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Metal sulfide minerals are assumed to form naturally at ambient conditions via reaction of a metallic element with (poly)sulfide ions, usually produced by microbes in oxygen-depleted environments. Recently, the formation of mercury sulfide (β-HgS) directly from linear Hg(II)-thiolate complexes (Hg(SR)2) in natural organic matter and in cysteine solutions was demonstrated under aerated conditions. Here, a detailed description of this non-sulfidic reaction is provided by computations at a high level of molecular-orbital theory. The HgS stoichiometry is obtained through the cleavage of the S-C bond in one thiolate, transfer of the resulting alkyl group (R') to another thiolate, and subsequent elimination of a sulfur atom from the second thiolate as a thioether (RSR'). Repetition of this mechanism leads to the formation of RS-(HgS)n-R chains which may self-assemble in parallel arrays to form cinnabar (α-HgS), or more commonly, quickly condense to four-coordinate metacinnabar (β-HgS). The mechanistic pathway is thermodynamically favorable and its predicted kinetics agrees with experiment. The results provide robust theoretical support for the abiotic natural formation of nanoparticulate HgS under oxic conditions and in the absence of a catalyst, and suggest a new route for the (bio)synthesis of HgS nanoparticles with improved technological properties.
The mammalian AlkB homologue-3 (AlkBH3) is a member of the dioxygenase family of enzymes that in humans is involved in DNA dealkylation repair. Because of its role in promoting tumor cell proliferation and metastasis of cancer, extensive efforts are being directed in developing selective inhibitors for AlkBH3. Here we report synthesis, screening and evaluation of panel of arylated indenone derivatives as new class of inhibitors of AlkBH3 DNA repair activity. An efficient synthesis of 2,3-diaryl indenones from 2,3-dibromo indenones was achieved via Suzuki-Miyaura cross-coupling. Using a robust quantitative assay, we have obtained an AlkBH3 inhibitor that display specific binding and competitive mode of inhibition against DNA substrate. Finally, we established that this compound could prevent the proliferation of lung cancer cell line and enhance sensitivity to DNA damaging alkylating agent.
Herein we report the development and biological screening of a bioorthogonal palladium-labile prodrug of the nucleoside analogue floxuridine, a potent antineoplastic drug used in the clinic to treat advanced cancers. N-propargylation of the N3 position of its uracil ring resulted in a vast reduction of its biological activity (~6,250-fold). Cytotoxic properties were bioorthogonally rescued in cancer cell culture by heterogeneous palladium chemistry both in normoxia and hypoxia. Within the same environment, the reported chemo-reversible prodrug exhibited up to 1,450-fold difference of cytotoxicity whether it was in the absence or presence of the extracellular palladium source, underlining the precise modulation of bioactivity enabled by this bioorthogonally-activated prodrug strategy.
DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signalling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks. Understanding the upstream signalling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation. Here we demonstrate that human cells have a previously unrecognized signalling mechanism for sensing damage induced by alkylation. We find that the alkylation repair complex ASCC (activating signal cointegrator complex) relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE (coupling of ubiquitin conjugation to ER degradation) domain of the subunit ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RING finger protein 113A (RNF113A) as the E3 ligase responsible for upstream ubiquitin signalling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy, which harbour a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a previously unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked trichothiodystrophy.
Thiiranium salts [Ad2 SR]+ X- (5, 8, 9, 11, 12; X- =Tf2 N- (Tf=CF3 SO2 ), SbCl6 - ) and seleniranium salts [Ad2 SeR]+ X- (14, 16, 17, 23-25; X- =Tf2 N- , BF4 - , CHB11 Cl11 - , SbCl6 - ) are synthesized from strained alkene bis(adamantylidene) (1). The disulfides and the diselenides (Me3 SiCH2 CH2 E)2 (4, 13), (tBuMe2 SiCH2 CH2 E)2 (7, 22), and (NCCH2 CH2 E)2 (10, 15; E=S, Se) have been used. The thiirenium salts [tBu2 C2 SR]+ X- (34) and [Ad2 C2 SR]+ X- (35, 36) are prepared from the bis-tert-butylacetylene (2) and bis-adamantyl-acetylene (3) with R=Me3 SiCH2 CH2 and tBuMe2 SiCH2 CH2 . Attempts to cleave off the groups Me3 SiCH2 CH2 , tBuMe2 SiCH2 CH2 , and NCCH2 CH2 resulted in thiiranes 27, 30. No selenirane Ad2 Se (33) is formed from seleniranium salts, instead cleavage to the alkene (1) and diselenide (13, 15) occurs. The thiirenium salt [Ad2 C2 SCH2 CH2 SiMe3 ]+ Tf2 N- (35) does not yield the thiirene Ad2 C2 S (37), the three-membered ring is cleaved, forming the alkyne (3) and disulfide (4). All compounds are characterized by ESI mass spectra, NMR spectra, and by quantum chemical calculations. Crystal structures of the salts 8, 12, 25, 17, 26, 36 and the thiiranes 27, 30 are presented.
