Drug-enzyme Interactions Research Articles

In-silico screening and analysis of missense SNPs in human CYP3A4/5 affecting drug-enzyme interactions of FDA-approved COVID-19 antiviral drugs

Single nucleotide polymorphisms (SNPs) represent the prevailing form of genetic variations observed in the human population. Such variations could alter the encoded enzymes’ activities. CYP3A4/5 enzymes are involved in metabolizing drugs, notably antivirals against SARS-CoV-2. In this work, we computationally investigated antiviral-enzyme interactions of CYP3A4/5 genetic variants. We also examined the deleterious impact of 751 missense single nucleotide polymorphisms (SNPs) within the CYP3A4/5 genes. An ensemble of bioinformatics tools, [SIFT, PolyPhen-2, cadd, revel, metaLr, mutation assessor, Panther, SNP&GO, PhD-SNP, SNAP, Meta-SNP, FATHMM, I-Mutant, MuPro, INPS, CONSURF, GPS 5.0, MusiteDeep and NetPhos], identified a total of 94 variants (47 SNPs in CYP3A4, 47 SNPs in CYP3A5) to potentially impact the structural integrity as well as the activity of the CYP3A4/5 enzymes. Molecular docking was done to recognize the structural stability and binding properties of the CYP3A4/5 protein isoforms with 3 FDA-approved antiviral drugs. Our findings indicated that the CYP3A4 gene variants; R418T, I335T and R130P and the CYP3A5 gene variants; I335T, L133P and R130Q are considered the most deleterious missense SNPs. These mutants potentially affect drug-enzyme binding and hence may alter therapeutic response. Cataloguing deleterious SNPs is essential for personalized gene-based pharmacotherapy.

A ubiquitin-mediated post-translational degradation of Cyp51A contributes to a novel azole resistance mode in Aspergillus fumigatus

The airborne fungus Aspergillus fumigatus is a major pathogen that poses a serious health threat to humans by causing aspergillosis. Azole antifungals inhibit sterol 14-demethylase (encoded by cyp51A), an enzyme crucial for fungal cell survival. However, the most common mechanism of azole resistance in A. fumigatus is associated with the mutations in cyp51A and tandem repeats in its promoter, leading to reduced drug-enzyme interaction and overexpression of cyp51A. It remains unknown whether post-translational modifications of Cyp51A contribute to azole resistance. In this study, we report that the Cyp51A expression is highly induced upon exposure to itraconazole, while its ubiquitination level is significantly reduced by itraconazole. Loss of the ubiquitin-conjugating enzyme Ubc7 confers resistance to multiple azole antifungals but hinders hyphal growth, conidiation, and virulence. Western blot and immunoprecipitation assays show that deletion of ubc7 reduces Cyp51A degradation by impairing its ubiquitination, thereby leading to drug resistance. Most importantly, the overexpression of ubc7 in common environmental and clinical azole-resistant cyp51A isolates partially restores azole sensitivity. Our findings demonstrate a non-cyp51A mutation-based resistance mechanism and uncover a novel role of post-translational modification in contributing to azole resistance in A. fumigatus.

Drug interaction with UDP-Glucuronosyltransferase (UGT) enzymes is a predictor of drug-induced liver injury.

DILI frequently contributes to the attrition of new drug candidates and is a common cause for the withdrawal of approved drugs from the market. Although some noncytochrome P450 (non-CYP) metabolism enzymes have been implicated in DILI development, their association with DILI outcomes has not been systematically evaluated. In this study, we analyzed a large data set comprising 317 drugs and their interactions in vitro with 42 non-CYP enzymes as substrates, inducers, and/or inhibitors retrieved from historical regulatory documents using multivariate logistic regression. We examined how these in vitro drug-enzyme interactions are correlated with the drugs' potential for DILI concern, as classified in the Liver Toxicity Knowledge Base database. Our study revealed that drugs that inhibit non-CYP enzymes are significantly associated with high DILI concern. Particularly, interaction with UDP-glucuronosyltransferases (UGT) enzymes is an important predictor of DILI outcomes. Further analysis indicated that only pure UGT inhibitors and dual substrate inhibitors, but not pure UGT substrates, are significantly associated with high DILI concern. Drug interactions with UGT enzymes may independently predict DILI, and their combined use with the rule-of-two model further improves overall predictive performance. These findings could expand the currently available tools for assessing the potential for DILI in humans.

