Interaction of human cytochrome P4503A4 with ritonavir analogs

Ritonavir is a HIV protease inhibitor that also potently inactivates cytochrome P450 3A4 (CYP3A4), a major human drug-metabolizing enzyme. To better understand the mechanism of ligand binding and to find strategies for improvement of the inhibitory potency of ritonavir, currently administered to enhance pharmacokinetics of other anti-HIV drugs that are quickly metabolized by CYP3A4, we compared the manner of CYP3A4 interaction with the drug and two analogs lacking either the heme-ligating thiazole nitrogen or the entire thiazole group. Based on the kinetic, mutagenesis and structural data, we conclude that: (i) the active site residue Arg212 assists binding of all investigated compounds and, thus, may play a more prominent role in metabolic transformation of xenobiotics than previously thought, (ii) peripheral binding of ritonavir limits the heme coordination rate and complicates the binding kinetics, (iii) association of ritonavir-like type II ligands is driven by heme coordination whereas hydrophobic forces define the binding mode, and (iv) substitution of one phenyl group in ritonavir with a smaller hydrophobic moiety could prevent steric clashing and, hence, increase the affinity and inhibitory potency of the drug.     

Intermittent preventive therapy for malaria: arguments in favour of artesunate and sulphamethoxypyrazine - pyrimethamine combination

The case of a 32-year-old Caucasian female with multi-drug resistant HIV-1 subtype B infection treated with a salvage regimen including maraviroc, raltegravir, etravirine and unboosted saquinavir who started atovaquone/proguanil prophylaxis, is reported. The potential interactions between atovaquone/proguanil and these anti-retroviral drugs are investigated. Pharmacokinetic analyses documented a marked increase in etravirine and saquinavir plasma concentrations (+55% and +274%, respectively), but not in raltegravir and maraviroc plasma concentrations. The evidence that atovaquone/proguanil significantly interacts with etravirine and saquinavir, but not with raltegravir and maraviroc, suggests that the mechanism of interaction is related to cytochrome P450.     

Synthesis and evaluation of inhibitors of cytochrome P450 3A (CYP3A) for pharmacokinetic enhancement of drugs

The HIV protease inhibitor ritonavir (RTV) is also a potent inhibitor of the metabolizing enzyme cytochrome P450 3A (CYP3A) and is clinically useful in HIV therapy in its ability to enhance human plasma levels of other HIV protease inhibitors (PIs). A novel series of CYP3A inhibitors was designed around the structural elements of RTV believed to be important to CYP3A inhibition, with general design features being the attachment of groups that mimic the P2–P3 segment of RTV to a soluble core. Several analogs were found to strongly enhance plasma levels of lopinavir (LPV), including 8, which compares favorably with RTV in the same model. Interestingly, an inverse correlation between in vitro inhibition of CYP3A and elevation of LPV was observed. The compounds described in this study may be useful for enhancing the pharmacokinetics of drugs that are metabolized by CYP3A.     

Analysis of clinically relevant substrates of CYP2B6 enzyme by computational methods

Mounting evidence thus far indicates that human cytochrome P450 2B6 (CYP2B6), an enzyme expressed at a relatively low level functionally, is primarily responsible for the metabolism of several clinically relevant drugs, including propofol, efavirenz, bupropion, mephobarbital, and the propofol analog 2,6-di-sec-butyl phenol. We used molecular dynamics and molecular docking methods to predict such interactions and to compare with experimentally measured metabolisms. Insight II and Discover Studio 2.5 were used to carry out the docking of these substrates into CYP2B6 to explore the critical residues and interaction energies of the complexes. Phe297, Glu301, Thr302 and Val367 were identified as major drug-binding residues, which is consistent with previous data on site-directed mutagenesis, crystallography structure, and from modeling and docking studies. In addition, our docking results suggest that nonpolar amino acid clusters and heme also participate in binding to mediate drug oxidative metabolism. The binding modes of the five clinically relevant substrates mentioned above for metabolism on CYP2B6 are presented.     

Analysis of binding modes of ligands to multiple conformations of CYP3A4

Cytochromes P450 (CYPs) are extremely versatile enzymes capable of catalyzing a vast number of compounds, and CYP3A4 is no exception metabolizing approximately half of the currently marketed drugs, besides endogenous compounds. To metabolize such a variety of compounds, CYP3A4 has to be extremely flexible, which makes interaction studies difficult. We employ a multi-conformational docking setup where conformations are generated by several molecular dynamics simulations to analyze the binding modes of various ligands, and the docking is considered successful if the ligand site of catalysis (SOC) is within 6.0 Å of the haem Fe. While docking with the X-ray structure proved unsuccessful with all ligands, the multi-conformational docking achieved successful binding of each ligand to at least one protein conformation. Analysis of the docked solutions highlights residues in the active site cavity that may have an important role in access, binding and stabilization of the ligand.     

