Novel hierarchical classification and visualization method for multiobjective optimization of drug properties: application to structure-activity relationship analysis of cytochrome P450 metabolism

In the lead optimization process, medicinal chemists must consider various chemical properties of active compounds, including ADME/Tox properties, and find the best compromise among these. This study presents a novel data mining method for multiobjective optimization of chemical properties, which consists of the hierarchical classification and visualization of multidimensional data. A hierarchical classification tree model is generated by an extension of recursive partitioning that utilizes averaged information gains for multiple objective variables as a quality-of-split criterion. All the hierarchically structured data objects are represented using a large-scale data visualization technique. The technique is an extension of HeiankyoView, which displays data objects as colored icons and group nodes as rectangular borders. Each icon is divided into subregions with different colors, so that it can present multidimensional data according to brightness of the colors. The proposed method was applied to the structure-activity relationship analysis for cytochrome P450 (CYP) substrates. The substrate specificity of six CYP isoforms was successfully delineated: e.g., CYP2C9 substrates are anionic compounds, while CYP2D6 substrates are cationic; and CYP2E1 substrates are smaller compounds, while CYP3A4 substrates are larger compounds.     

Modeling of complexes between cytochrome P450 1A2 and substrates

Based on the 2HI4 complex taken from the PDB database, molecular docking of 17 substrates in the cytochrome P450 1A2 cavity is performed using the 3D-QSAR algorithm CiS. The arrangement of substrate molecules in the 1A2 isoform cavity is considered, the orientation of the molecular reaction centers relative to heme is analyzed, and the character of interaction between substrates and active site aminoacid residues is investigated. The structures of the modeled complexes allow us to explain metabolic pathways in demethylation reactions and some reactions of hydroxylation, which enables an application of the CiS algorithm to predict metabolic pathways.     

Investigations into the analysis and modeling of the cytochrome P450 cycle

The main focus of our research is to explore the fundamental dynamics of the mechanism of the cytochrome P450 (CYP450) cycle. For this purpose we propose a system-theoretical approach, a time-dependent metabolic control analysis (tdMCA), to the analysis and quantitative modeling of the CYP450 catalytic pathway. This provides theoretical enlightenment for us to assess the transient response of the system to perturbations. In addition, the robustness of the cycle has also been observed, where perturbations elicit very weak responses and the system quickly recovers to the steady state (in an average of 10(-5) s). The tdMCA also shows that the two electron transfers to the cycle have different impacts on the system, and the cycle is more sensitive to the first electron than to the second one. Knowing the dynamics of transient fluctuations, the robustness of the cycle, and the effects from the key interim steps, one has a deeper understanding of the catalytic mechanism of cytochrome P450.     

Engineering and assaying of cytochrome P450 biocatalysts

Cytochrome P450s constitute a highly fascinating superfamily of enzymes which catalyze a broad range of reactions. They are essential for drug metabolism and promise industrial applications in biotechnology and biosensing. The constant search for cytochrome P450 enzymes with enhanced catalytic performances has generated a large body of research. This review will concentrate on two key aspects related to the identification and improvement of cytochrome P450 biocatalysts, namely the engineering and assaying of these enzymes. To this end, recent advances in cytochrome P450 development are reported and commonly used screening methods are surveyed.     

细胞色素P450的电化学研究进展

细胞色素P450的电化学研究从一个侧面反映了为使细胞色素P450达到工业催化剂的最终目的人们所作的不懈努力。本文从细胞色素P450在电极上的电子转移研究,隧道扫描显微镜的微观成像研究和使用电极作为细胞色素P450的电子给体从而实现细胞色素P450底物转化三方面,评述了近年来细胞色素P450的电化学研究进展。     

Application of a generic fast gradient liquid chromatography tandem mass spectrometry method for the analysis of cytochrome P450 probe substrates

A liquid chromatography tandem mass spectrometry (LC/MS/MS) method has been developed for the fast routine analysis of selected CYP450 probe substrate metabolites in microsomal incubations, with no sample pretreatment. This has allowed fast and simple assessment of the potential effects which drug candidates may or may not have on the metabolism of specific CYP450 probe substrates, providing information which can then be used to rationalize in vivo interaction studies required in the clinic. This methodology takes advantage of fast gradient chromatography as a generic means of sample separation and analysis. It provides high throughput analysis compared to conventional gradient HPLC, with no significant loss in chromatographic performance. © 1998 John Wiley & Sons, Ltd.     

