Lipid composition affects the thermal stability of cytochrome P450 3A4 in nanodiscs.

Nanodiscs (NDs), self-assembled lipid bilayers encircled by membrane scaffold proteins (MSPs), offer a versatile platform for the reconstitution of membrane proteins for structural and biochemical investigations. Saturated, isoprenoid lipids are commonly found in thermophiles and have been associated with thermotolerance. To test whether these lipids confer additional stability on ND-incorporated membrane proteins, this study focuses on the thermal stability of human cytochrome P450 3A4 (CYP3A4) inside NDs composed of different phosphocholine lipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). NDs were characterized using size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) and densitometric SDS-PAGE. CYP3A4-DPhPC-NDs were found to comprise three MSP copies instead of the canonical dimer, as reported before for the empty NDs. Rapid, thermally induced unfolding of CYP3A4 inside NDs measured using circular dichroism and differential scanning fluorimetry (nanoDSF) revealed that the CYP3A4 melting temperature was dependent on ND composition. In POPC and DMPC-CYP3A4-NDs the melting temperature was comparable to CYP3A4 without NDs (59 °C). CYP3A4 in DPhPC-NDs showed an increase in melting temperature of 4 °C. Decline in CYP3A4 integrity as well as ND aggregation and disintegration occur at similar rates for all membrane types when subjected to exposure at 37 °C for several hours. The POPC and DMPC- CYP3A4-NDs show significant lipid loss over time, which is not observed for DPhPC-NDs. The results demonstrate that thermally induced denaturation of protein-NDs is a complex, multifaceted process, which is not represented well by rapid thermal unfolding experiments.

Application of machine learning models for property prediction to targeted protein degraders.

Machine learning (ML) systems can model quantitative structure-property relationships (QSPR) using existing experimental data and make property predictions for new molecules. With the advent of modalities such as targeted protein degraders (TPD), the applicability of QSPR models is questioned and ML usage in TPD-centric projects remains limited. Herein, ML models are developed and evaluated for TPDs' property predictions, including passive permeability, metabolic clearance, cytochrome P450 inhibition, plasma protein binding, and lipophilicity. Interestingly, performance on TPDs is comparable to that of other modalities. Predictions for glues and heterobifunctionals often yield lower and higher errors, respectively. For permeability, CYP3A4 inhibition, and human and rat microsomal clearance, misclassification errors into high and low risk categories are lower than 4% for glues and 15% for heterobifunctionals. For all modalities, misclassification errors range from 0.8% to 8.1%. Investigated transfer learning strategies improve predictions for heterobifunctionals. This is the first comprehensive evaluation of ML for the prediction of absorption, distribution, metabolism, and excretion (ADME) and physicochemical properties of TPD molecules, including heterobifunctional and molecular glue sub-modalities. Taken together, our investigations show that ML-based QSPR models are applicable to TPDs and support ML usage for TPDs' design, to potentially accelerate drug discovery.

Driving forces and large scale affinity calculations for piperine/γ-cyclodxetrin complexes: Mechanistic insights from umbrella sampling simulation and DFT calculations.

Piperine (PiP), a bioactive molecule, exhibits numerous health benefits and is frequently employed as a co-delivery agent with various phytomedicines (e.g., curcumin) to enhance their bioavailability. This is attributed to PiP's inhibitory activity against drug-metabolizing proteins, notably CYP3A4. Nevertheless, PiP encounters solubility challenges addressed in this study using cyclodextrins (CDs). Specifically, γ-CD and its derivatives, Hydroxypropyl-γ-CD (HP-γ-CD), and Octakis (6-O-sulfo)-γ-CD (Octakis-S-γ-CD), were employed to form supramolecular complexes with PiP. The conformational space of the complexes was assessed through 1 µs molecular dynamics simulations and umbrella sampling. Additionally, quantum mechanical calculations using wB97X-D dispersion-corrected DFT functional and 6-311 + G(d,p) basis set were conducted on the complexes to examine the thermodynamics and kinetic stability. Results indicated that Octakis-S-γ-CD exhibits superior host capabilities for PiP, with the most favorable complexation energy (-457.05 kJ/mol), followed by HP-γ-CD (-249.16 kJ/mol). Furthermore, two conformations of the Octakis-S-γ-CD/PiP complex were explored to elucidate the optimal binding orientation of PiP within the binding pocket of Octakis-S-γ-CD. Supramolecular chemistry relies significantly on non-covalent interactions. Therefore, our investigation extensively explores the critical atoms involved in these interactions, elucidating the influence of substituted groups on the stability of inclusion complexes. This comprehensive analysis contributes to emphasizing the γ-CD derivatives with improved host capacity.