Discovery of new antibiotics using AI-guided spectroscopy and 3D drug-protein computer simulation technologies to combat MDR bacteria-associated mortality

Multidrug-resistant (MDR), extensively drug-resistant (XDR), and totally drug-resistant bacteria can cause sepsis and death in patients due to their ability to inactivate most antibiotics, including ampicillin, tetracycline, streptomycin, chloramphenicol, erythromycin, and ciprofloxacin. This paper aims to review recent advancements in synthetic antibiotics, lantibiotics, and phytoantibiotics and to present our research on phytoantibiotics, specifically focusing on CU1 and NU2. While third- and fifth-generation synthetic antibiotics such as meropenem, moxifloxacin, amikacin, and tigecycline are currently relied upon for treating MDR infections, research is underway to develop peptide antibiotics known as lantibiotics (e.g., nisins, bacteriocins, and salivaricins). Lantibiotics such as nisin-A and salivaricin-B have demonstrated efficacy in curing numerous MDR infections, while phytochemicals such as artemisinin and quinine have shown effectiveness against chloroquine-resistant Plasmodium falciparum infections (malaria). In our study, we utilized techniques such as mass spectroscopy, nuclear magnetic resonance, and Fourier transform infrared spectroscopy in conjunction with artificial intelligence (AI) and computer simulation technologies to determine the structure of phytochemicals. Our results revealed that CU1, derived from Cassia fistula bark ethanol extract, exhibits potent antibiotic activity against XDR bacteria by targeting the RNA polymerases of Escherichia coli and Mycobacterium tuberculosis. Consequently, our MDR-Cure extract containing CU1 represents a promising antibacterial Ayurvedic medicine specifically tailored for skin and nail infections. Similarly, NU2 poly-fluorophosphate-glycosides from Suregada multiflora roots ethanol extract exhibited strong inhibitory effects on XDR bacteria by targeting DNA topoisomerase I. Recently, many cyclic peptide antibiotics have been synthesized in vitro using computer-guided AI technologies to predict 3D drug-enzyme interactions and are currently undergoing clinical trials. Our ultimate goal is to combat XDR bacteria-associated deaths, which are predicted to escalate as we approach 2050.

Fluoropyrimidine Toxicity: the Hidden Secrets of DPYD.

Fluoropyrimidine-induced toxicity is a main limitation of therapy. Currently, polymorphisms in the DPYD gene, which encodes the 5-FU activation enzyme dihydropyrimidine dehydrogenase (DPD), are used to adjust the dosage and prevent toxicity. Despite the predictive value of DPYD genotyping, a great proportion of fluoropyrimidine toxicity cannot be solely explained by DPYD variations. We herein summarize additional sources of DPD enzyme activity variability, spanning from epigenetic regulation of DPYD expression, factors potentially inducing protein modifications, as well as drug-enzyme interactions that contribute to fluoropyrimidine toxicity. While seminal in vitro studies provided evidence that DPYD promoter methylation downregulates DPD expression, the association of DPYD methylation with fluoropyrimidine toxicity was not replicated in clinical studies. Different non-coding RNA molecules, such as microRNA, piwi-RNAs, circular-RNAs and long non-coding RNAs, are involved in post-transcriptional DPYD regulation. DPD protein modifications and environmental factors affecting enzyme activity may also add a proportion to the pooled variability of DPD enzyme activity. Lastly, DPD-drug interactions are common in therapeutics, with the most well-characterized paradigm the withdrawal of sorivudine due to fluoropyrimidine toxicity deaths in 5-FU treated cancer patients; a mechanism involving DPD severe inhibition. DPYD polymorphisms are the main source of DPD variability. A study on DPYD epigenetics (both transcriptionally and post-transcriptionally) holds promise to provide insights into molecular pathways of fluoropyrimidine toxicity. Additional post-translational DPD modifications, as well as DPD inhibition by other drugs, may explain a proportion of enzyme activity variability. Therefore, there is still a lot we can learn about the DPYD/DPD fluoropyrimidine-induced toxicity machinery.