Palladium-activated prodrug therapy is an experimental therapeutic approach that relies on the unique chemical properties and biocompatibility of heterogeneous palladium catalysis to enable the spatially-controlled in vivo conversion of a biochemically-stable prodrug into its active form. This strategy, which would allow inducing local activation of systemically administered drug precursors by mediation of an implantable activating device made of Pd(0), has been proposed by our group as a way to reach therapeutic levels of the active drug in the affected tissue/organ while reducing its systemic toxicity. In the seminal study of such an approach, we reported that propargylation of the N1 position of 5-fluorouracil suppressed the drug's cytotoxic properties, showed high stability in cell culture and facilitated the bioorthogonal restoration of the drug's pharmacological activity in the presence of extracellular Pd(0)-functionalized resins. To provide additional insight on the properties of this system, we have investigated different N1-alkynyl derivatives of 5-fluorouracil and shown that the presence of substituents near the triple bond influence negatively on its sensitivity to palladium catalysis under biocompatible conditions. Comparative studies of the N1- vs. the N3-propargyl derivatives of 5-fluorouracil revealed that masking each or both positions equally led to inactive derivatives (>200-fold reduction of cytotoxicity relative to the unmodified drug), whereas the depropargylation process occurred faster at the N1 position than at the N3, thus resulting in greater toxigenic properties in cancer cell culture.
Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C(29) and C(28)) yielding cholesterol (C(27)). The final step of this dealkylation pathway involves desmosterol reductase (DHCR24)-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735). Following PCR-based cloning of the cDNA (1.6 kb) and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD-dependent reaction.Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250 kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation.
Tamoxifen (TAM) is extensively used for the treatment and prevention of breast cancer. Associated with TAM treatment is a two- to eightfold increase in risk of endometrial cancer. To understand the mechanisms associated with this increased risk several pathways for TAM metabolism and DNA adduct formation have been studied. The purpose of this study was to investigate the role of peroxidase enzymes in the metabolism of TAM and its activation to form DNA adducts. Using advanced tandem mass spectrometry we have investigated the peroxidase-mediated metabolism of TAM. Incubation of TAM with horseradish peroxidase (HRP) and H(2)O(2) produced multiple metabolites. Electrospray ionization-MS/MS analysis of the metabolites demonstrated a peak at 301.3m/z with daughter ions at 183.0, 166.9, 128.9, and 120.9m/z, which identified the metabolite as metabolite E (ME). The levels of ME were significantly inhibited by the addition of ascorbic acid to the incubation mixture. Co-incubation of either TAM or ME and DNA with HRP and H(2)O(2) produced three DNA adducts with a RAL of 1.97±0.01×10(-7) and 8.45±2.7×10(-7). Oxidation of ME with MnO(2) produced metabolite E quinone methide (MEQM). Furthermore, incubation of either TAM or ME with HRP and H(2)O(2) resulted in formation of MEQM. Reaction of calf thymus DNA with MEQM produced three DNA adducts with a RAL of 9.8±1.0×10(-7). Rechromatography analyses indicated that DNA adducts 1, 2, and 3 formed in the HRP activation of either TAM or ME were the same as those formed by the chemical reaction of DNA with MEQM. The results of these studies demonstrate that peroxidase enzymes can both metabolize TAM to form the primary metabolite ME and activate ME to a quinone methide intermediate, which reacts with DNA to form adducts. It is possible that peroxidase enzymes or peroxidase-like activity in endometrium could contribute to the formation of DNA damage and genotoxic effects in endometrium after TAM administration.