Intracellular transport and localization of microsomal cytochrome P450

The cytochrome P450 (P450) enzymes are mainly localized to the endoplasmic reticulum (ER), where they function within catalytic complexes metabolizing xenobiotics and some endogenous substrates. However, certain members of families 1–3 were also found in other subcellular compartments, such as mitochondria, plasma membrane, and lysosomes. The physiological function of these enzymes in non-ER locations is not known, although plasma-membrane-associated P450s have been described to be catalytically active and to participate in immune-mediated reactions with autoantibody formation that can trigger drug-induced hepatitis. Several retention/retrieval mechanisms are active in the ER retention of the P450s and inverse integration of the translated P450 into the ER membrane appears to be responsible for transport to the plasma membrane. Furthermore, hydrophilic motifs in the NH₂-terminal part have been suggested to be important for mitochondrial import. Phosphorylation of P450s has been described to be important for increased rate of degradation as well as for targeting into mitochondria. It was also suggested that the mitochondria-targeted P450s from families 1–3 could be active in drug metabolism using an alternative electron transport chain. In this review we present an update of the field emphasizing studies concerning localization, posttranslational modification, such as phosphorylation, and intracellular transport of microsomal P450s.     

An evaluation of molecular models of the cytochrome P450 Streptomyces griseolus enzymes P450SU1 and P450SU2

Summary P450SU1 and P450SU2 are herbicide-inducible bacterial cytochrome P450 enzymes from Streptomyces griseolus. They have two of the highest sequence identities to camphor hydroxylase (P450cam from Pseudomonas putida), the cytochrome P450 with the first known crystal structure. We have built several models of these two proteins to investigate the variability in the structures that can occur from using different modeling protocols. We looked at variability due to alignment methods, backbone loop conformations and refinement methods. We have constructed two models for each protein using two alignment algorithms, and then an additional model using an identical alignment but different loop conformations for both buried and surface loops. The alignments used to build the models were created using the Needleman-Wunsch method, adapted for multiple sequences, and a manual method that utilized both a dotmatrix search matrix and the Needleman-Wunsch method. After constructing the initial models, several energy minimization methods were used to explore the variability in the final models caused by the choice of minimization techniques. Features of cytochrome P450cam and the cytochrome P450 superfamily, such as the ferredoxin binding site, the heme binding site and the substrate binding site were used to evaluate the validity of the models. Although the final structures were very similar between the models with different alignments, active-site residues were found to be dependent on the conformations of buried loops and early stages of energy minimization. We show which regions of the active site are the most dependent on the particular methods used, and which parts of the structures seem to be independent of the methods.     

Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3

The sesquiterpenoids are a large class of naturally occurring compounds with biological functions and desirable properties. Oxidation of the sesquiterpene (+)-valencene by wild type and mutants of P450cam from Pseudomonas putida, and of P450BM-3 from Bacillus megaterium, have been investigated as a potential route to (+)-nootkatone, a fine fragrance. Wild type P450cam did not oxidise (+)-valencene but the mutants showed activities up to 9.8 nmol (nmol P450)(-1) min(-1), with (+)-trans-nootkatol and (+)-nootkatone constituting >85% of the products. Wild type P450BM-3 and mutants had higher activities (up to 43 min(-1)) than P450cam but were much less selective. Of the many products, cis- and trans-(+)-nootkatol, (+)-nootkatone, cis-(+)-valencene-1,10-epoxide, trans-(+)-nootkaton-9-ol, and (+)-nootkatone-13S,14-epoxide were isolated from whole-cell reactions and characterised. The selectivity patterns suggest that (+)-valencene has one binding orientation in P450cam but multiple orientations in P450BM-3.     