High-resolution landscape of an antibiotic binding site

Antibiotic binding sites are located in important domains of essential enzymes and have been extensively studied in the context of resistance mutations; however, their study is limited by positive selection. Using multiplex genome engineering1 to overcome this constraint, we generate and characterize a collection of 760 single-residue mutants encompassing the entire rifampicin binding site of Escherichiacoli RNA polymerase (RNAP). By genetically mapping drug–enzyme interactions, we identify an alpha helix where mutations considerably enhance or disrupt rifampicin binding. We find mutations in this region that prolong antibiotic binding, converting rifampicin from a bacteriostatic to bactericidal drug by inducing lethal DNA breaks. The latter are replication dependent, indicating that rifampicin kills by causing detrimental transcription–replication conflicts at promoters. We also identify additional binding site mutations that greatly increase the speed of RNAP.Fast RNAP depletes the cell of nucleotides, alters cell sensitivity to different antibiotics and provides a cold growth advantage. Finally, by mapping natural rpoB sequence diversity, we discover that functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature.

Virtual screening and biological evaluation to identify pharmaceuticals potentially causing hypertension and hypokalemia by inhibiting steroid 11β-hydroxylase

Several drugs were found after their market approval to unexpectedly inhibit adrenal 11β-hydroxylase (CYP11B1)-dependent cortisol synthesis. Known side-effects of CYP11B1 inhibition include hypertension and hypokalemia, due to a feedback activation of adrenal steroidogenesis, leading to supraphysiological concentrations of 11-deoxycortisol and 11-deoxycorticosterone that can activate the mineralocorticoid receptor. This results in potassium excretion and sodium and water retention, ultimately causing hypertension. With the risk known but usually not addressed in preclinical evaluation, this study aimed to identify drugs and drug candidates inhibiting CYP11B1. Two conceptually different virtual screening methods were combined, a pharmacophore based and an induced fit docking approach. Cell-free and cell-based CYP11B1 activity measurements revealed several inhibitors with IC50 values in the nanomolar range. Inhibitors include retinoic acid metabolism blocking agents (RAMBAs), azole antifungals, α2-adrenoceptor ligands, and a farnesyltransferase inhibitor. The active compounds share a nitrogen atom embedded in an aromatic ring system. Structure activity analysis identified the free electron pair of the nitrogen atom as a prerequisite for the drug-enzyme interaction, with its pKa value as an indicator of inhibitory potency. Another important parameter is drug lipophilicity, exemplified by etomidate. Changing its ethyl ester moiety to a more hydrophilic carboxylic acid group dramatically decreased the inhibitory potential, most likely due to less efficient cellular uptake. The presented work successfully combined different in silico and in vitro methods to identify several previously unknown CYP11B1 inhibitors. This workflow facilitates the identification of compounds that inhibit CYP11B1 and therefore pose a risk for inducing hypertension and hypokalemia.

MOZART, a QSAR Multi-Target Web-Based Tool to Predict Multiple Drug-Enzyme Interactions.

Developing models able to predict interactions between drugs and enzymes is a primary goal in computational biology since these models may be used for predicting both new active drugs and the interactions between known drugs on untested targets. With the compilation of a large dataset of drug-enzyme pairs (62,524), we recognized a unique opportunity to attempt to build a novel multi-target machine learning (MTML) quantitative structure-activity relationship (QSAR) model for probing interactions among different drugs and enzyme targets. To this end, this paper presents an MTML-QSAR model based on using the features of topological drugs together with the artificial neural network (ANN) multi-layer perceptron (MLP). Validation of the final best model found was carried out by internal cross-validation statistics and other relevant diagnostic statistical parameters. The overall accuracy of the derived model was found to be higher than 96%. Finally, to maximize the diffusion of this model, a public and accessible tool has been developed to allow users to perform their own predictions. The developed web-based tool is public accessible and can be downloaded as free open-source software.