The structural basis for homotropic and heterotropic cooperativity of midazolam metabolism by human cytochrome P450 3A4

Human cytochrome P450 3A4 (CYP3A4) metabolizes a significant portion of clinically relevant drugs and often exhibits complex steady-state kinetics that can involve homotropic and heterotropic cooperativity between bound ligands. In previous studies, the hydroxylation of the sedative midazolam (MDZ) exhibited homotropic cooperativity via a decrease in the ratio of 1'-OH-MDZ to 4-OH-MDZ at higher drug concentrations. In this study, MDZ exhibited heterotropic cooperativity with the antiepileptic drug carbamazepine (CBZ) with characteristic decreases in the 1'-OH-MDZ to 4-OH-MDZ ratios. To unravel the structural basis of MDZ cooperativity, we probed MDZ and CBZ bound to CYP3A4 using longitudinal T(1) nuclear magnetic resonance (NMR) relaxation and molecular docking with AutoDock 4.2. The distances calculated from longitudinal T(1) NMR relaxation were used during simulated annealing to constrain the molecules to the substrate-free X-ray crystal structure of CYP3A4. These simulations revealed that either two MDZ molecules or an MDZ molecule and a CBZ molecule assume a stacked configuration within the CYP3A4 active site. In either case, the proton at position 4 of the MDZ molecule was closer to the heme than the protons of the 1'-CH(3) group. In contrast, molecular docking of a single molecule of MDZ revealed that the molecule was preferentially oriented with the 1'-CH(3) position closer to the heme than position 4. This study provides the first detailed molecular analysis of heterotropic and homotropic cooperativity of a human cytochrome P450 from an NMR-based model. Cooperativity of ligand binding through direct interaction between stacked molecules may represent a common motif for homotropic and heterotropic cooperativity.     

Effect of Ethanol on the Metabolic Characteristics of HIV-1 Integrase Inhibitor Elvitegravir and Elvitegravir/Cobicistat with CYP3A: An Analysis Using a Newly Developed LC-MS/MS Method

Elvitegravir (EVG), an integrase inhibitor for the treatment HIV infection, is increasingly becoming the part of first-line antiretroviral therapy (ART) regimen. EVG is mainly metabolized through cytochrome P450 (CYP) 3A4. Previously, we have shown that ethanol alters ART-CYP3A4 interactions with protease inhibitors thereby altering their metabolisms. However, as EVG is a fairly new class of drug, its kinetic characteristics and the effect of ethanol on EVG-CYPP3A4 interaction is poorly understood. In this study, we characterized EVG and cobicistat (COBI)-boosted EVG metabolism in human microsomes followed by ethanol-EVG, ethanol-COBI-EVG interaction with CYP3A. First, we developed and validated a simple, sensitive, and robust liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the quantification of EVG in the human liver microsomes. The lower limit of quantification for the drug was at 0.003 μM (1.34ng/ml). Extraction yield, matrix effects, drug stability, and calibration curves for the proposed method were validated according to the FDA guidelines. Time dependent kinetics data showed that 20mM ethanol decreases the apparent half-life of EVG degradation by ~50% compared to EVG alone. Our substrate kinetic results revealed that ethanol mildly decreases the catalytic efficiency for EVG metabolism. Inhibition studies demonstrated that EVG inhibits CYP3A4, and 20 mM ethanol causes a decrease in the IC₅₀ of EVG. However, in the presence of COBI we were unable to determine these parameters effectively because COBI, being a strong inhibitor of CYP3A4, blocked the EVG/ethanol-CYP3A4 interactions. Docking studies predicted a shift of EVG or COBI binding to the active site of CYP3A4 in the presence of ethanol. Taken together, these results suggest that ethanol interacts with microsomal CYP3A and alters EVG-CYP3A4 interaction thereby altering EVG metabolism and inhibition of CYP3A4 by EVG. This finding has clinical significance because alcohol use is highly prevalent in HIV population, and there are no separate guidelines for these patients while they are on ART medication.     

