A novel microplate-based HPLC-fluorescence assay for determination of NADPH-cytochrome P450 reductase activity

A 96‐well microplate‐based HPLC endpoint assay is described for the determination of NADPH‐cytochrome P450 reductase (CPR) activity. Novel sampling of NADPH into microplates was optimized. Separation was performed on a Zorbax Eclipse XDB‐C₁₈ analytical 4.6 × 150 mm, 5 µm column. To validate the method, recombinant human NADPH‐P450 reductase and microsomes with cytochrome P450 CYP1A1 were used. The mobile phase consisted of 80% acetonitrile and 20% water at a flow‐rate of 0.8 mL/min. The CPR activity was quantified using NADPH fluorescence at λ_Ex = 340 nm and λ_Em = 450 nm. Enzymatic activity was directly proportional to the decrease in NADPH fluorescence. This analytical process enables a highly sensitive endpoint determination for reductase activity in vitro and monitoring of the consumption of NADPH in enzymatic reactions. The method avoids the use of substrates and of organic solvents that may affect CPR and cytochrome P450 activity. In the reaction, molecular oxygen served as a proton source. The method can substitute spectrophotometric detection methods for its accuracy, high reproducibility (~100%) and sensitivity. The lower limit of detection, shown using the Agilent 1200 aparatus, is in the 250 nmol range. In addition, using this method it is possible to set up reactions in a high‐throughput format. Copyright © 2011 John Wiley & Sons, Ltd.     

Enzyme–Substrate Complementarity Governs Access to a Cationic Reaction Manifold in the P450BM3‐Catalysed Oxidation of Cyclopropyl Fatty Acids

The products of cytochrome P450_BM3‐catalysed oxidation of cyclopropyl‐containing dodecanoic acids are consistent with the presence of a cationic reaction intermediate, which results in efficient dehydrogenation of the rearranged probes by the enzyme. These results highlight the importance of enzyme–substrate complementarity, with a cationic intermediate occurring only when the probes used begin to diverge from ideal substrates for this enzyme. This also aids in reconciling literature reports supporting the presence of cationic intermediates with certain cytochrome P450 enzyme/substrate pairs.     

Dual‐Functional Small Molecules for Generating an Efficient Cytochrome P450BM3 Peroxygenase

We report a unique strategy for the development of a H₂O₂‐dependent cytochrome P450BM3 system, which catalyzes the monooxygenation of non‐native substrates with the assistance of dual‐functional small molecules (DFSMs), such as N‐(ω‐imidazolyl fatty acyl)‐l ‐amino acids. The acyl amino acid group of DFSM is responsible for bounding to enzyme as an anchoring group, while the imidazolyl group plays the role of general acid–base catalyst in the activation of H₂O₂. This system affords the best peroxygenase activity for the epoxidation of styrene, sulfoxidation of thioanisole, and hydroxylation of ethylbenzene among those P450–H₂O₂ system previously reported. This work provides the first example of the activation of the normally H₂O₂‐inert P450s through the introduction of an exogenous small molecule. This approach improves the potential use of P450s in organic synthesis as it avoids the expensive consumption of the reduced nicotinamide cofactor NAD(P)H and its dependent electron transport system. This introduces a promising approach for exploiting enzyme activity and function based on direct chemical intervention in the catalytic process.     

Selective Enzymatic Oxidation of Silanes to Silanols

Compared to the biological world's rich chemistry for functionalizing carbon, enzymatic transformations of the heavier homologue silicon are rare. We report that a wild‐type cytochrome P450 monooxygenase (P450_BM3 from Bacillus megaterium, CYP102A1) has promiscuous activity for oxidation of hydrosilanes to give silanols. Directed evolution was applied to enhance this non‐native activity and create a highly efficient catalyst for selective silane oxidation under mild conditions with oxygen as the terminal oxidant. The evolved enzyme leaves C?H bonds present in the silane substrates untouched, and this biotransformation does not lead to disiloxane formation, a common problem in silanol syntheses. Computational studies reveal that catalysis proceeds through hydrogen atom abstraction followed by radical rebound, as observed in the native C?H hydroxylation mechanism of the P450 enzyme. This enzymatic silane oxidation extends nature's impressive catalytic repertoire.     