Does dexamethasone inhibit glucose oxidase: an analysis in kinetics and molecular study.

Glucose oxidase is an enzyme that is widely used in biosensors, especially kits for measuring blood sugar. Many diabetics use this type of kit to determine their blood sugar level. Aspergillus niger is the most important source of glucose oxidase for use in biosensors. Diabetes causes secondary diseases in patients for which medications are prescribed to improve them. Dexamethasone, a corticosteroid, is one of the drugs prescribed to diabetics to cure some secondary diseases. In this study, the effect of this drug on glucose oxidase was investigated from a kinetic and molecular point of view. In this study, the kinetics of drug binding to the enzyme was measured and the type of inhibition was determined by Lineweaver-Burk plot. The Ki value of the drug was determined by drawing the secondary curve. Using fluorescence spectrophotometry and molecular docking, the binding of the drug to the enzyme was confirmed. The results showed that the drug inhibits the enzyme non-competitively. Determining the kinetics parameters of the drug-enzyme interaction showed that the drug acts as a potent inhibitor. Study at the molecular level by fluorescence spectrophotometer showed that the drug attachment alters the enzyme conformation to more compaction. In silico results showed that the drug is placed between two helices that are outside the active site and binds to the enzyme by three hydrogen bonds. The result of this study is useful because it suggests that in diabetic patients taking dexamethasone, the amount of glucose declared by the kit may not be real due to the inhibition of glucose oxidase.

Kinetic Characteristics of Curcumin and Germacrone in Rat and Human Liver Microsomes: Involvement of CYP Enzymes.

Curcumin and germacrone, natural products present in the Zingiberaceae family of plants, have several biological properties. Among these properties, the anti-NSCLC cancer action is noteworthy. In this paper, kinetics of the two compounds in rat liver microsomes (RLMs), human liver microsomes (HLMs), and cytochrome P450 (CYP) enzymes (CYP3A4, 1A2, 2E1, and 2C19) in an NADPH-generating system in vitro were evaluated by UP-HPLC–MS/MS (ultrahigh-pressure liquid chromatography–tandem mass spectrometry). The contents of four cytochrome P450 (CYP) enzymes, adjusting by the compounds were detected using Western blotting in vitro and in vivo. The t1/2 of curcumin was 22.35 min in RLMs and 173.28 min in HLMs, while 18.02 and 16.37 min were gained for germacrone. The Vmax of curcumin in RLMs was about 4-fold in HLMs, meanwhile, the Vmax of germacrone in RLMs was similar to that of HLMs. The single enzyme t1/2 of curcumin was 38.51 min in CYP3A4, 301.4 min in 1A2, 69.31 min in 2E1, 63.01 min in 2C19; besides, as to the same enzymes, t1/2 of germacrone was 36.48 min, 86.64 min, 69.31 min, and 57.76 min. The dynamic curves were obtained by reasonable experimental design and the metabolism of curcumin and germacrone were selected in RLMs/HLMs. The selectivities in the two liver microsomes differed in degradation performance. These results meant that we should pay more attention to drugs in clinical medication–drug and drug–enzyme interactions.

In vitro and in silico interactions of antiulcer, glucocorticoids and urological drugs on human carbonic anhydrase I and II isozymes.

Carbonic anhydrases (CAs, Enzyme Commission 4.2.1.1) convert carbon dioxide to bicarbonate in metabolism and use Zn2+ ions as a cofactor for their catalytic activity. The activators or inhibitors of CA-I and CA-II, which are the most abundant CA isozymes in erythrocytes, have pharmacological applications in medicine. So, investigation of drug-protein interaction of these isozymes is significant. On this basis, the objective of this study was to clarify the primer effects of widely used drugs on the activity of human CA-I and CA-II enzymes and elucidate the inhibition mechanism through molecular docking studies. For this aim isozymes were purified from human erythrocytes by affinity chromatography technique. Then inhibition profiles of antiulcer, glucocorticoids, and urological drugs were investigated. As a result, while budesonide had the highest inhibitory potency on hydratase activity of hCA-I with the IC50 of 0.08mM, levofloxacin showed the highest inhibition effect on hCA-II with the IC50 of 0.886mM. The most effective inhibitor on the esterase activity of isozymes was found as fluticasone propionate with the Ki values of 0.0365±0.016mM and 0.054±0.018mM respectively. However, by molecular docking study, it was estimated that budesonide showed maximum inhibition potency for both isozymes with the free binding energy of -7.58 and -6.97kcal/mol, respectively. Consequently, it was observed that some of the drugs studied did not show any inhibitory effect. Drug-enzyme interactions were also estimated by molecular docking. This study could contribute to the discovery of new drug candidates and as well as target proteins.