Metabolism, Pharmacogenetics, and Metabolic Drug–Drug Interactions of Antipsychotic Drugs

1. Antipsychotic drugs are extensively metabolised by cytochrome P450 (CYP) enzymes.2. Dispositions of a number of antipsychotic drugs have been shown to cosegregate with polymorphism of CYP2D6.3. Metabolic drug–drug interactions have frequently been observed when antipsychotics are coadministered with other drugs.4. Many antipsychotic drugs are converted to active metabolites which can contribute to the therapeutic or side effects of the parent drug.5. Information concerning the individual CYP isoenzymes involved in the metabolism of antipsychotic drugs is important for the safe clinical use of this group of drugs.     

Exploration of the binding of proton pump inhibitors to human P450 2C9 based on docking and molecular dynamics simulation

Human P450 protein CYP2C9 is one of the major drug-metabolizing isomers, contributing to the oxidation of 16% of the drugs currently in clinical use. To examine the interaction mechanisms between CYP2C9 and proton pump inhibitions (PPIs), we used molecular docking and molecular dynamics (MD) simulation methods to investigate the conformations and interactions around the binding sites of PPIs/CYPP2C9. Results from molecular docking and MD simulations demonstrate that nine PPIs adopt two different conformations (extended and U-bend structures) at the binding sites and position themselves far above the heme of 2C9. The presence of PPIs changes the secondary structures and residue flexibilities of 2C9. Interestingly, at the binding sites of all PPI–CYP2C9 complexes except for Lan/CYP2C9, there are hydrogen-bonding networks made of PPIs, water molecules, and some residues of 2C9. Moreover, there are strong hydrophobic interactions at all binding sites for PPIs/2C9, which indicate that electrostatic interactions and hydrophobic interactions appear to be important for stabilizing the binding sites of most PPIs/2C9. However, in the case of Lan/2C9, the hydrophobic interactions are more important than the electrostatic interactions for stabilizing the binding site. In addition, an interesting conformational conversion from extended to U-bend structures was observed for pantoprazole, which is attributed to an H-bond interaction in the binding pocket, an internal π–π stacking interaction, and an internal electrostatic interaction of pantoprazole.     

Metabolism and Excretion of Mood Stabilizers and New Anticonvulsants

1. The mood stabilizers lithium, carbamazepine (CBZ), and valproate (VPA), have differing pharmacokinetics, structures, mechanisms of action, efficacy spectra, and adverse effects. Lithium has a low therapeutic index and is renally excreted and hence has renally-mediated but not hepatically-mediated drug–drug interactions.2. CBZ has multiple problematic drug–drug interactions due to its low therapeutic index, metabolism primarily by a single isoform (CYP3A3/4), active epoxide metabolite, susceptibility to CYP3A3/4 or epoxide hydrolase inhibitors, and ability to induce drug metabolism (via both cytochrome P450 oxidation and conjugation). In contrast, VPA has less prominent neurotoxicity and three principal metabolic pathways, rendering it less susceptible to toxicity due to inhibition of its metabolism. However, VPA can increase plasma concentrations of some drugs by inhibiting metabolism and increase free fractions of certain medications by displacing them from plasma proteins.3. Older anticonvulsants such as phenobarbital and phenytoin induce hepatic metabolism, may produce toxicity due to inhibition of their metabolism, and have not gained general acceptance in the treatment of primary psychiatric disorders.4. The newer anticonvulsants felbamate, lamotrigine, topiramate, and tiagabine have different hepatically-mediated drug–drug interactions, while the renally excreted gabapentin lacks hepatic drug–drug interactions but may have reduced bioavailability at higher doses.5. Investigational anticonvulsants such as oxcarbazepine, vigabatrin, and zonisamide appear to have improved pharmacokinetic profiles compared to older agents.6. Thus, several of the newer anticonvulsants lack the problematic drug-drug interactions seen with older agents, and some may even (based on their mechanisms of action and preliminary preclinical and clinical data) ultimately prove to have novel psychotropic effects.     

Effect of Methamphetamine on Spectral Binding,Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4

Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δA_max and K_D of 0.016±0.001 and 204±18 μM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δA_max (0.004±0.0003 vs. 0.0068±0.0001) and K_D (1.42±0.36 vs.2.93±0.08 μM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δA_max (0.0038±0.0003 vs. 0.0055±0.0003) and K_D (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced K_D in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir-boosted antiretroviral therapy, in HIV-1-infected individuals who abuse methamphetamine.     