Asymmetric Enzymatic Synthesis of Allylic Amines: A Sigmatropic Rearrangement Strategy

Sigmatropic rearrangements, while rare in biology, offer opportunities for the efficient and selective synthesis of complex chemical motifs. A “P411” serine‐ligated variant of cytochrome P450_BM3 has been engineered to initiate a sulfimidation/[2,3]‐sigmatropic rearrangement sequence in whole E. coli cells, a non‐natural function for any enzyme, providing access to enantioenriched, protected allylic amines. Five mutations in the enzyme substantially enhance its activity toward this new function, demonstrating the evolvability of the catalyst toward challenging nitrene transfer reactions. The evolved catalyst additionally performs the highly enantioselective imidation of non‐allylic sulfides.     

Engineering of P450pyr Hydroxylase for the Highly Regio‐ and Enantioselective Subterminal Hydroxylation of Alkanes

Terminal‐selective cytochrome P450pyr has been successfully engineered through directed evolution for the subterminal hydroxylation of alkanes with excellent regio‐ and enantioselectivity. A sensitive colorimetric high‐throughput screening (HTS) assay was developed for the measurement of both the regioselectivity and the enantioselectivity of a hydroxylation reaction. By using the HTS assay and iterative saturation mutagenesis, sextuple‐mutant P450pyrSM1 was created for the hydroxylation of n‐octane ( 1 ) to give (S)‐2‐octanol ( 2 ) with 98 % ee and >99 % subterminal selectivity. The engineered P450 is the first enzyme for this type of highly selective alkane hydroxylation, being useful for the C? H activation and functionalization of alkanes and the preparation of enantiopure alcohols. Molecular modeling provided structure‐based understanding of the fully altered regioselectivity and the excellent enantioselectivity. Another sextuple‐mutant P450pyrSM2 catalyzed the hydroxylation of propylbenzene ( 3 ) to afford (S)‐1‐phenyl‐2‐propanol ( 4 ) with 95 % ee and 98 % subterminal selectivity.     

Reconstitution of the Cytb5–CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy

Cytochrome P450s (P450s) are a superfamily of enzymes responsible for the catalysis of a wide range of substrates. Dynamic interactions between full‐length membrane‐bound P450 and its redox partner cytochrome b₅ (cytb₅) have been found to be important for the enzymatic activity of P450. However, the stability of the circa 70 kDa membrane‐bound complex in model membranes renders high‐resolution structural NMR studies particularly difficult. To overcome these challenges, reconstitution of the P450–cytb₅ complex in peptide‐based nanodiscs, containing no detergents, has been demonstrated, which are characterized by size exclusion chromatography and NMR spectroscopy. In addition, NMR experiments are used to identify the binding interface of the P450–cytb₅ complex in the nanodisc. This is the first successful demonstration of a protein–protein complex in a nanodisc using NMR structural studies and should be useful to obtain valuable structural information on membrane‐bound protein complexes.     

Improved Cyclopropanation Activity of Histidine‐Ligated Cytochrome P450 Enables the Enantioselective Formal Synthesis of Levomilnacipran

Engineering enzymes capable of modes of activation unprecedented in nature will increase the range of industrially important molecules that can be synthesized through biocatalysis. However, low activity for a new function is often a limitation in adopting enzymes for preparative‐scale synthesis, reaction with demanding substrates, or when a natural substrate is also present. By mutating the proximal ligand and other key active‐site residues of the cytochrome P450 enzyme from Bacillus megaterium (P450‐BM3), a highly active His‐ligated variant of P450‐BM3 that can be employed for the enantioselective synthesis of the levomilnacipran core was engineered. This enzyme, BM3‐Hstar, catalyzes the cyclopropanation of N,N‐diethyl‐2‐phenylacrylamide with an estimated initial rate of over 1000 turnovers per minute and can be used under aerobic conditions. Cyclopropanation activity is highly dependent on the electronic properties of the P450 proximal ligand, which can be used to tune this non‐natural enzyme activity.     