An in silico investigation of the toxicological effects and biological activities of 3-phenoxybenzoic acid and its metabolite products.

We aimed to elucidate the toxic effects and biological activities of 3-phenoxybenzoic acid (3PBA) and its metabolite products.Numerous in silico methods were used to identify the toxic effects and biological activities of 3PBA, including PASS online, molecular docking, ADMETlab 2.0, ADMESWISS, MetaTox, and molecular dynamic simulation.Ten metabolite products were identified via Phase II reactions (O-glucuronidation, O-sulfation, and methylation).All of the investigated compounds were followed by Lipinski's rule, indicating that they were stimulants or inducers of hazardous processes.Because of their high gastrointestinal absorption and ability to reach the blood-brain barrier, the studied compounds' physicochemical and pharmacokinetic properties matched existing evidence of harmful effects, including haematemesis, reproductive dysfunction, allergic dermatitis, toxic respiration, and neurotoxicity.The studied compounds have been linked to the apoptotic pathway, the reproductivity system, neuroendocrine disruptors, phospholipid-translocating ATPase inhibitors, and JAK2 expression.An O-glucuronidation metabolite product demonstrated higher binding affinity and interaction with CYP2C9, CYP3A4, caspase 3, and caspase 8 than 3PBA and other metabolite products, whereas metabolite products from methylation were predominant and more toxic.Our in silico findings partly meet the 3Rs principle by minimizing animal testing before more study is needed to identify the detrimental effects of 3PBA on other organs (liver, kidneys).Future research directions may involve experimental validation of in silico predictions, elucidation of molecular mechanisms, and exploration of therapeutic interventions.These findings contribute to our understanding of the toxicological profile of 3PBA and its metabolites, which has implications for risk assessment and regulatory decisions.

Key properties & pharmacokinetics of 3PBA & its metabolites were reportedMetabolite products from methylation were predominant and more toxicMain toxics: haematemesis, reproductive dysfunction, toxic respiration, dermatitis.

Interaction of CYP3A4 with the inhibitor cobicistat: Structural and mechanistic insights and comparison with ritonavir.

Cobicistat is a derivative of ritonavir marketed as a pharmacoenhancer for anti-HIV therapy. This study investigated the interaction of cobicistat with the target protein, drug-metabolizing cytochrome P450 3A4 (CYP3A4), at the molecular level using spectral, kinetic, functional, and structural approaches. It was found that, similar to ritonavir, cobicistat directly coordinates to the heme via the thiazole nitrogen but its affinity and the binding rate are 2-fold lower: 0.030 µM and 0.72 s-1, respectively. The newly determined 2.5 Å crystal structure of cobicistat-bound CYP3A4 suggests that these changes arise from the inability of cobicistat to H-bond to the active site S119 and establish multiple stabilizing contacts with the F-F' connecting fragment, which becomes disordered upon steric clashing with the bulky morpholine moiety. Nonetheless, cobicistat inhibits recombinant CYP3A4 as potently as ritonavir (IC50 of 0.24 µM vs 0.22 µM, respectively) due to strong ligation to the heme and formation of extensive hydrophobic/aromatic interactions via the phenyl side-groups. To get insights into the inhibitory mechanism, the K257 residue, known to be solely and irreversibly modified by the reactive ritonavir metabolite, was substituted with alanine. Neither this nor control K266A mutation changed the extent of time-dependent inhibition of CYP3A4 by cobicistat and ritonavir, suggesting the existence of alternative inactivation mechanism(s). More importantly, K257 was found to be functionally important and contributed to CYP3A4 allosterism, possibly by modulating protein-ligand interactions through conformational dynamics.

