Inhibition Effect of Okanin Toward Human Cytochrome P450 3A4 and 2D6 with Multi-spectroscopic Studies and Molecular Docking.

Okanin, a major flavonoid of a popular herb tea, Coreopsis tinctoria Nutt., showed strong inhibition on CYP3A4 and CYP2D6. The strong interaction between okanin and CYPs were determined by enzyme kinetics, multispectral technique and molecular docking. The inhibition type of two enzymes, CYP3A4 and CYP2D6, by okanin are mixed and non-competitive inhibition type, respectively. The IC50 values and the binding constant of okanin to CYP3A4 can be deduced that the interaction was stronger than that of CYP2D6. The Conformations of CYP3A4 and CYP2D6 were changed by okanin. The evidence from fluorescence measurement along with molecular docking verified that these two CYPs were bound with okanin by hydrogen bonds and hydrophobic forces. Our investigation suggested that okanin may lead to interactions between herb and drug by inhibiting CYP3A4 and CYP2D6 activities, thus its consumption should be taken with caution.

How does multiple substrate binding lead to substrate inhibition of CYP2D6 metabolizing dextromethorphan? A theoretical study.

CYP2D6 is one of the most important metalloenzymes involved in the biodegradation of many drug molecules in the human body. It has been found that multiple substrate binding can lead to substrate inhibition of CYP2D6 metabolizing dextromethorphan (DM), but the corresponding theoretical mechanism is rarely reported. Therefore, we chose DM as the probe and performed molecular dynamics simulations and quantum mechanical calculations on CYP2D6-DM systems to investigate the mechanism of how the multiple substrate binding leads to the substrate inhibition of CYP2D6 metabolizing substrates. According to our results, three gate residues (Arg221, Val374, and Phe483) for the catalytic pocket are determined. We also found that the multiple substrate binding can lead to substrate inhibition by reducing the stability of CYP2D6 binding DM and increasing the reactive activation energy of the rate-determining step. Our findings would help to understand the substrate inhibition of CYP2D6 metabolizing the DM and enrich the knowledge of the drug-drug interactions for the cytochrome P450 superfamily.

The regioselectivity of the interaction between dextromethorphan and CYP2D6.

CYP2D6 is an important enzyme of the cytochrome P450 superfamily, and catalyzes nearly 25% of the drugs sold in the market. For decades, the interactions and metabolism between CYP2D6 and substrates have been a hot topic. However, the key factors of the catalytic regioselectivity for CYP2D6 still remain controversial. Here, we construct four systems to explore the interaction between dextromethorphan (DM) and CYP2D6. A new binding mode of CYP2D6 is defined, and two key residues (residue Asp301 and residue Glu216) are discovered working simultaneously to stabilize the DM at the reactive site by forming water bridge hydrogen bonds when CYP2D6 binds DM. Our results also indicate that the substrate concentration could mediate the binding mode between the substrate and CYP2D6 by decreasing the volume of the catalytic pocket, which is not conducive to the O-demethylation of DM but benefits the N-demethylation of DM. These results could shed light on the process of CYP2D6 binding to the substrate, and help to better understand the regioselectivity of CYP2D6 catalyzing the substrates.

A Conserved Allosteric Site on Drug-Metabolizing CYPs: A Systematic Computational Assessment.

Cytochrome P450 enzymes (CYPs) are the largest group of enzymes involved in human drug metabolism. Ligand tunnels connect their active site buried at the core of the membrane-anchored protein to the surrounding solvent environment. Recently, evidence of a superficial allosteric site, here denoted as hotspot 1 (H1), involved in the regulation of ligand access in a soluble prokaryotic CYP emerged. Here, we applied multi-scale computational modeling techniques to study the conservation and functionality of this allosteric site in the nine most relevant mammalian CYPs responsible for approximately 70% of drug metabolism. In total, we systematically analyzed over 44 µs of trajectories from conventional MD, cosolvent MD, and metadynamics simulations. Our bioinformatic analysis and simulations with organic probe molecules revealed the site to be well conserved in the CYP2 family with the exception of CYP2E1. In the presence of a ligand bound to the H1 site, we could observe an enlargement of a ligand tunnel in several members of the CYP2 family. Further, we could detect the facilitation of ligand translocation by H1 interactions with statistical significance in CYP2C8 and CYP2D6, even though all other enzymes except for CYP2C19, CYP2E1, and CYP3A4 presented a similar trend. As the detailed comprehension of ligand access and egress phenomena remains one of the most relevant challenges in the field, this work contributes to its elucidation and ultimately helps in estimating the selectivity of metabolic transformations using computational techniques.