The relationship between the redox reaction of camphor‐induced cytochrome p‐450 and its activity

The relationship between the redox reaction of camphor‐induced cytochrome P‐450 (P‐450_cam) and its activity was measured by using cyclic voltammetry. The redox potential of P‐450_cam solution shifted to the lower side of the potential by binding of substrate, and the change was proportional to the amount of the substrate binding to the protein. The substrate binding was inhibited at the low concentration of oxygen in the reaction solution. The reaction product, hydroxycamphor, was observed in the reaction mixture by gas chromatography/mass spectroscopy. However, hydroxycamphor was not observed at an oxygen concentration of about a tenth part of the saturated one. The shift of redox potential of P‐450_cam solution corresponded to the substrate specificity of the activity. These results suggest that the redox reaction of P‐450_cam related to the substrate‐binding to the protein and its activity. Furthermore, the present system was very simple and speedy for the measurement of the activity. Copyright © 1999 John Wiley & Sons, Ltd.     

Influence of Moringa oleifera on pharmacokinetic disposition of rifampicin using HPLC‐PDA method: a pre‐clinical study

The influence of active fraction isolated from pods of an indigenous plant, Moringa oleifera (MoAF) was studied on the pharmacokinetic profile of the orally administered frontline anti‐tuberculosis drug rifampicin (20 mg/kg b.w.) in Swiss albino mice. The antibiotic rifampicin alone and in combination with MoAF (0.1 mg/kg b.w.) was administered orally and heparanized blood samples were collected from the orbital plexus of mice for plasma separation at 0, 1, 2, 3, 4 and 5 h, post treatment. Plasma rifampicin concentration, pharmacokinetic parameters and drug metabolizing enzyme (cytochrome P‐450) activity were determined. The pharmacokinetic data revealed that MoAF‐treated animals had significantly increased rifampicin plasma concentration, C_max, K_el, t_½(a), t_½(el), K_a and AUC as well as inhibited rifampicin‐induced cytochrome P‐450 activity. In conclusion, the result of this study suggested that the bioavailability‐enhancing property of MoAF may help to lower the dosage level and shorten the treatment course of rifampicin. Copyright © 2010 John Wiley & Sons, Ltd.     

A high‐throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography–tandem mass spectrometry

A sensitive and high‐throughput inhibition screening liquid chromatography–mass spectrometry (LC‐MS/MS) method was developed and validated for the simultaneous quantification of five probe metabolites (7‐hydroxycoumarin, CYP2A6; 4‐hydroxytolbutamide, CYP2C9; 4′‐hydroxymephenytoin, CYP2C19; α‐hydroxymetoprolol, CYP2D6; and 1‐hydroxymidazolam, CYP3A4) for in vitro cytochrome P450 activity determination in human liver microsome and recombinant. All the metabolites and the internal standard, tramadol, were separated on a Waters 2695 series liquid chromatograph with a Phenomenex Luna C₁₈ column (150 × 2.0 mm, 5 µm). Quality control samples and a positive control CYP inhibitor were included in the method. The IC₅₀ values determined for typical CYP inhibitors were reproducible and in agreement with the literature. The method was selective and showed good accuracy (99.13–103.37%), and inter‐day (RSD < 6.20%) and intra‐day (RSD < 6.13%) precision. Also, the incubation extracts of the sample were stable at room temperature (20 °C) for 48 h and for 96 h in the autosampler (4 °C). The presented method is the first HPLC‐MS/MS method of this combination for simultaneous detection of the five metabolites 7‐hydroxycoumarin, 4‐hydroxytolbutamide, 4′‐hydroxymephenytoin, α‐hydroxymetoprolol and 1‐hydroxymidazolam in a single‐run process. It is possible that the high‐quality and ‐throughput cocktail provides suitable information in drug discovery and screening for new drug entities. Copyright © 2013 John Wiley & Sons, Ltd.     

Selective Enzymatic Oxidation of Silanes to Silanols

Compared to the biological world's rich chemistry for functionalizing carbon, enzymatic transformations of the heavier homologue silicon are rare. We report that a wild-type cytochrome P450 monooxygenase (P450_BM3 from Bacillus megaterium, CYP102A1) has promiscuous activity for oxidation of hydrosilanes to give silanols. Directed evolution was applied to enhance this non-native activity and create a highly efficient catalyst for selective silane oxidation under mild conditions with oxygen as the terminal oxidant. The evolved enzyme leaves C−H bonds present in the silane substrates untouched, and this biotransformation does not lead to disiloxane formation, a common problem in silanol syntheses. Computational studies reveal that catalysis proceeds through hydrogen atom abstraction followed by radical rebound, as observed in the native C−H hydroxylation mechanism of the P450 enzyme. This enzymatic silane oxidation extends nature's impressive catalytic repertoire.     