Mozart, a Qsar Multi-Target Web Based Tool to Predict Multiple Drug-Enzyme Interactions

Mozart, a Qsar Multi-Target Web Based Tool to Predict Multiple Drug-Enzyme Interactions

Systems Biology Approaches to Enzyme Kinetics.

Intracellular drug metabolism involves transport, bioactivation, conjugation, and other biochemical steps. The dynamics of these steps are each dependent on a number of other cellular factors that can ultimately lead to unexpected behavior. In this review, we discuss the confounding processes and coupled reactions within bioactivation networks that require a systems-level perspective in order to fully understand the time-varying behavior. When converting known in vitro characteristics of drug-enzyme interactions into descriptions of cellular systems, features such as substrate availability, cell-to-cell variability, and intracellular redox state, deserve special focus. Two examples are provided. First, a model of hydrogen peroxide clearance during chemotherapy treatment serves as a basis to discuss an example of sensitivity analysis. Second, an example of doxorubicin bioactivation is used for discussing points of consideration when constructing and analyzing network models of drug metabolism.

An Update on the Pharmacological Usage of Curcumin: Has it Failed in the Drug Discovery Pipeline?

The pharmacological propensities of curcumin have been reported in a plethora of pre-clinical and clinical studies. However, innate attributes account for extremely low oral bioavailability which impedes its development as a therapeutic agent. Regardless, these drawbacks have not deterred researchers from optimizing its potentials. This review discussed the pharmacokinetic properties of curcumin relative to its outlook as a lead compound in drug discovery. Also, we highlighted therapeutic strategies that have expedited improvements in curcumin oral bioavailability and delivery to target sites over the years. Recent implementations of these strategies were also covered. More research efforts should be directed towards investigating the pharmacokinetic impacts of these novel curcumin formulations in human clinical studies since inter-species disparities could limit the accuracies of animal studies. We envisaged that integrative-clinical research would help determine 'actual' improvements in curcumin pharmacokinetics coupled with suitable administrative routes, optimal dosing, and drug-enzyme or drug-drug interactions. In addition, this could help determine formulations for achieving higher systemic exposure of parent curcumin thereby providing a strong impetus towards the development of curcumin as a drug candidate in disease treatment.