Exploration of the binding of curcumin analogues to human P450 2C9 based on docking and molecular dynamics simulation

Molecular docking and molecular dynamics (MD) simulations are used to investigate the interactions of curcumin analogues (CAs) with human cytochrome P450 2 C9 (CYP2C9 or 2 C9) and the conformations of their binding sites. In order to examine conformations of CAs/2 C9 and interaction characteristics of their binding sites, RMSDs, RMSFs, and B-factors are computed, and electrostatic and hydrophobic interactions between CAs and 2 C9 are analyzed and discussed. Results demonstrate that the most CAs studied lie 4~15 ? above the heme of CYP2C9. The presence of CAs makes some residues in bound CYP2C9s become more flexible. In the binding sites of A0/2 C9 and C0/2 C9, the formation of H-bond networks (or CA-water-residue bridges) enhances the interactions between CAs and 2 C9. The stronger inhibitory effects of A0, B0, and C0 on 2 C9 can be ascribed to stronger electrostatic and hydrophobic interactions in the binding sites of CAs/2 C9.     

3D structure modeling of cytochrome P450 2C19 and its implication for personalized drug design

Cytochrome P450 2C19 (CYP2C19) is a member of the cytochrome P-450 enzyme superfamily and plays an important role in the metabolism of drugs. In order to gain insights for developing personalized drugs, the 3D (dimensional) structure of CYP2C19 has been developed based on the crystal structure of CYP2C9 (PDB code 1R90), and its structure-activity relationship with the ligands of CEC, Fluvoxamine, Lescol, and Ticlopidine investigated through the structure-activity relationship approach. By means of a series of docking studies, the binding pockets of CYP2C19 for the four compounds are explicitly defined that will be very useful for conducting mutagenesis studies, providing insights into personalization of drug treatments and stimulating novel strategies for finding desired personalized drugs.     

Hyperforin and its analogues inhibit CYP3A4 enzyme activity

Literature indicates that herb-drug interaction of St. John's wort is largely due to increased metabolism of the co-administered drugs that are the substrates of cytochrome P450 (CYP) 3A4 enzyme, alteration of the activity and/or expression of the enzyme. The major St. John's wort constituents, acylphloroglucinols, were evaluated for their effects on CYP3A4 enzyme activity to investigate their roles in herb-drug interaction. Hyperforin and four oxidized analogues were isolated from the plant and fully characterized by mass spectral and NMR analysis. These acylphloroglucinols inhibited activity of CYP3A4 enzyme potently in the fluorometric assay using the recombinant enzyme. Furoadhyperforin (IC(50) 0.072 microM) was found to be the most potent inhibitor of CYP3A4 enzyme activity, followed by furohyperforin isomer 1 (IC(50) 0.079 microM), furohyperforin isomer 2 (IC(50) 0.23 microM), hyperforin (IC(50) 0.63 microM) and furohyperforin (IC(50) 1.3 microM). As the acylphloroglucinols are potent inhibitors of the CYP3A4 enzyme, their modulation of the enzyme activity is unlikely to be involved in increased drug metabolism by St. John's wort.     

Structure–activity relationships of diamine inhibitors of cytochrome P450 (CYP) 3A as novel pharmacoenhancers,part I: Core region

Ritonavir (RTV), an HIV-1 protease inhibitor (PI), is also a potent mechanism-based inhibitor of human cytochrome P450 3A (CYP3A) and has been widely prescribed as a pharmacoenhancer. As a boosting agent for marketed PIs, it reduces pill burden, and improves compliance. Removal of the hydroxyl group from RTV reduces, but does not eliminate HIV PI activity and does not affect CYP3A inhibition. Herein we report the discovery of a novel series of CYP3A inhibitors that are devoid of antiviral activity. The synthesis and evaluation of analogs with extensive modifications of the 1,4-diamine core along with the structure activity relationships with respect to anti-HIV activity, CYP3A inhibitory activity, selectivity against other CYP enzymes and the human pregnane X receptor (PXR) will be discussed.     