The Fe‐N‐C Nanozyme with Both Accelerated and Inhibited Biocatalytic Activities Capable of Accessing Drug–Drug Interactions

Emerging as a cost‐effective and robust enzyme mimic, nanozymes have drawn increasing attention with broad applications ranging from cancer therapy to biosensing. Developing nanozymes with both accelerated and inhibited biocatalytic properties in a biological context is intriguing to peruse more advanced functions of natural enzymes, but remains challenging, because most nanozymes are lack of enzyme‐like molecular structures. By re‐visiting and engineering the well‐known Fe‐N‐C electrocatalyst that has a heme‐like Fe‐N_x active sites, herein, it is reported that Fe‐N‐C could not only catalyze drug metabolization but also had inhibition behaviors similar to cytochrome P450 (CYP), endowing it a potential replacement of CYP for preliminary evaluation of massive potential chemicals, drug dosing guide, and outcome prediction. In addition, in contrast to electrocatalysts, the highly graphitic framework of Fe‐N‐C may not be obligatory for a competitive CYP‐like activity.     

Electrochemistry of Cytochrome P450 2B6 on Electrodes Modified with Zirconium Dioxide Nanoparticles and Platin Components

The direct electrochemical and electrocatalytic behavior of the immobilized cytochrome P450 2B6 (CYP2B6) on zirconium dioxide nanoparticles (ZrO₂) was investigated. The film of nano‐structured ZrO₂ that incorporated cytochrome P450 2B6 (CYP2B6) with colloidal paltin, which was stabilized by poly‐lysine (Pt‐PLL), was prepared on glassy carbon electrodes. In anaerobic solutions, the immobilized CYP2B6 exhibited a reversible electron transfer between the heme electroactive center of CYP2B6 and electrodes with a formal potential of ?(0.449±0.004) V at pH 7.4. In air‐saturated solutions, an increased bioelectrocatalytic reduction current could be obtained with the CYP2B6‐modified electrode with the addition of anticancer drugs, such as lidocaine. This leads to the construction of disposable biosensors for drugs by utilizing the electrochemical activity and catalytic reactions of the immobilized CYP2B6.     

A rapid ultra‐performance liquid chromatography/tandem mass spectrometric methodology for the in vitro analysis of Pooled and Cocktail cytochrome P450 assays

Drug‐drug interaction evaluations of new pharmaceutical candidates are critical to preventing drug withdrawal and are routinely determined through the use of cytochrome P450 assays. The measurement of the effect of test compounds on the metabolism of known substrates allows for the determination of specific CYP450 isoenzyme inhibition and calculation of IC50 values. A sensitive, high‐throughput ultra‐performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method is presented for the evaluation of CYP450 inhibition. The assay was performed using a cocktail of probe substrates and the results were compared to those obtained with the more time‐consuming methodology utilizing individual substrates. The use of a high‐resolution, sub‐2 µm particle, LC system allowed for a high‐throughput assay of just 1 min. The extra resolution of the UPLC/MS/MS system allowed for the complete resolution of the analytes, with a fast switching MS for comprehensive data collection. The CYP450 inhibition results obtained using the substrate cocktail approach were found to be essentially identical to those obtained using individual substrates. Copyright © 2009 John Wiley & Sons, Ltd.     

A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs

Structural interactions that enable electron transfer to cytochrome‐P450 (CYP450) from its redox partner CYP450‐reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane‐bound functional complex to reveal interactions between the full‐length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome‐b₅ (cyt‐b₅), Arg 125 on the C‐helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein–protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.     

