-methyltransferase (NNMT) is a cytosolic enzyme catalyzing the transfer of a methyl group from S-adenosyl-methionine (SAM) to nicotinamide (Nam). It is expressed in many tissues including the liver, adipose tissue, and skeletal muscle. Its expression in several cancer cell lines has been widely discussed in the literature, and recent work established a link between NNMT expression and metabolic diseases. Here we describe our approach to identify potent small molecule inhibitors of NNMT featuring different binding modes as elucidated by X-ray crystallographic studies.

The inhibition of CYP1A2, CYP2C9, and CYP2D6 by pterostilbene in human liver microsomes.

This study used human liver microsomes to assess pterostilbene's effect on the metabolic activity of cytochrome P450 (CYP) 1A2, CYP2C9, and CYP2D6. The metabolism of their substrates (phenacetin, tolbutamide, and dextromethorphan) was assayed by quantifying their relevant metabolites by HPLC. The IC50 value was used to express the strength of inhibition, and the value of a volume per dose index (VDI) was used to indicate the metabolic ability of the enzyme. In this study, pterostilbene inhibited CYP1A2, CYP2C9, and CYP2D6's metabolic activities in vitro. CYP2C9's activity was most significantly inhibited by pterostilbene; its IC50 value was 0.12±0.04 µM. The IC50 value of CYP1A2 and CYP2D6 was 56.3±10.4 µM and 62.33±11.4 µM, respectively. The finding that suggests that pterostilbene has the potential to interact with CYP2C9 substrates in vivo. These results warrant clinical studies to assess the in vivo significance of these interactions.