Epoxidation of olefins by hydroperoxo-ferric cytochrome P450

The T252A mutant of cytochrome P450cam is unable to form the oxoferryl "active oxygen" intermediate, as judged by its inability to hydroxylate its normal substrate, camphor. In the present study, we demonstrate that T252A P450cam is nonetheless able to epoxidize olefins, due to the action of a second oxidant. However, as shown in earlier radiolytic studies and by the ability of T252A to reduce dioxygen to hydrogen peroxide, the mutant retains the ability to form the hydroperoxo-ferric reaction cycle intermediate. The present results provide strong evidence that hydroperoxo-ferric P450 can serve as a second electrophilic oxidant capable of olefin epoxidation.     

Mechanisms of reaction in cytochrome P450: Hydroxylation of camphor in P450cam

The fundamental nature of reactivity in cytochrome P450 enzymes is currently controversial. Modelling of bacterial P450cam has suggested an important role for the haem propionates in the catalysis, though this finding has been questioned. Understanding the mechanisms of this enzyme family is important both in terms of basic biochemistry and potentially in the prediction of drug metabolism. We have modelled the hydroxylation of camphor by P450cam, using combined quantum mechanics/molecular mechanics (QM/MM) methods. A set of reaction pathways in the enzyme was determined. We were able to pinpoint the source of the discrepancies in the previous results. We show that when a correct ionization state is assigned to Asp297, no spin density appears on the haem propionates and the protein structure in this region remains preserved. These results indicate that the haem propionates are not involved in catalysis.     

Catalytic oxidations of steroid substrates by artificial cytochrome p-450 enzymes

Catalysts comprising manganese-porphyrins carrying cyclodextrin binding groups are able to perform hydroxylations with substrate selectivity and regio- and stereoselectivity and high catalytic turnovers. The geometries of the catalyst/substrate complexes override intrinsic substrate reactivities, permitting attack on geometrically accessible saturated carbons of steroids in the presence of secondary carbinol groups and carbon-carbon double bonds, as in enzymatic reactions. Selective hydroxylations of steroid carbon 9 positions are of particular practical interest.     

Molecular modeling of cytochrome P450 3A4

The three-dimensional structure of human cytochrome P450 3A4 was modeled based on crystallographic coordinates of four bacterial P450s: P450 BM-3, P450cam, P450terp, and P450eryF. The P450 3A4 sequence was aligned to those of the known proteins using a structure-based alignment of P450 BM-3, P450cam, P450terp, and P450eryF. The coordinates of the model were then calculated using a consensus strategy, and the final structure was optimized in the presence of water. The P450 3A4 model resembles P450 BM-3 the most, but the B helix is similar to that of P450eryF, which leads to an enlarged active site when compared with P450 BM-3, P450cam, and P450terp. The 3A4 residues equivalent to known substrate contact residues of the bacterial proteins and key residues of rat P450 2B1 are located in the active site or the substrate access channel. Docking of progesterone into the P450 3A4 model demonstrated that the substrate bound in a 6-orientation can interact with a number of active site residues, such as 114, 119, 301, 304, 305, 309, 370, 373, and 479, through hydrophobic interactions. The active site of the enzyme can also accommodate erythromycin, which, in addition to the residues listed for progesterone, also contacts residues 101, 104, 105, 214, 215, 217, 218, 374, and 478. The majority of 3A4 residues which interact with progesterone and/or erythromycin possess their equivalents in key residues of P450 2B enzymes, except for residues 297, 480 and 482, which do not contact either substrate in P450 3A4. The results from docking of progesterone and erythromycin into the enzyme model make it possible to pinpoint residues which may be important for 3A4 function and to target them for site-directed mutagenesis.     

Oxidizing species in the mechanism of cytochrome P450

This review discusses the mechanisms of oxygen activation by cytochrome P450 enzymes, the possible catalytic roles of the various iron--oxygen species formed in the catalytic cycle, and progress in understanding the mechanisms of hydrocarbon hydroxylation, heteroatom oxidation, and olefin epoxidation. The focus of the review is on recent results, but earlier work is discussed as appropriate. The literature through to February 2002 is surveyed, and 175 referenced are cited.     