Mechanistic insights on nsSNPs on binding site of renin and cytochrome P450 proteins: A computational perceptual study for pharmacogenomics evaluation.

Past several decades, therapeutic investigations lead to the discovery of numerous antihypertensive drugs. Although it has been proved for their potency, altered efficacy is common norms in several conditions due to genetic variations. Cytochrome P450 plays a crucial role in drug metabolism and responsible for the pharmacokinetic and pharmacodynamic properties of the drug molecules. Here, we report the deleterious point mutations in the genes associated with the altered response of antihypertensive drug molecules and their metabolizers. Missense variants were filtered as potential nonsynonymous single nucleotide polymorphisms among the available data for the target genes (REN, CYP2D6, CYP3A4). The key objective of the work is to identify the deleterious single nucleotide polymorphisms (SNPs) responsible for the drug response and metabolism for the application of personalized medication. The molecular docking studies revealed that Aliskiren and other clinically approved drug molecules have a high binding affinity with both wild and mutant structures of renin, CYP2D6, and CYP3A4 proteins. The docking (Glide XP) score was observed to have in the range of -8.896 to -11.693 kcal/mol. The molecular dynamics simulation studies were employed to perceive the structural changes and conformational deviation through various analyses. Each studied SNPs was observed to have disparate scoring in the binding affinity to the specific drug molecules. As a prospective plan, we assume this study might be applied to identify the risky SNPs associated with hypertension from the patients to recommend the suitable drug for personalized hypertensive treatment. Further, extensive clinical pharmacogenomics studies are required to support the findings.

Conformational Landscape of Cytochrome P450 Reductase Interactions.

Oxidative reactions catalyzed by Cytochrome P450 enzymes (CYPs), which constitute the most relevant group of drug-metabolizing enzymes, are enabled by their redox partner Cytochrome P450 reductase (CPR). Both proteins are anchored to the membrane of the endoplasmic reticulum and the CPR undergoes a conformational change in order to interact with the respective CYP and transfer electrons. Here, we conducted over 22 microseconds of molecular dynamics (MD) simulations in combination with protein-protein docking to investigate the conformational changes necessary for the formation of the CPR-CYP complex. While some structural features of the CPR and the CPR-CYP2D6 complex that we highlighted confirmed previous observations, our simulations revealed additional mechanisms for the conformational transition of the CPR. Unbiased simulations exposed a movement of the whole protein relative to the  membrane, potentially to facilitate interactions with its diverse set of redox partners. Further, we present a structural mechanism for the susceptibility of the CPR to different redox states based on the flip of a glycine residue disrupting the local interaction network that maintains inter-domain proximity. Simulations of the CPR-CYP2D6 complex pointed toward an additional interaction surface of the FAD domain and the proximal side of CYP2D6. Altogether, this study provides novel structural insight into the mechanism of CPR-CYP interactions and underlying conformational changes, improving our understanding of this complex machinery Cytochrome P450 reductase; CPR; conformational; dynamicsrelevant for drug metabolism.

Tryptophan-75 Is a Low-Energy Channel-Gating Residue that Facilitates Substrate Egress/Access in Cytochrome P450 2D6.

CYP2D6 is a major drug metabolizing enzyme with a buried active site. Channels leading to the active site from various enzyme surfaces are believed to facilitate ligand egress and access to the active site. The present study used molecular dynamics (MD) and in vitro studies with CYP2D6*1 and a Trp75-to-Ala mutant to examine channel gating in CYP2D6 by Trp75. MD simulations measured energy landscapes of Trp75 conformations and simulated substrate passage within channel 2b using bufuralol as a model substrate. Trp75 alternated between multiple stable states that supported substrate transport along channel 2b with low-energy barriers between states (∼ -1 kcal/mol). Trp75 conformations were stabilized primarily by hydrogen bonding between Trp75 and Glu222, Asn226, Ala225, or Gln72. Energy barriers were low between Trp75 conformations, allowing Trp75 to easily move between various conformations over time and to function in both binding to and moving substrates in the 2b channel of CYP2D6. Michaelis-Menten kinetic studies completed with purified enzyme in a reconstituted system showed overall reduced enzyme efficiency for metabolism of bufuralol and dextromethorphan by the Trp75Ala mutant compared with CYP2D6*1. In stopped-flow measurements, k off for dextromethorphan was decreased in the absence of Trp75. Our results support a role for Trp75 in substrate shuttling to the active site of CYP2D6. SIGNIFICANCE STATEMENT: Using combined molecular dynamics and in vitro assays, this study shows for the first time a role for Trp75 as a channel entrance gating residue in the mechanism of substrate binding/unbinding in CYP2D6. Energy landscapes derived from molecular dynamics were used to quantitate the strength of gating, and kinetics assays showed the impact on enzyme efficiency and k off of a Trp75Ala mutation.