CYP3A Mediates an Unusual C(sp2)-C(sp3) Bond Cleavage via Ipso-Addition of Oxygen in Drug Metabolism.

Mammalian cytochrome P450 drug-metabolizing enzymes rarely cleave carbon-carbon (C-C) bonds and the mechanisms of such cleavages are largely unknown. We identified two unusual cleavages of non-polar, unstrained C(sp2)-C(sp3) bonds in the FDA-approved tyrosine kinase inhibitor pexidartinib that are mediated by CYP3A4/5, the major human phase I drug metabolizing enzymes. Using a synthetic ketone, we rule out the Baeyer-Villiger oxidation mechanism that is commonly invoked to address P450-mediated C-C bond cleavages. Our studies in 18O2 and H2 18O enriched systems reveal two unusual distinct mechanisms of C-C bond cleavage: one bond is cleaved by CYP3A-mediated ipso-addition of oxygen to a C(sp2) site of N-protected pyridin-2-amines, and the other occurs by a pseudo-retro-aldol reaction after hydroxylation of a C(sp3) site. This is the first report of CYP3A-mediated C-C bond cleavage in drug metabolism via ipso-addition of oxygen mediated mechanism. CYP3A-mediated ipso-addition is also implicated in the regioselective C-C cleavages of several pexidartinib analogs. The regiospecificity of CYP3A-catalyzed oxygen ipso-addition under environmentally friendly conditions may be attractive and inspire biomimetic or P450-engineering methods to address the challenging task of C-C bond cleavages.

Unraveling the Ligand-Binding Sites of CYP3A4 by Molecular Dynamics Simulations with Solvent Probes.

Cytochrome P450 3A4 (CYP3A4) is one of the most important drug-metabolizing enzymes in the human body and is well known for its complicated, atypical kinetic characteristics. The existence of multiple ligand-binding sites in CYP3A4 has been widely recognized as being capable of interfering with the active pocket through allosteric effects. The identification of ligand-binding sites other than the canonical active site above the heme is especially important for understanding the atypical kinetic characteristics of CYP3A4 and the intriguing association between the ligand and the receptor. In this study, we first employed mixed-solvent molecular dynamics (MixMD) simulations coupled with the online computational predictive tools to explore potential ligand-binding sites in CYP3A4. The MixMD approach demonstrates better performance in dealing with the receptor flexibility compared with other computational tools. From the sites identified by MixMD, we then picked out multiple sites for further exploration using ensemble docking and conventional molecular dynamics (cMD) simulations. Our results indicate that three extra sites are suitable for ligand binding in CYP3A4, including one experimentally confirmed site and two novel sites.

Assessing the role of residue Phe108 of cytochrome P450 3A4 in allosteric effects of midazolam metabolism.

Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of more drugs in clinical use than any other xenobiotic-metabolizing enzyme. CYP3A4-mediated drug metabolism is usually allosterically modulated by substrate concentration (homotropic allostery) and other drugs (heterotropic allostery), exhibiting unusual kinetic profiles and regiospecific metabolism. Recent studies suggest that residue Phe108 (F108) of CYP3A4 may have an important role in drug metabolism. In this work, residue mutations were coupled with well-tempered metadynamics simulations to assess the importance of F108 in the allosteric effects of midazolam metabolism. Comparing the simulation results of the wild-type and mutation systems, we identify that the π-π interaction and steric effect between the F108 side chain and midazolam is favorable for the stable binding of substrate in the active site. F108 also plays an important role in the transition of substrate binding mode, which mainly induces the transition of substrate binding mode by forming π-π interactions with multiple aromatic rings of the substrate. Moreover, the side chain of F108 is closely related to the radius and depth of the 2a and 2f channels, and F108 may further regulate drug metabolism by affecting the pathway, orientation, or time of substrate entry into the CYP3A4 active site or product egress from the active site. Altogether, we suggest that F108 affects drug metabolism and regulatory mechanisms by affecting substrate binding stability, binding mode transition, and channel characteristics of CYP3A4. Our findings could promote the understanding of complicated allosteric mechanisms in CYP3A4-mediated drug metabolism, and the knowledge could be used for drug development and disease treatment.