Rapid and accurate liquid chromatography and tandem mass spectrometry method for the simultaneous quantification of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes

The hepatic cytochrome P450 enzymes play a central role in the biotransformation of endogenous and exogenous substances. A sensitive high‐throughput liquid chromatography with tandem mass spectrometry assay was developed and validated for the simultaneous quantification of the products of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes. After the substrates were incubated separately, the samples were pooled and analyzed by liquid chromatography with tandem mass spectrometry using an electrospray ionization source in the positive and negative ion modes. The method exhibited linearity over a broad concentration range, insensitivity to matrix effects, and high accuracy, precision, and stability. The novel method was successfully applied to study the kinetics of phenacetin‐O deethylation, coumarin‐7 hydroxylation, bupropion hydroxylation, taxol‐6 hydroxylation, omeprazole‐5 hydroxylation, dextromethorphan‐O demethylation, tolbutamide‐4 hydroxylation, chlorzoxazone‐6 hydroxylation, testosterone‐6β hydroxylation, and midazolam‐1 hydroxylation in rat liver microsomes.     

Cytochrome P450 reaction phenotyping and inhibition and induction studies of pinostrobin in human liver microsomes and hepatocytes

Pinostrobin (PI, 5‐hydroxy‐7‐methoxyflavanone) is a natural flavonoid known for its rich pharmacological activities. The objective of this study was to identify the human liver cytochrome P450 (CYP450) isoenzymes involved in the metabolism of PI. A single hydoxylated metabolite was obtained from PI after an incubation with pooled human liver microsomes (HLMs). The relative contributions of different CYP450s were evaluated using CYP450‐selective inhibitors in HLMs and recombinant human CYP450 enzymes, and the results revealed the major involvement of CYP1A2, CYP2C9 and CYP2E1 in PI metabolism. We also evaluated the ability of PI to inhibit and induce human cytochrome P450 enzymes in vitro . High‐performance liquid chromatography and liquid chromatography–tandem mass spectrometry analytical techniques were used to estimate the enzymatic activities of seven drug‐metabolizing CYP450 isozymes in vitro . In HLMs, PI did not inhibit CYP 1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 (IC₅₀ > 100 μm ). In the induction studies, PI had minimal effects on CYP1A2, CYP2B6and CYP3A4 activity. Based on these results, PI would not be expected to cause clinically significant CYP450 inhibition or induction.     

Hydrogen‐Bonding Interactions Trigger a Spin‐Flip in Iron(III) Porphyrin Complexes

A key step in cytochrome P450 catalysis includes the spin‐state crossing from low spin to high spin upon substrate binding and subsequent reduction of the heme. Clearly, a weak perturbation in P450 enzymes triggers a spin‐state crossing. However, the origin of the process whereby enzymes reorganize their active site through external perturbations, such as hydrogen bonding, is still poorly understood. We have thus studied the impact of hydrogen‐bonding interactions on the electronic structure of a five‐coordinate iron(III) octaethyltetraarylporphyrin chloride. The spin state of the metal was found to switch reversibly between high (S=⁵/₂) and intermediate spin (S=³/₂) with hydrogen bonding. Our study highlights the possible effects and importance of hydrogen‐bonding interactions in heme proteins. This is the first example of a synthetic iron(III) complex that can reversibly change its spin state between a high and an intermediate state through weak external perturbations.     

Mechanistic studies of oxene reactions with organic substrates: Reaction paths on MNDO enthalpy surfaces—models for cytochrome P450 oxidations

In a systematic study, the characteristics of triplet oxene models for alkane, alkene, chloroalkane, and aryl oxidations by the cytochrome P450s have been examined using the semiempirical molecular orbital method MNDO and the formalism of statistical mechanics. Specific model substrates chosen were: methane, ethylene, propene, carbon tetrachloride, chloroform, and toluene. It was found that transition state geometries and activation entropies were reliably predicted, but that an empirical factor was necessary to correct overestimation of activation enthalpies. It was determined that both hydroxylations and epoxidation initiated by a O(³P) atom are nonconcerted; and that oxidations of C? Cl bonds (halosylations) occur by a two-step mechanism similar to hydroxylation. It is shown that the radical mechanisms derived from these studies are consistent with a range of observed properties of cytochrome P450 reactions and provide reasonable explanations for secondary deuterium isotope effects and substituent effects in cytochrome P450 epoxidation of styrenes, suicide inactivation of a P450 enzyme by ethylene, and the characteristics of aerobic CCl₄ and CHCl₃ metabolism. A triplet oxene mechanism for the initial steps of aromatic epoxidation and hydroxylation is also discussed.