Development and validation of a microsomal online cytochrome P450 bioreactor coupled to solid-phase extraction and reversed-phase liquid chromatography

The development and validation of an online cytochrome P450 (CYP)-based bioreactor coupled to automated solid-phase extraction (SPE) and gradient HPLC separation is described. The analytical method was checked on intra- and inter-day repeatability of the ethoxyresorufin-O-demethylation (EROD) reaction with CYP 1Al/1A2 containing beta-NF induced rat liver microsomes as an enzyme source. These experiments showed that CYP activity was linearly decreased with 16% over an 11 h period. Inter-day measurements had a CV of 9.1%. Furthermore, Km and Vmax values of the EROD reaction, measured with the bioreactor, were 2.72 +/- 0.46 microM and 7.9 +/- 0.5 nmol/min/mg protein, respectively. These were in good correspondence with Km and Vmax values, measured with standard batch assay, which amounted 0.66 +/- 0.08 microM and 6.4 +/- 0.2 nmol/min/mg protein respectively. In conclusion the newly developed analytical method can be used effectively and at a microliter scale for online generation, extraction and separation of metabolites.     

DNA-mediated assembly of cytochrome P450 BM3 subdomains

Cytochrome P450 BM3 is a versatile enzyme, which holds great promise for applications in biocatalysis and biomedicine. We here report on the generation of a hybrid DNA-protein device based on the two subdomains of BM3, the reductase domain BMR and the porphyrin domain BMP. Both subdomains were fused genetically to the HaloTag protein, a self-labeling enzyme, allowing for the bioconjugation with chloroalkane-modified oligonucleotides. The subdomain-DNA-chimeras could be reassembled by complementary oligonucleotides, thus leading to reconstitution of the monooxygenase activity of BM3 holoenzyme, as demonstrated by conversion of the reporter substrate 12-pNCA. Arrangement of the two chimeras on a switchable DNA scaffold allowed one to control the distance between both subdomains, as indicated by the DNA-dependent activity of the holoenzyme. Furthermore, a switchable chimeric device was constructed, in which monooxygenase activity could be turned off by DNA strand displacement. This study demonstrates that P450 BM3 engineering and strategies of DNA nanotechnology can be merged to open up novel ways for the development of novel screening systems or responsive catalysts with potential applications in drug delivery.     

Structural fine-tuning of a multifunctional cytochrome P450 monooxygenase

AurH is a unique cytochrome P450 monooxygenase catalyzing the stepwise formation of a homochiral oxygen heterocycle, a key structural and pharmacophoric component of the antibiotic aureothin. The exceptional enzymatic reaction involves a tandem oxygenation process including a regio- and stereospecific hydroxylation, followed by heterocyclization. For the structural and biochemical basis of this unparalleled sequence, four crystal structures of AurH variants in different conformational states and in complex with the P450 inhibitor ancymidol were solved, which represent the first structures of the CYP151A group. Structural data in conjunction with computational docking, site-directed mutagenesis, and chemical analyses unveiled a switch function when recognizing the two substrates, deoxyaureothin and the hydroxylated intermediate, thus allowing the second oxygenation-heterocyclization step. Furthermore, we were able to modify the chemo- and regioselectivity of AurH, yielding mutants that catalyze the regioselective six-electron transfer of a nonactivated methyl group to a carboxylic acid via hydroxyl and aldehyde intermediates.     

Stochastic simulations of the cytochrome P450 catalytic cycle

Cytochrome P450 enzymes involve a complex reaction cycle which has been described, for the first time, by a stochastic simulation of the system in the present work. A series of models are developed for a basic catalytic cycle, employing a set of microscopic rate constants for the oxidation of p-alkoxyacylanilides catalyzed by the cytochrome P450 1A2. By analyzing the effects of low concentrations of enzyme and substrate on the system, and the dependence of the system on several rate constants, it is discovered that the system evolves along relatively stable patterns from its initial state, as indicated from different runs of simulations. Strong fluctuations appear at the entrance and exit of the pathway, with very weak fluctuations in the middle sections of the cycle. Although noises are apparent when the reactant populations are very low, basically, the fundamental feature of the P450 cycle based on a microscopic view is that it is deterministic in nature. Meanwhile, the mathematical models we developed are qualitatively validated by a comparison with those experimental results of the P450 cycle. The findings of this work will be helpful for a further deeper understanding of the catalytic mechanism of cytochrome P450 enzymes.