Human cytochrome P450 3A7 binding four copies of its native substrate dehydroepiandrosterone 3-sulfate.

Human fetal cytochrome P450 3A7 (CYP3A7) is involved in both xenobiotic metabolism and the estriol biosynthetic pathway. Although much is understood about cytochrome P450 3A4 and its role in adult drug metabolism, CYP3A7 is poorly characterized in terms of its interactions with both categories of substrates. Herein, a crystallizable mutated form of CYP3A7 was saturated with its primary endogenous substrate dehydroepiandrosterone 3-sulfate (DHEA-S) to yield a 2.6 Å X-ray structure revealing the unexpected capacity to simultaneously bind four copies of DHEA-S. Two DHEA-S molecules are located in the active site proper, one in a ligand access channel, and one on the hydrophobic F'-G' surface normally embedded in the membrane. While neither DHEA-S binding nor metabolism exhibit cooperative kinetics, the current structure is consistent with cooperativity common to CYP3A enzymes. Overall, this information suggests that mechanism(s) of CYP3A7 interactions with steroidal substrates are complex.

Interaction of CYP3A4 with caffeine: First insights into multiple substrate binding.

Human cytochrome P450 3A4 (CYP3A4) is a major drug-metabolizing enzyme that shows extreme substrate promiscuity. Moreover, its large and malleable active site can simultaneously accommodate several substrate molecules of the same or different nature, which may lead to cooperative binding and allosteric behavior. Due to difficulty of crystallization of CYP3A4-substrate complexes, it remains unknown how multiple substrates can arrange in the active site. We determined crystal structures of CYP3A4 bound to three and six molecules of caffeine, a psychoactive alkaloid serving as a substrate and modulator of CYP3A4. In the ternary complex, one caffeine binds to the active site suitably for C8-hydroxylation, most preferable for CYP3A4. In the senary complex, three caffeine molecules stack parallel to the heme with the proximal ligand poised for 3-N-demethylation. However, the caffeine stack forms extensive hydrophobic interactions that could preclude product dissociation and multiple turnovers. In both complexes, caffeine is also bound in the substrate channel and on the outer surface known as a peripheral site. At all sites, aromatic stacking with the caffeine ring(s) is likely a dominant interaction, while direct and water-mediated polar contacts provide additional stabilization for the substrate-bound complexes. Protein-ligand interactions via the active site R212, intrachannel T224, and peripheral F219 were experimentally confirmed, and the latter two residues were identified as important for caffeine association. Collectively, the structural, spectral, and mutagenesis data provide valuable insights on the ligand binding mechanism and help better understand how purine-based pharmaceuticals and other aromatic compounds could interact with CYP3A4 and mediate drug-drug interactions.

Nanodisc-embedded cytochrome P450 P3A4 binds diverse ligands by distributing conformational dynamics to its flexible elements.

Cytochrome P450 3A4 (CYP3A4) metabolizes a wide range of drugs and toxins. Interactions of CYP3A4 with ligands are difficult to predict due to promiscuity and conformational flexibility. To better understand CYP3A4 conformational responses to ligands we use hydrogen deuterium exchange mass spectrometry (HDX-MS) to investigate the effect of ligands on nanodisc-embedded CYP3A4. For a subset of CYP3A4-ligand complexes, differences in the low-frequency modes derived by principal component analyses of molecular dynamics trajectories mirrored the HDX-MS results. The effects of ligands are distributed to flexible elements of CYP3A4 between stretches of secondary structure. The largest effects occur in the F- and G-helices, where most ligands increase the flexibility of the F-helix and connecting loops and decrease the flexibility of the C-term of the G-helix. Most ligands affect the E-F-G, CD and HI regions of the protein. Ligand-dependent differences are observed in the A"-A' loop, BC region, E-helix, K-ß1 region, proximal loop, and C-term loop. Correlated HDX responses were observed in the CD region and the C-term of the G-helix that were most pronounced for Type II ligands. Collectively, the HDX and molecular dynamics results suggest that CYP3A4 accommodates diverse binding partners by propagating local backbone fluctuations from the binding site onto the flexible regions of the enzyme via long-range interactions that are differentially modulated by ligands. In contrast to the paradigm wherein ligands decrease protein dynamics at their binding site, a wide range of ligands modestly increase CYP3A4 dynamics throughout the protein including effects remote from the active site.