A bioorthogonal organometallic reaction is a biocompatible transformation undergone by a synthetic material exclusively through the mediation of a non-biotic metal source; a selective process used to label biomolecules and activate probes in biological environs. Here we report the in vitro bioorthogonal generation of 5-fluorouracil from a biologically inert precursor by heterogeneous Pd(0) catalysis. Although independently harmless, combined treatment of 5-fluoro-1-propargyl-uracil and Pd(0)-functionalized resins exhibits comparable antiproliferative properties to the unmodified drug in colorectal and pancreatic cancer cells. Live-cell imaging and immunoassay studies demonstrate that the cytotoxic activity of the prodrug/Pd(0)-resin combination is due to the in situ generation of 5-fluorouracil. Pd(0)-resins can be carefully implanted in the yolk sac of zebrafish embryos and display excellent biocompatibility and local catalytic activity. The in vitro efficacy shown by this masking/activation strategy underlines its potential to develop a bioorthogonally activated prodrug approach and supports further in vivo investigations.
The Mag1 and Tpa1 proteins from budding yeast (Saccharomyces cerevisiae) have both been reported to repair alkylation damage in DNA. Mag1 initiates the base excision repair pathway by removing alkylated bases from DNA, and Tpa1 has been proposed to directly repair alkylated bases as does the prototypical oxidative dealkylase AlkB from Escherichia coli However, we found that in vivo repair of methyl methanesulfonate (MMS)-induced alkylation damage in DNA involves Mag1 but not Tpa1. We observed that yeast strains without tpa1 are no more sensitive to MMS than WT yeast, whereas mag1-deficient yeast are ∼500-fold more sensitive to MMS. We therefore investigated the substrate specificity of Mag1 and found that it excises alkylated bases that are known AlkB substrates. In contrast, purified recombinant Tpa1 did not repair these alkylated DNA substrates, but it did exhibit the prolyl hydroxylase activity that has also been ascribed to it. A comparison of several of the kinetic parameters of Mag1 and its E. coli homolog AlkA revealed that Mag1 catalyzes base excision from known AlkB substrates with greater efficiency than does AlkA, consistent with an expanded role of yeast Mag1 in repair of alkylation damage. Our results challenge the proposal that Tpa1 directly functions in DNA repair and suggest that Mag1-initiated base excision repair compensates for the absence of oxidative dealkylation of alkylated nucleobases in budding yeast. This expanded role of Mag1, as compared with alkylation repair glycosylases in other organisms, could explain the extreme sensitivity of Mag1-deficient S. cerevisiae toward alkylation damage.
The oxidative C-H bond activation mediated by heme and nonheme enzymes and related biomimetics is one of the most interesting processes in bioinorganic and oxidative chemistry. However, the mechanisms of these reactions are still elusive and controversy due to the involvement of highly reactive metal-oxo intermediates with multiple spin states, despite extensive experimental efforts, especially for the N-dealkylation of N,N-dialkyalinines. In this work, we employed multistate density functional theory (MSDFT) and the Kohn-Sham DFT to investigate the mechanism of N-demethylation of N,N-dimethyalinines oxidized by the reaction intermediate FeIV(O)(N4Py)(ClO4)2. The Kohn-Sham DFT study demonstrated that the reaction proceeds via a rate-limiting hydrogen atom transfer (HAT) step and a subsequent barrier-free oxygen rebound step to form the carbinol product. The MSDFT investigation on the first C-H activation further showed that this step is an initial hydrogen atom abstraction that is highly correlated between CEPT and HAT, i.e., both CEPT and HAT processes make significant contributions to the mechanism before reaching the diabatic crossing point, then the valence bond character of the adiabatic ground state is switched to the CEPT product configuration. The findings from this work may be applicable to other hydrogen abstraction process.
Cytochrome P450 enzymes catalyze a wide range of reactions in plant metabolism. Besides their physiological functions on primary and secondary metabolites, P450s are also involved in herbicide detoxification via hydroxylation or dealkylation. Ginseng as a perennial plant offers more sustainable solutions to herbicide resistance.