P0016EFFECT OF GEMFIBROZIL ON KYNURENINE AMINOTRANSFERASE ACTIVITY AND KYNURENIC ACID PRODUCTION IN RAT KIDNEY

Abstract Background and Aims Hypertriglyceridemia is the most common lipid disorder in chronic kidney disease. Lifestyle changes and fibrates administration are main methods of lowering triglycerides’ level. PPARα (peroxisome proliferator-activated receptor-α) activation is the primary mechanism of action of fibrates. A tryptophan metabolite, kynurenic acid (KYNA), is produced from L-kynurenine (L-KYN) by kynurenine aminotransferases (KATs). KAT I and KAT II isoenzymes are the best analyzed KATs. KYNA acts predominantly as a nonselective antagonist of ionotropic glutamatergic receptors, particularly N-methyl-D-aspartate (NMDA) type, which are highly expressed in the kidney. Natriuretic and hypotensive effects of KYNA are well described. Diet rich in fatty acids was reported to elevate central and peripheral KYNA level. The goal of presented study was to analyze the influence of gemfibrozil, one of the fibrates given to treat hypertriglyceridemia, on KYNA production and the activity of KAT isoenzymes: KAT I and KAT II, in rat kidney in vitro. Additionally, the molecular docking of gemfibrozil to KAT I and KAT II structures was performed to study drug-enzyme interaction. On the final step the microarray datamining was carried out to investigate whether gemfibrozil affects the expression of KAT-coding genes. Method The effect of gemfibrozil on KYNA synthesis together with KAT I and KAT II activity was tested in rat kidney homogenates in vitro after 2 hours incubation in the presence of L-KYN and gemfibrozil. The drug was examined at the concentration of 1 µM, 10 µM, 50 µM, 100 µM, 500 µM and 1 mM. Production of KYNA was analyzed using the high performance liquid chromatography (HPLC) with fluorometric detector. Results Gemfibrozil at 100 µM, 500 µM and 1 mM decreased KYNA production in kidney homogenates in vitro to 66% (P < 0.05), 58% (P < 0.01) and 41% (P < 0.01) of control value, respectively. At 100 µM, 500 µM and 1 mM concentration gemfibrozil lowered renal KAT I activity in vitro to 68% (P < 0.05), 56% (P < 0.01) and 52% (P < 0.01) of control value, respectively. Moreover, gemfibrozil at 500 µM and 1 mM concentration decreased kidney KAT II activity in vitro to 47% (P < 0.001) and 26% (P < 0.001) of control value, respectively. Results of the molecular docking suggested that gemfibrozil may affect the active site of both KAT I and KAT II. Publicly available microarray datasets suggested that the expression of KAT-coding genes does not change after gemfibrozil administration. Conclusion Gemfibrozil decreases KYNA production in rat kidney in vitro through inhibition of KAT I and KAT II isoenzymes. Presented results indicate a novel mechanism of gemfibrozil’s action in the kidney. Its potential role in nephrotoxicity needs verification in upcoming studies.

The quasi-irreversible inactivation of cytochrome P450 enzymes by paroxetine: a computational approach.

The mechanism-based inactivation (MBI) of P450 by paroxetine was investigated by computational analysis. The drug-enzyme interactions were figured out through studying energy profiles of three competing mechanisms. The potency of paroxetine as P450's inhibitor was estimated based on the availability of two active sites for the MBI in the paroxetine structure. The inactivation by the amino site of paroxetine mainly proceeds via the hydrogen atom transfer pathway because of the lower energy demand of its rate determining step. In addition, the low-spin state is the predominant route in the MBI at the methylenedioxo active site as a result of being rebound barrier-free mechanism. Our comparative investigation showed that inactivation at the secondary amine is thermodynamically more favorable because of the lower energy barrier of the dehydration mechanism of the hydroxylated paroxetine complex than its methylenedioxo counterpart. The results of docking analysis coincided with the outputs of DFT calculations since the docking pose with the lowest binding affinity is that for conformation with polar interaction between the amino group of paroxetine and the oxo moiety of P450's active site. Assessment of the molecular dynamics simulations trajectories revealed the favorable interaction of paroxetine with P450.

Design of antimalarial agents based on pyrimidine derivatives as methionine aminopeptidase 1b inhibitor: Molecular docking, quantitative structure activity relationships, and molecular dynamics simulation studies

AbstractMalaria is a parasitic disease mainly caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale and rarely by animal plasmodium such as Plasmodium knowlesi. Despite the worldwide endorsement of eradicating malaria, the mortality and morbidity of malaria is still high, especially in children of resource‐poor regions of the world. In the current study, a series of 2‐pyridyl pyrimidine derivatives with P. falciparum methionine aminopeptidase 1b (MA1b) inhibitory activity were subjected to quantitative structure, relationship activities, molecular docking, and molecular dynamics simulation to identify the ideal physicochemical characteristics and the drug–receptor interactions of potential antimalarial compounds. The structures were built using HyperChem. A docking study was performed using AutoDock. The obtained quantitative structure activity relationships model has showed that the descriptors MLOGP, E2p, Mor24u, PW2, and H8m mainly affect the antimalarial activity of the series of ligands. The residues His 277, Fe 370, Asp 240, Trp 320, and Tyr 260 of MA1b play a very important role in the drug–enzyme interaction by creating hydrogen bond, π‐cation, and π‐π interactions. To give a complete impression about the pyrimidine derivatives, compound 7 was subjected to 1,000 ps molecular dynamics (MD) simulations. The best structure based on our study was suggested for a future new drug with potential antimalarial compounds.