Unraveling the Abnormal Molecular Mechanism of Suicide Inhibition of Cytochrome P450 3A4.

Suicide inhibition of the CYP3A4 enzyme by a drug inactivates the enzyme in the drug biotransformation process and often shows safety concerns about the drug. Despite extensive experimental studies, the abnormal molecular mechanism of a suicide inhibitor that forms a covalent bond with the residue far away from the catalytically active center of CYP3A4 inactivating the enzyme remains elusive. Here, the authors used molecular simulation approaches to study in detail how diquinone methide (DQR), the metabolite product of raloxifene, unbinds from CYP3A4 and inactivates the enzyme at the atomistic level. The results clearly indicate that in one of the intermediate states formed in its unbinding process, DQR covalently binds to Cys239, a residue far away from the catalytically active center of CYP3A4, and hinders the substrate from entering or leaving the enzyme. This work therefore provides an unprecedented way of clarifying the abnormal mechanism of suicide inhibition of the CYP3A4 enzyme.

Molecular Insights into the Heterotropic Allosteric Mechanism in Cytochrome P450 3A4-Mediated Midazolam Metabolism.

Cytochrome P450 3A4 (CYP3A4) is the main P450 enzyme for drug metabolism and drug-drug interactions (DDIs), as it is involved in the metabolic process of approximately 50% of drugs. A detailed mechanistic elucidation of DDIs mediated by CYP3A4 is commonly believed to be critical for drug optimization and rational use. Here, two typical probes, midazolam (MDZ, substrate) and testosterone (TST, allosteric effector), are used to investigate the molecular mechanism of CYP3A4-mediated heterotropic allosteric interactions, through conventional molecular dynamics (cMD) and well-tempered metadynamics (WT-MTD) simulations. Distance monitoring shows that TST can stably bind in two potential peripheral sites (Site 1 and Site 2) of CYP3A4. The binding of TST at these two sites can induce conformational changes in CYP3A4 flexible loops on the basis of conformational analysis, thereby promoting the transition of the MDZ binding mode and affecting the ratio of MDZ metabolites. According to the results of the residue interaction network, multiple allosteric communication pathways are identified that can provide vivid and applicable insights into the heterotropic allostery of TST on MDZ metabolism. Comparing the regulatory effects and the communication pathways, the allosteric effect caused by TST binding in Site 2 seems to be more pronounced than in Site 1. Our findings could provide a deeper understanding of CYP3A4-mediated heterotropic allostery at the atomic level and would be helpful for rational drug use as well as the design of new allosteric modulators.

Midazolam as a Probe for Heterotropic Drug-Drug Interactions Mediated by CYP3A4.

Human cytochrome P450 CYP3A4 is involved in the processing of more than 35% of current pharmaceuticals and therefore is responsible for multiple drug-drug interactions (DDI). In order to develop a method for the detection and prediction of the possible involvement of new drug candidates in CYP3A4-mediated DDI, we evaluated the application of midazolam (MDZ) as a probe substrate. MDZ is hydroxylated by CYP3A4 in two positions: 1-hydroxy MDZ formed at lower substrate concentrations, and up to 35% of 4-hydroxy MDZ at high concentrations. The ratio of the formation rates of these two products (the site of metabolism ratio, SOM) was used as a measure of allosteric heterotropic interactions caused by effector molecules using CYP3A4 incorporated in lipid nanodiscs. The extent of the changes in the SOM in the presence of effectors is determined by chemical structure and is concentration-dependent. MD simulations of CYP3A4 in the lipid bilayer suggest that experimental results can be explained by the movement of the F-F' loop and concomitant changes in the shape and volume of the substrate-binding pocket. As a result of PGS binding at the allosteric site, several residues directly contacting MDZ move away from the substrate molecule, enabling the repositioning of the latter for minor product formation.