Presence of the antidepressant fluoxetine in different water bodies has raised significant concerns due to its detrimental effects on non-targeted organisms, especially on fish. When seeking for an appropriate technology able to remove fluoxetine residue from a complex water matrix, special attention needs to be paid to the elimination of the neurophysiological activity that eventually lies behind the noxious effects of the parent compound. Our aim was to probe the applicability of advanced oxidation techniques for this purpose using in situ generated free radical system based on OH-initiated peroxyl radical-mediated processes. By performing product analysis experiments along with quantum chemical calculations, the most probable reaction paths were analyzed including aromatic hydroxylation, defluorination, O-dealkylation and C-dealkylation. The candidates for neurophysiological activity were further investigated by molecular docking. The hydroxylated derivatives are well accommodated in the binding pocket of the corresponding protein, suggesting that these compounds may retain the activity of the parent compound. From a worst-case perspective, we suggest that prolonged treatment needs to be applied to further transform hydroxylated derivatives.
1. We investigated how microsomal cytochrome P450 mono-oxygenase (Cyp450 MO) is regulated in cultured porcine aortic endothelial cells. The hypothesis that a Cyp450 MO-derived metabolite links Ca2+ store depletion and Ca2+ entry was studied further. 2. Microsomal Cyp450 MO was monitored fluorometrically by dealkylation of 1-ethoxypyrene-3,6,8-tris-(dimethyl-sulphonamide; EPSA) in saponin permeabilized cells or in subcellular compartments. Endothelial Ca2+ signalling was measured by a standard fura-2 technique, membrane potential was determined with the potential-sensitive fluorescence dye, bis-(1,3-dibutylbarbituric acid) pentamethine oxonol (DiBAC4(5)) and tyrosine kinase was quantified by measuring the phosphorylation of a immobilized substrate with a horseradish peroxidase labelled phosphotyrosine specific antibody. 3. Depletion of cellular Ca2+ pools with inositol 1,4,5-trisphosphate (IP3), thapsigargin or cyclopiazonic acid activated microsomal Cyp450 MO. Similar to direct Ca2+ store depletion, chelating of intramicrosomal Ca2+ with oxalate stimulated Cyp450 MO activity, while changing cytosolic free Ca2+ failed to influence Cyp450 MO activity. These data indicate that microsomal Cyp450 MO is activated by depletion of IP3-sensitive stores. 4. Besides the common cytochrome P450 inhibitors, econazole, proadifen and miconazole, thiopentone sodium and methohexitone inhibited Cyp450 MO in a concentration-dependent manner. The physiological substrate of Cyp450 MO, arachidonic acid, inhibited EPSA dealkylation. In contrast to most other cytochrome P450 inhibitors used in this study, thiopentone sodium did not directly interfere with Ca2+ entry pathways, membrane hyperpolarization due to K+ channel activation or tyrosine kinase activity. 5. Inhibition of Cyp450 MO by thiopentone sodium diminished Ca2+/Mn2+ entry to Ca2+ store depletion by 43%, while it did not interfere with intracellular Ca2+ release by IP3 or thapsigargin. 6. Cyp450 MO inhibition with thiopentone sodium diminished autacoid-induced membrane hyperpolarization. 7. Induction of Cyp450 MO with dexamethasone/clofibrate for 72 h yielded increases in thapsigargin-induced Cyp450 MO activity (by 35%), Ca2+/Mn2+ entry (by 105%) and membrane hyperpolarization (by 40%). 8. The Cyp450 MO-derived compounds, 11,12 and 5,6-epoxyeicosatrienoic acids (EETs) yielded membrane hyperpolarization, insensitive to thiopentone sodium. 9. These data demonstrate that endothelial Cyp450 MO is activated by Ca2+ store depletion and Cyp450 MO produced compounds that hyperpolarize endothelial cells. 10. The data presented and our previous findings indicate that Cyp450 MO plays a crucial role in the regulation of store-operated Ca2+ influx. We propose that Cyp450 MO-derived EETs constitute a signal for Ca2+ entry activation and increase the driving force for Ca2+ entry by membrane hyperpolarization in porcine aortic endothelial cells.