G-quadruplex structures bind to EZ-Tn5 transposase.

Next generation DNA sequencing and analysis of amplicons spanning the pharmacogene CYP2D6 suggested that the Nextera transposase used for fragmenting and providing sequencing priming sites displayed a targeting bias. This manifested as dramatically lower sequencing coverage at sites in the amplicon that appeared likely to form G-quadruplex structures. Since secondary DNA structures such as G-quadruplexes are abundant in the human genome, and are known to interact with many other proteins, we further investigated these sites of low coverage. Our investigation revealed that G-quadruplex structures are formed in vitro within the CYP2D6 pharmacogene at these sites, and G-quadruplexes can interact with the hyperactive Tn5 transposase (EZ-Tn5) with high affinity. These findings indicate that secondary DNA structures such as G-quadruplexes may represent preferential transposon integration sites and provide additional evidence for the role of G-quadruplex structures in transposition or viral integration processes.

Mechanism-based inactivation of cytochrome P450 2D6 by Notopterol.

Notopterol (NOT) is a major bioactive ingredient extracted from the rhizomes of either Notopterygium incisum Ting ex H. T. Chang or N. forbesii Boiss (Qianghuo in Chinese), a botanical drug that was adopted as a traditional Chinese medicine. NOT is suggested to show analgesic and anti-inflammatory effects in clinical practice. The inhibitory effects of NOT on human cytochrome P450 enzymes were investigated in the present study. Our results indicate that NOT inhibited the activity of CYP2D6 in a time-, concentration- and NADPH-dependent manner. The values of KI and kinact were 10.8 µM and 0.62 min-1, respectively. The calculated kobs at 10 µM was 0.29 min-1, above the 0.02 min-1 risk level. After incubation with NOT at 10 µM for 9 min, approximately 92% of CYP2D6 activity was inhibited. Such loss of enzyme activity was not restored through dialysis, which indicates that the observed enzyme inhibition was irreversible. Partition ratio of the inactivation was approximately 29. Quinidine, a competitive CYP2D6 inhibitor, demonstrated protection on enzymes against the NOT-induced inactivation, but such protection was not found in incubation systems fortified with glutathione or catalase/superoxide dismutase. Additionally, CYP3A4 was observed to function as an enzyme mainly involved in the biotransformation of NOT. Taken together, these findings indicate that NOT served as a mechanism-based inactivator of CYP2D6, meanwhile, those observed effects may induce the latent drug-drug interactions. The metabolic activation of NOT may be the key to trigger the inactivation of the enzyme.

Variantes alélicas *3, *4, *5 y *6 del gen CYP2D6 en una muestra de pacientes cubanos con esquizofrenia / Aallelic variants *3, *4, *5 and *6 of gene CYP2D6 in a sample of Cuban patients with schizophrenia

Introducción: El gen CYP2D6 codifica la proteína de igual nombre, enzima principal del complejo de citocromos P-450 en la fase I del metabolismo de muchos medicamentos ampliamente usados en la práctica clínica, entre los que se encuentran los neurolépticos. Las variantes alélicas que implican una función nula de esta enzima: *3, *4, *5, y *6, predicen entre el 93-97,5(percent) de los posibles fenotipos metabolizadores lentos, que implican acumulación de concentraciones tóxicas y, por consiguiente, mayor riesgo de reacciones adversas. Por otro lado, hay estudios que reportan una baja frecuencia de metabolizadores lentos en pacientes con esquizofrenia. Objetivo: Evaluar la asociación entre las variantes no funcionales del gen CYP2D6 con la esquizofrenia en pacientes cubanos. Métodos: Se realizó un estudio de casos y controles. En el grupo de los casos se incluyeron 212 pacientes con esquizofrenia, y en los controles 326 voluntarios sanos. Resultados: Las frecuencias de los alelos CYP2D6 con actividad enzimática nula fueron similares entre los casos y los controles. No se detectó una diferencia estadísticamente significativa de metabolizadores lentos entre los pacientes con esquizofrenia ni en los voluntarios sanos…(AU)

Metabolism of Benzalkonium Chlorides by Human Hepatic Cytochromes P450.