Investigation of the molecular and mechanistic basis for the regioselective metabolism of midazolam by cytochrome P450 3A4.

Cytochrome P450 3A4 (CYP3A4) is the most important P450 enzyme for drug metabolism and drug-drug interaction, due to it being responsible for the biotransformation of approximately 50% of clinically used drugs. Advance knowledge of the molecular and mechanistic basis of CYP3A4 regioselective metabolism is beneficial for understanding the production of metabolites, and may allow personalized metabolic pathways or designing pathway-specific therapeutics. In this work, we focus on investigating the ligand-receptor interactions, substrate conformational transition, and key factors regulating the specificity of metabolic pathways using midazolam (MDZ) as a probe. Here, three types of substrate-binding conformations related to the diversity of MDZ metabolites are identified. The results also suggest that an allosteric site for MDZ is located near the F'-helix, A-anchor, and C-terminal loop of CYP3A4. The presence of an effector in the allosteric site can accelerate the conformational transition of the substrate via modulating a "sandwich" structure, and may affect the proportion of metabolites at high substrate concentration. We hope that the results can improve the understanding of the CYP3A4 structure and function, and provide a new perspective for drug development.

Preparative Production of Functionalized (N- and O-Heterocyclic) Polycyclic Aromatic Hydrocarbons by Human Cytochrome P450 3A4 in a Bioreactor.

Polycyclic aromatic hydrocarbons (PAHs) and their N- and O-containing derivatives (N-/O-PAHs) are environmental pollutants and synthetically attractive building blocks in pharmaceuticals. Functionalization of PAHs can be achieved via C-H activation by cytochrome P450 enzymes (e.g., P450 CYP3A4) in an environmentally friendly manner. Despite its broad substrate scope, the contribution of CYP3A4 to metabolize common PAHs in humans was found to be small. We recently showcased the potential of CYP3A4 in whole-cell biocatalysis with recombinant yeast Komagataella phaffii (Pichia pastoris) catalysts for the preparative-scale synthesis of naturally occurring metabolites in humans. In this study, we aimed at exploring the substrate scope of CYP3A4 towards (N-/O)-PAHs and conducted a bioconversion experiment at 10 L scale to validate the synthetic potential of CYP3A4 for the preparative-scale production of functionalized PAH metabolites. Hydroxylated products were purified and characterized using HPLC and NMR analysis. In total, 237 mg of fluorenol and 48 mg of fluorenone were produced from 498 mg of fluorene, with peak productivities of 27.7 µmol/L/h for fluorenol and 5.9 µmol/L/h for fluorenone; the latter confirmed that CYP3A4 is an excellent whole-cell biocatalyst for producing authentic human metabolites.

Discovery and Characterization of Potent Dual P-Glycoprotein and CYP3A4 Inhibitors: Design, Synthesis, Cryo-EM Analysis, and Biological Evaluations.

Targeted concurrent inhibition of intestinal drug efflux transporter P-glycoprotein (P-gp) and drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4) is a promising approach to improve oral bioavailability of their common substrates such as docetaxel, while avoiding side effects arising from their pan inhibitions. Herein, we report the discovery and characterization of potent small molecule inhibitors of P-gp and CYP3A4 with encequidar (minimally absorbed P-gp inhibitor) as a starting point for optimization. To aid in the design of these dual inhibitors, we solved the high-resolution cryo-EM structure of encequidar bound to human P-gp. The structure guided us to prudently decorate the encequidar scaffold with CYP3A4 pharmacophores, leading to the identification of several analogues with dual potency against P-gp and CYP3A4. In vivo, dual P-gp and CYP3A4 inhibitor 3a improved the oral absorption of docetaxel by 3-fold as compared to vehicle, while 3a itself remained poorly absorbed.

Physicochemical characterization of paclitaxel prodrugs with cytochrome 3A4 to correlate solubility and bioavailability implementing molecular docking and simulation studies.