Fenebrutinib is an orally available Bruton tyrosine kinase inhibitor. It is currently in multiple phase III clinical trials for the management of B-cell tumors and autoimmune disorders. Elementary in-silico studies were first performed to predict susceptible sites of metabolism and structural alerts for toxicities by StarDrop WhichP450™ module and DEREK software; respectively. Fenebrutinib metabolites and adducts were characterized in-vitro in rat liver microsomes (RLM) using MS3 method in Ion Trap LC-MS/MS. Formation of reactive and unstable intermediates was explored using potassium cyanide (KCN), glutathione (GSH) and methoxylamine as trapping nucleophiles to capture the transient and unstable iminium, 6-iminopyridin-3(6H)-one and aldehyde intermediates, respectively, to generate a stable adducts that can be investigated and analyzed using mass spectrometry. Ten phase I metabolites, four cyanide adducts, five GSH adducts and six methoxylamine adducts of fenebrutinib were identified. The proposed metabolic reactions involved in formation of these metabolites are hydroxylation, oxidation of primary alcohol to aldehyde, n-oxidation, and n-dealkylation. The mechanism of reactive intermediate formation of fenebrutinib can provide a justification of the cause of its adverse effects. Formation of iminium, iminoquinone and aldehyde intermediates of fenebrutinib was characterized. N-dealkylation followed by hydroxylation of the piperazine ring is proposed to cause the bioactivation to iminium intermediates captured by cyanide. Oxidation of the hydroxymethyl group on the pyridine moiety is proposed to cause the generation of reactive aldehyde intermediates captures by methoxylamine. N-dealkylation and hydroxylation of the pyridine ring is proposed to cause formation of iminoquinone reactive intermediates captured by glutathione. FBB and several phase I metabolites are bioactivated to fifteen reactive intermediates which might be the cause of adverse effects. In the future, drug discovery experiments utilizing this information could be performed, permitting the synthesis of new drugs with better safety profile. Overall, in silico software and in vitro metabolic incubation experiments were able to characterize the FBB metabolites and reactive intermediates using the multistep fragmentation capability of ion trap mass spectrometry.
During the past decades, there have been continuous attempts in the prediction of metabolism mediated by cytochrome P450s (CYP450s) 3A4, 2D6, and 2C9. However, it has indeed remained a huge challenge to accurately predict the metabolism of xenobiotics mediated by these enzymes. To address this issue, microsomal metabolic reaction system (MMRS)-a novel concept, which integrates information about site of metabolism (SOM) and enzyme-was introduced. By incorporating the use of multiple feature selection (FS) techniques (ChiSquared (CHI), InfoGain (IG), GainRatio (GR), Relief) and hybrid classification procedures (Kstar, Bayes (BN), K-nearest neighbours (IBK), C4.5 decision tree (J48), RandomForest (RF), Support vector machines (SVM), AdaBoostM1, Bagging), metabolism prediction models were established based on metabolism data released by Sheridan et al. Four major biotransformations, including aliphatic C-hydroxylation, aromatic C-hydroxylation, N-dealkylation and O-dealkylation, were involved. For validation, the overall accuracies of all four biotransformations exceeded 0.95. For receiver operating characteristic (ROC) analysis, each of these models gave a significant area under curve (AUC) value >0.98. In addition, an external test was performed based on dataset published previously. As a result, 87.7% of the potential SOMs were correctly identified by our four models. In summary, four MMRS-based models were established, which can be used to predict the metabolism mediated by CYP3A4, 2D6, and 2C9 with high accuracy.