Benzalkonium chlorides (BACs) are widely used as disinfectants in cleaning products, medical products, and the food processing industry. Despite a wide range of reported toxicities, limited studies have been conducted on the metabolism of these compounds in animal models and none in human-derived cells or tissues. In this work, we report on the metabolism of BACs in human liver microsomes (HLM) and by recombinant human hepatic cytochrome P450 (CYP) enzymes. BAC metabolism in HLM was NADPH-dependent and displayed apparent half-lives that increased with BAC alkyl chain length (C10 < C12 < C14 < C16), suggesting enhanced metabolic stability of the more lipophilic, longer chain BACs. Metabolites of d7-benzyl labeled BAC substrates retained all deuteriums and there was no evidence of N-dealkylation. Tandem mass spectrometry fragmentation of BAC metabolites confirmed that oxidation occurs on the alkyl chain region. Major metabolites of C10-BAC were identified as ω-hydroxy-, (ω-1)-hydroxy-, (ω, ω-1)-diol-, (ω-1)-ketone-, and ω-carboxylic acid-C10-BAC by liquid chromatography-mass spectrometry comparison with synthetic standards. In a screen of hepatic CYP isoforms, recombinant CYP2D6, CYP4F2, and CYP4F12 consumed substantial quantities of BAC substrates and produced the major microsomal metabolites. The use of potent pan-CYP4 inhibitor HET0016, the specific CYP2D6 inhibitor quinidine, or both confirmed major contributions of CYP4- and CYP2D6-mediated metabolism in the microsomal disappearance of BACs. Kinetic characterization of C10-BAC metabolite formation in HLM demonstrated robust Michaelis-Menten kinetic parameters for ω-hydroxylation (Vmax = 380 pmol/min/mg, Km = 0.69 µM) and (ω-1)-hydroxylation (Vmax = 126 pmol/min/mg, Km = 0.13 µM) reactions. This work illustrates important roles for CYP4-mediated ω-hydroxylation and CYP2D6/CYP4-mediated (ω-1)-hydroxylation during the hepatic elimination of BACs, an environmental contaminant of emerging concern. Furthermore, we demonstrate that CYP-mediated oxidation of C10-BAC mitigates the potent inhibition of cholesterol biosynthesis exhibited by this short-chain BAC.

Spontaneous Ligand Access Events to Membrane-Bound Cytochrome P450 2D6 Sampled at Atomic Resolution.

The membrane-anchored enzyme Cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of around 25% of marketed drugs and its metabolic performance shows a high interindividual variation. While it was suggested that ligands access the buried active site of the enzyme from the membrane, no proof from unbiased simulations has been provided to support this hypothesis. Laboratory experiments fail to capture the access process which is suspected to influence binding kinetics. Here, we applied unbiased molecular dynamics (MD) simulations to investigate the access of ligands to wild-type CYP2D6, as well as the allelic variant CYP2D6*53. In multiple simulations, substrates accessed the active site of the enzyme from the protein-membrane interface to ultimately adopt a conformation that would allow a metabolic reaction. We propose the necessary steps for ligand access and the results suggest that the increased metabolic activity of CYP2D6*53 might be caused by a facilitated ligand uptake.

Inhibition of Hepatic CYP2D6 by the Active N-Oxide Metabolite of Sorafenib.

The multikinase inhibitor sorafenib (SOR) is used to treat patients with hepatocellular and renal carcinomas. SOR undergoes CYP-mediated biotransformation to a pharmacologically active N-oxide metabolite (SNO) that has been shown to accumulate to varying extents in individuals. Kinase inhibitors like SOR are frequently coadministered with a range of other drugs to improve the efficacy of anticancer drug therapy and to treat comorbidities. Recent evidence has suggested that SNO is more effective than SOR as an inhibitor of CYP3A4-mediated midazolam 1'-hydroxylation. CYP2D6 is also reportedly inhibited by SOR. The present study assessed the possibility that SNO might contribute to CYP2D6 inhibition. The inhibition kinetics of CYP2D6-mediated dextromethorphan O-demethylation were analyzed in human hepatic microsomes, with SNO found to be ~ 19-fold more active than SOR (Kis 1.8 ± 0.3 µM and 34 ± 11 µM, respectively). Molecular docking studies of SOR and SNO were undertaken using multiple crystal structures of CYP2D6. Both molecules mediated interactions with key amino acid residues in putative substrate recognition sites of CYP2D6. However, a larger number of H-bonding interactions was noted between the N-oxide moiety of SNO and active site residues that account for its greater inhibition potency. These findings suggest that SNO has the potential to contribute to pharmacokinetic interactions involving SOR, perhaps in those individuals in whom SNO accumulates.