Prodrugs are biologically inactive drug molecules that may be developed through rational drug design with an objective to improve a drug's pharmaceutical and pharmacokinetic properties. Paclitaxel, a highly potent anticancer drug, is directed against many cancers like breast cancer, ovarian cancer, lung cancer, head and neck tumors, non-small cell lung cancer, and Kaposi's sarcoma, etc. Along with its excellent antitumor activity the drug had a major limitation of low water solubility. To overcome this limitation of this nanomolar active drug many prodrugs were formed in the past. Though increase in the solubility of the drug was obtained but that may or may not account for its increase in bioavailability. CYP3A4 liver enzymes are responsible for the metabolism of fifty percent of the drugs and are major metabolizing enzyme for paclitaxel. Phosphate prodrugs are well known to account the insolubility of many drugs and thus increasing their bioavailability also. In this study, we calculated the ADMET properties of a dataset of twenty phosphate prodrugs of paclitaxel. On the basis of reflection of three favourable properties, ten prodrugs were chosen for further docking studies against CYP3A4. Finally, three prodrugs showing unfavourable binding affinities were selected for Molecular Dynamics Simulations and from this in-silico study we identified that all the three selected prodrugs were unstable as compared to the paclitaxel. The instability of these prodrugs showed their lesser interaction with the CYP3A4 and hence contributing more towards its bioavailability. Thus the three suggested prodrugs those were studied in-silico for oral bioavailability can be further validated for gastrointestinal cancer.Communicated by Ramaswamy H. Sarma.

On the inhibition of cytochrome P450 3A4 by structurally diversified flavonoids.

Cytochrome P450 3A4 (CYP3A4) is the most versatile enzyme involved in drug metabolism. The time-dependent inhibition of CYP3A4 by acacetin, apigenin, chrysin, and pinocembrin was experimentally detected, but not entirely elaborated so far. Thus, a two-level QM/MM (Quantum Mechanics/Molecular Mechanics) model is developed to yield insights into the receptor-flavonoid recognition at the molecular scale. Active site residues and the flavonoid are modelled using SCC-DFTB-D (QM level), while the rest of the complex is treated using AMBER force field (MM level). QM/MM binding free energies are well correlated with experimental data, indicating the largest inhibitory effect of chrysin on enzyme activity at a submicromolar concentration. Consequently, quercetin (QUE) and flavopiridol (FLP) are observed as representative examples of structurally different flavonoids. The inhibition parameters for QUE and FLP are evaluated using the well-calibrated QM/MM strategy, thereby aiding to quantitatively conceive the functional behavior of the whole family of flavonoids. A kinetic threshold for further assessment of the drug-drug interactions underlying the time-dependent inhibition of CYP3A4 by flavonoids is explored.Communicated by Ramaswamy H. Sarma.

Cytotoxicity evaluation and metabolism of hepatotoxicity components of Euodiae Fructus in L02 cells.

Euodiae Fructus (EF), the dried unripe scented fruit of Euodia rutaecarpa (Juss.) Benth., was reported to show anti-hypertensive, antitumor, and anti-obesity effects. The main alkaloids of EF were reported as the reason for toxicity of EF by metabolic activation majority through CYP3A. Up till the present moment, the cytotoxicity mechanisms of EF have not yet to be fully clarified. For the purposes of this article, the influence of CYP3A inducer and inhibitor on cytotoxicity of EF and metabolism in L02 cells of five alkaloids related to toxicity of EF were evaluated. The results indicated that CYP3A inducer aggravated the toxicity and CYP3A inhibitor alleviated the toxicity. UPLC-Q-Exactive-MS was used for the identification of five alkaloids of EF in L02 cells. A total of 13 metabolites were detected in L02 cells. In general, five alkaloids were widely metabolized in L02 cells such as oxygenation, demethylation, dehydrogenation, and etc. In addition, oxygenation was the main metabolic pathway. It was inferred that the toxicity of EF was closely related to the CYP3A and the metabolic intermediate might be one of the reasons for the toxicity of EF. Hence, the choice of optimal dose might be critical to avoid the adverse reactions owing to combination of EF and CYP3A inducer.