Recently, a number of fentanyl analogs have been implicated in overdose deaths in Europe and in the US. So far, little is known of the molecular behavior of the structurally related subgroup; the alicyclic fentanyls. In this study, reference standards of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and 2,2,3,3-tetramethylcyclopropyl fentanyl (TMCPF) at a final concentration of 5 µM were incubated with cryopreserved human hepatocytes (1 × 106 cells/mL) for 0, 1, 3 and 5 h. The metabolites formed were identified by liquid chromatography-quadrupole time-of-flight mass spectrometry analysis. The most abundant biotransformation found was N-dealkylation (formation of normetabolites) and oxidation of the alicyclic rings. As ring size increased, the significance of N-dealkylation decreased in favor of alicyclic ring oxidation. An example of this was cyclopropyl fentanyl, with a three-carbon ring, whose normetabolite covered 82% of the total metabolic peak area and no oxidation of the alicyclic ring was observed. In contrast, TMCPF, with a seven-carbon ring structure, rendered as much as 85% of its metabolites oxidized on the alicyclic ring. Other biotransformations found included oxidation of the piperidine ethyl moiety and/or the phenethyl substructure, glucuronidation as well as amide hydrolysis to form metabolites identical to despropionyl fentanyl. Taken together, this study provides a base for understanding the metabolism of a number of structurally related fentanyl analogs formed upon intake.
Metoprolol is used for phenotyping of cytochrome P450 (CYP) 2D6, a CYP isoform considered not to be inducible by inducers of the CYP2C, CYP2B, and CYP3A families such as rifampicin. While assessing CYP2D6 activity under basal conditions and after pre-treatment with rifampicin in vivo, we surprisingly observed a drop in the metoprolol/α-OH-metoprolol clearance ratio, suggesting CYP2D6 induction. To study this problem, we performed in vitro investigations using HepaRG cells and primary human hepatocytes (before and after treatment with 20 μM rifampicin), human liver microsomes, and CYP3A4-overexpressing supersomes. While mRNA expression levels of CYP3A4 showed a 15- to 30-fold increase in both cell models, mRNA of CYP2D6 was not affected by rifampicin. 1'-OH-midazolam formation (reflecting CYP3A4 activity) increased by a factor of 5-8 in both cell models, while the formation of α-OH-metoprolol increased by a factor of 6 in HepaRG cells and of 1.4 in primary human hepatocytes. Inhibition studies using human liver microsomes showed that CYP3A4, 2B6, and 2C9 together contributed 19.0 ± 2.6% (mean ± 95%CI) to O-demethylation, 4.0 ± 0.7% to α-hydroxylation, and 7.6 ± 1.7% to N-dealkylation of metoprolol. In supersomes overexpressing CYP3A4, metoprolol was α-hydroxylated in a reaction inhibited by the CYP3A4-specific inhibitor ketoconazole, but not by the CYP2D6-specific inhibitor quinidine. We conclude that metoprolol is not exclusively metabolized by CYP2D6. CYP3A4, 2B6, and 2C9, which are inducible by rifampicin, contribute to α-hydroxylation, O-demethylation, and N-dealkylation of metoprolol. This contribution is larger after CYP induction by rifampicin but is too small to compromise the usability of metoprolol α-hydroxylation for CYP2D6 phenotyping.
E. Coli AlkB catalyzes the direct dealkylation of various alkylated bases in damaged DNA. The diffusion of molecular oxygen to the active site in AlkB is an essential step for the oxidative dealkylation activity. Despite detailed studies on the stepwise oxidation mechanism of AlkB, there is no conclusive picture of how O2 molecules reach the active site of the protein. Yu et al. (Nature, 439, 879) proposed the existence of an intra-molecular tunnel based on their initial crystal structures of AlkB. We have employed computational simulations to investigate possible migration pathways inside AlkB for O2 molecules. Extensive molecular dynamics (MD) simulations, including explicit ligand sampling and potential of mean force (PMF) calculations, have been performed to provide a microscopic description of the O2 delivery pathway in AlkB. Analysis of intra-molecular tunnels using the CAVER software indicates two possible pathways for O2 to diffuse into the AlkB active site. Explicit ligand sampling simulations suggests that only one of these tunnels provides a viable route. The free energy path for an oxygen molecule to travel along each of these tunnels has been determined with AMBER and AMOEBA. Both PMFs indicate passive transport of O2 from the surface of the protein. However, the inclusion of explicit polarization shows a very large barrier for diffusion of the co-substrate out of the active site, compared with the non-polarizable potential. In addition, our results suggest that the mutation of a conserved residue along the tunnel, Y178, has dramatic effects on the dynamics of AlkB and on the transport of O2 along the tunnel.
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