The Binding Mechanisms and Inhibitory Effect of Intravenous Anesthetics on AChE In Vitro and In Vivo: Kinetic Analysis and Molecular Docking.

Inhibitors of acetylcholinesterase (AChE), which have an important role in the prevention of excessive AChE activity and β-amyloid (Aβ) formation are widely used in the symptomatic treatment of Alzheimer's disease (AD). The inhibitory effect of anesthetic agents on AChE was determined by several approaches, including binding mechanisms, molecular docking and kinetic analysis. Inhibitory effect of intravenous anesthetics on AChE as in vitro and in vivo have been discovered. The midazolam, propofol and thiopental have shown competitive inhibition type (midazolam > propofol > thiopental) and Ki values were found to be 3.96.0 ± 0.1, 5.75 ± 0.12 and 29.65 ± 2.04µM, respectively. The thiopental and midazolam showed inhibition effect on AChE in vitro, whereas they showed activation effect in vivo when they are combined together. The order of binding of the drugs to the active site of the 4M0E receptor was found to be midazolam > propofol > thiopental. This study on anesthetic agents that are now widely used in surgical applications, have provided a molecular basis for investigating the drug-enzyme interactions mechanism. In addition, the study is important in understanding the molecular mechanism of inhibitors that are effective in the treatment of AD.

In vitro metabolism of magnolol and honokiol in rat liver microsomes and their interactions with seven cytochrome P substrates.

Magnolol and honokiol are the main active components of Magnolia officinalis Rehd. et Wils. The study of their interactions with liver microsomes is very important for the clinical safety of M. officinalis Rehd. et Wils. The main metabolites of magnolol and honokiol in rat liver microsomes were investigated using ultrahigh-performance liquid chromatography/mass spectrometry and their possible structures were identified. In addition, cytochrome P450 (CYP450) isoenzymes of the major rat metabolites of magnolol and honokiol were identified using a specific inhibitor. This study suggests that the CYP2E1 subtype is responsible for the oxidation of magnolol and honokiol terminal double bonds to epoxy metabolites. CYP3A4 appears to be the major subtype responsible for further hydrolytic metabolism, while CYP1A2 may promote decarboxylation of the metabolites. CYP2A6 may be the main subtype responsible for the hydrogenation of magnolol (p< 0.05). This study demonstrated that different CYP450 enzyme isoforms showed different activities in the in vitro metabolism of magnolol and honokiol in rat liver microsomes. It has certain practical applications in that we should pay attention to drug-drug interactions in clinical medications and also to drug-enzyme interactions.

Interaction of drugs amlodipine and paroxetine with the metabolizing enzyme CYP2B4: a molecular dynamics simulation study.

The interactions of the drugs amlodipine and paroxetine, which are prescribed respectively for treatment of hypertension and depression, with the metabolizing enzyme cytochrome CYP2B4 as the drug target, have been studied by molecular dynamics (MD) simulation. Poly ethylene glycol was used to control the drugs' interactions with each other and with the target CYP2B4. Thirteen simulation systems were carefully designed, and the results obtained from MD simulations indicated that amlodipine in the PEGylated form prescribed with paroxetine in the nonPEGylated form promotes higher cytochrome stability and causes fewer fluctuations as the drugs approach the target CYP2B4 and interact with it. The simulation results led us to hypothesize that the combination of the drugs with a specific drug ratio, as proposed in this work, manifests more effective diffusivity and less instability while metabolizing with enzyme CYP2B4. Also, the active residues in the CYP2B4 enzyme that interact with the drugs were determined by MD simulation, which were consistent with the reported experimental results. Graphical Abstract Efficient drug-enzyme interactions, as a result of PEGylation.