In vitro inhibition of human CYP2D6 by the chiral pesticide fipronil and its metabolite fipronil sulfone: Prediction of pesticide-drug interactions.

Fipronil is a chiral insecticide employed worldwide in crops, control of public hygiene and control of veterinary pests. Humans can be exposed to fipronil through occupational, food, and environmental contamination. Therefore, the risk assessment of fipronil in humans is important to protect human health. Fipronil sulfone is the major metabolite formed during fipronil metabolism by humans. Since the CYP450 enzymes are the main ones involved in drug metabolism, the evaluation of their inhibition by fipronil and its main metabolite is important to predict drug-pesticide interactions. The aim of this work was to investigate the inhibition effects of rac-fipronil, S-fipronil, R-fipronil and fipronil sulfone on the main human CYP450 isoforms. The results showed that CYP2D6 is the only CYP450 isoform inhibited by these xenobiotics. In addition, no enantioselective differences were observed in the inhibition of CYP450 isoforms by fipronil and its individuals' enantiomers. Rac-fipronil, S-fipronil and R-fipronil are moderate CYP2D6 inhibitors showing a competitive inhibition profile. On the other hand, the metabolite fipronil sulfone showed to be a strong inhibitor of CYP2D6 also by competitive inhibition. These results highlight the importance of metabolite evaluation on pesticide safety since the metabolism of fipronil into fipronil sulfone increases the risk of pesticide-drug interactions for drugs metabolized by CYP2D6.

Functional and structural characterisation of common cytochrome P450 2D6 allelic variants-roles of Pro34 and Thr107 in catalysis and inhibition.

One major source of inter-individual variability in drug pharmacokinetics is genetic polymorphism of the cytochrome P450 (CYP) genes. This study aimed to elucidate the enzyme kinetic and molecular basis for altered activity in three major alleles of CYP2D6, namely CYP2D6*2, CYP2D6*10 and CYP2D6*17. The E. coli-expressed allelic variants were examined using substrate (venlafaxine and 3-cyano-7-ethoxycoumarin[CEC]) and inhibitor (quinidine, fluoxetine, paroxetine, terbinafine) probes in enzyme assays as well as molecular docking. The kinetics data indicated that R296C and S486T mutations in CYP2D6*2 have caused enhanced ligand binding (enhanced intrinsic clearance for venlafaxine and reduced IC50 for quinidine, paroxetine and terbinafine), suggesting morphological changes within the active site cavity that favoured ligand docking and binding. Mutations in CYP2D6*10 and CYP2D6*17 tended to cause deleterious effect on catalysis, with reduced clearance for venlafaxine and CEC. Molecular docking indicated that P34S and T107I, the unique mutations in the alleles, have negatively impacted activity by affecting ligand access and binding due to alteration of the substrate access channel and active site morphology. IC50 values however were quite variable for quinidine, fluoxetine and terbinafine, and a general decrease in IC50 was observed for paroxetine, suggesting ligand-specific altered susceptibility to inhibition in the alleles. This study indicates that CYP2D6 allele selectivity for ligands was not solely governed by changes in the active site architecture induced by the mutations, but that the intrinsic properties of the substrates and inhibitors also played vital role.

Insight of Captagon Abuse by Chemogenomics Knowledgebase-guided Systems Pharmacology Target Mapping Analyses.

Captagon, known by its genetic name Fenethylline, is an addictive drug that complicates the War on Drugs. Captagon has a strong CNS stimulating effect than its primary metabolite, Amphetamine. However, multi-targets issues associated with the drug and metabolites as well as its underlying mechanisms have not been fully defined. In the present work, we applied our established drug-abuse chemogenomics-knowledgebase systems pharmacology approach to conduct targets/off-targets mapping (SP-Targets) investigation of Captagon and its metabolites for hallucination addiction, and also analyzed the cell signaling pathways for both Amphetamine and Theophylline with data mining of available literature. Of note, Amphetamine, an agonist for trace amine-associated receptor 1 (TAAR1) with enhancing dopamine signaling (increase of irritability, aggression, etc.), is the main cause of Captagon addiction; Theophylline, an antagonist that blocks adenosine receptors (e.g. A2aR) in the brain responsible for restlessness and painlessness, may attenuate the behavioral sensitization caused by Amphetamine. We uncovered that Theophylline's metabolism and elimination could be retarded due to competition and/or blockage of the CYP2D6 enzyme by Amphetamine; We also found that the synergies between these two metabolites cause Captagon's psychoactive effects to act faster and far more potently than those of Amphetamine alone. We carried out further molecular docking modeling and molecular dynamics simulation to explore the molecular interactions between Amphetamine and Theophylline and their important GPCRs targets, including TAAR1 and adenosine receptors. All of the systems pharmacology analyses and results will shed light insight into a better understanding of Captagon addiction and future drug abuse prevention.