Induction of cytochrome P450 3A by Shexiang Baoxin Pill and its main components

The expression of cytochrome P450 is regulated by both endogenous factors and xenobiotics including chemical drugs and natural medicines. Induction on cytochrome P450 can reduce the therapeutic efficacy from drugs inactivated by this enzyme system, but may increase the efficacy or lead to intoxication for prodrugs. Shexiang Baoxin Pill (SBP) is a traditional Chinese medicine widely used for the treatment of angina pectoris and myocardial infarction in China and other oriental countries. To assess the potential of SBP to alter the activity and expression of cytochrome P450 3A (CYP3A) extensively involved in drug metabolism, we investigated the enzyme-inducing effects of SBP in HepG2 cells and in rats. The results showed that treatment with SBP increased the enzyme activity, mRNA levels and protein expression of CYP3A4 in a concentration-dependent manner in HepG2 cells. Moreover, treatment with SBP enhanced the activities and mRNA expressions of CYP3A1 and CYP3A2 ex vivo in rats. Furthermore, we utilized HepG2 cell line to identify individual components in SBP as potential inducers of CYP3A4. It was found that bufalin, cinobufagin, and resibufogenin were novel CYP3A4 inducers. Among them, bufalin and cinobufagin significantly promoted the CYP3A4 enzyme activity, mRNA and protein levels, with the maximal induction challenging or exceeding that of the induction by rifampicin, indicating that they might play a critical role in CYP3A4 enzyme-inducing effects of SBP. In addition, the metabolic studies with specific inhibitors of CYP isoforms suggested that the three CYP3A4 inducers in SBP are also the substrates for the enzyme. Overall, our results show that SBP contains constituents that can potently induce CYP3A and suggest that this traditional Chinese medicine should be examined clinically for potential drug metabolic interactions.     

Charge-dependent sidedness of cytochrome P450 forms studied by quartz crystal microbalance and atomic force microscopy

Quartz crystal microbalance (QCM) resonance measurements were used to examine the surface charge characteristics of cytochrome P450 forms and the influence of charge on the docking of redox partners like cytochrome b5. The distal surface of cytochrome P450 (CYP)101 (pI = 4.5), relative to the heme, is fairly anionic, as is the proximal surface. The latter, however, also has two cationic clusters. A considerably greater extent of CYP101 binding was seen to the cationic, polyethylene-surfaced resonators. CYP2B4 (pI = 8.5) preferentially bound to the polyanionic, polystyrene sulfonate-surfaced resonators. Cytochrome b5 is an acidic protein that had a preferential binding to the poly(ethyleneimine (PEI)-surfaced resonators. When binding to CYP2B4-surfaced films, cytochrome b5 preferentially bound to those cytochrome P450 molecules that were adsorbed to cationic (PEI) films. It is suggested that adsorption of CYP2B4 to an anionic poly(styrenesulfonate) (PSS) surface is with cationic clusters that include the cytochrome b5 docking domain. This diminishes the extent of docking of the cytochrome b5. In contrast, when CYP2B4 is adsorbed to a cationic film the proximal surface with the cytochrome b5-docking site is available for cytochrome b5 binding. A film of the polycation PEI was adsorbed to the silver QCM surface. It formed polymer islands when viewed with atomic force microscopy. Polyanionic PSS was adsorbed intermittently with the PEI. By the third and fourth layer of polyions the polymer islands were essentially merged and protein adsorption as a fourth or fifth layer formed a nearly continuous film. CYP101 was seen to adsorb as globules with a molecular diameter of about 10 nm. CYP2B4 adsorbed to the polyionic films had a slightly elliptical globular shape, also with a molecular diameter of about 10 nm.     

Rhodococcus sp. R04细胞色素P450单加氧酶及CYP125A18与唑类药物的互作分析

红球菌 (Rhodococcus sp.) R04基因组有15种细胞色素P450单加氧酶,其中CYP125A18与结核分枝杆菌 (Mycobacterium tuberculosis) 和马红球菌 (Rhodococcus equi) 的CYP125有较高同源性。利用NCBI蛋白质数据库搜索同源序列,对Rhodococcus sp. R04的15种CYP450一级结构序列进行比对和系统发育分析;对CYP125A18基因进行了克隆表达,并用紫外分光光度法对蛋白质的光谱学特性以及与唑类药物互作情况进行分析。实验结果表明,Rhodococcus sp. R04 15种CYP450均含有保守的氨基酸序列和铁血红素催化中心。SDS-PAGE分析表明,CYP125A18分子量约为50 kD,CYP125A18还原态和CO结合后与CYP125A18氧化态的差示光谱表现为典型的CYP450光谱特性。CYP125A18与底物4-胆甾烯-3-酮结合后,血红素铁全部转变为高自旋状态;与唑类药物滴定后发生了II型光谱转变。解离常数表明,7种唑类药物与CYP125A18的亲和力由强到弱依次为酮康唑、益康唑、4-苯基咪唑、氟康唑、4-甲基-2-苯基咪唑、克霉唑、甲硝唑。上述发现对研究CYP125代谢胆固醇具有重要意义,同时为疾病耐药性研究及药物选择提供数据和理论支持。