Regioselective hydroxylation of an antiarrhythmic drug, propafenone, mediated by rat liver cytochrome P450 2D2 differs from that catalyzed by human P450 2D6.

1. Propafenone, an antiarrhythmic drug, is a typical human cytochrome P450 (P450) 2D6 substrate used in preclinical studies. Here, propafenone oxidation by mammalian liver microsomes was investigated in vitro. 2. Liver microsomes from humans and marmosets preferentially mediated propafenone 5-hydroxylation, minipig, rat and mouse livers primarily mediated 4'-hydroxylation, but cynomolgus monkey and dog liver microsomes differently mediated N-despropylation. 3. Quinine, ketoconazole or anti-P450 2D antibodies suppressed propafenone 4'/5-hydroxylation in human and rat liver microsomes. Pretreatments with ß-naphthoflavone or dexamethasone increased N-despropylation in rat livers. 4. Recombinant rat P450 2D2 efficiently catalysed propafenone 4'-hydroxylation in a substrate inhibition manner, comparable to rat liver microsomes, while human P450 2D6 displayed propafenone 5-hydroxylation. Human and rat P450 1A, 2C and 3A enzymes mediated propafenone N-despropylation with high capacities. 5. Carbon-4' of propafenone docked favourably into the active site of P450 2D2 based on an in silico model; in contrast, carbon-5 of propafenone docked into human P450 2D6. 6. These results suggest that the major roles of individual P450 2D enzymes in regioselective hydroxylations of propafenone differ between human and rat livers, while the minor roles of P450 1A, 2C and 3A enzymes for propafenone N-despropylation are similar in livers of both species.

Mechanism-based inactivation of cytochrome P450 2D6 by chelidonine.

Chelidonine (CHE) is a major bioactive constituent of greater celandine, a plant used in traditional herbal medicines. CHE has widely been used as an analgesic in clinical settings. We evaluated the inhibitory effects of CHE on human cytochrome P450 enzymes. CHE produced time-, concentration-, and NADPH-dependent inhibition of CYP2D6, with K I and k inact values of 20.49 µM and 11.05 min -1 , respectively. Approximately 76% of CYP2D6 activity was suppressed after 9 minute incubation with CHE (50 µM). The loss of enzyme activity was not restored following dialysis. The estimated partition ratio of the inactivation was about 156. Quinidine, a competitive inhibitor of CYP2D6, attenuated the CHE-mediated enzyme inactivation, while glutathione and catalase/superoxide dismutase did not markedly ameliorate the inhibitory effect. Upon oxidation using potassium ferricyanide, the 15.1% activity of CYP2D6 was restored. These findings indicate that CHE acted as a mechanism-based inactivator of CYP2D6 and the observed effects may induce potential drug-drug interactions.

Microsecond MD simulations of human CYP2D6 wild-type and five allelic variants reveal mechanistic insights on the function.

Characterization of cytochrome P450 2D6 (CYP2D6) and the impact of the major identified allelic variants on the activity of one of the most dominating drug-metabolising enzymes is essential to increase drug safety and avoid adverse reactions. Microsecond molecular dynamics simulations have been performed to capture the dynamic signatures of this complex enzyme and five allelic variants with diverse enzymatic activity. In addition to the apo simulations, three substrates (bufuralol, veliparib and tamoxifen) and two inhibitors (prinomastat and quinidine) were included to explore their influence on the structure and dynamical features of the enzyme. Our results indicate that the altered enzyme activity can be attributed to changes in the hydrogen bonding network within the active site, and local structural differences in flexibility, position and shape of the binding pocket. In particular, the increased (CYP2D6*53) or the decreased (CYP2D6*17) activity seems to be related to a change in dynamics of mainly the BC loop due to a modified hydrogen bonding network around this region. In addition, the smallest active site volume was found for CYP2D6*4 (no activity). CYP2D6*2 (normal activity) showed no major differences in dynamic behaviour compared to the wild-type.