Exploring CYP1A1 as anticancer target: homology modeling and in silico inhibitor design

Microsomal cytochrome P450 family 1 enzymes has great importance in the bioactivation of mutagens. P450 catalyzed reactions involve a wide range of substrates, and this versatality is reflected in a structural diversity, evident in the active sites of available P450 structures. This structure offers a template to study further structure-function relationships of alternative substrates and other cytochrome P450 family 1 members. In this paper, we document a homology model of CYP P450 1A1 from Homo sapiens, developed on the basis of template crystal structure of human microsomal P450 1a2 in complex with inhibitor (PDB Id: 2HI4). Homology modeling is performed at the programs, both in the commercial and public realms. We tried to explore CYP1A1 as a potential target for anticancer chemotherapy. To gain an insight into the binding of ligands with enzyme, protein-ligand complex was developed by including information about the known ligand as spatial restraints and optimizing the mutual interactions between the ligand and the binding site. Active site characterization and the study for involvement of specific aminoacids in binding with ligand, facilitates structure based inhibitor design. This study should prove useful in the design and development of potential novel anticancer agents. Figure The refined structure of homology model of CYP1A1     

Tight-binding inhibition by alpha-naphthoflavone of human cytochrome P450 1A2

Human cytochrome P450 (P450) enzymes exhibit remarkable diversity in their substrate specificities, participating in oxidation reactions of a wide range of xenobiotic drugs. Previously, we reported that alpha-naphthoflavone (ANF) is bound to the recombinant P450 1A2 tightly and stabilizes an overall enzyme conformation. The present study is designed to determine the type of P450 1A2 inhibition exerted by ANF, using two different substrates of P450 1A2, 7-ethoxycoumarin (EOC) and 7-ethoxyresorufin (EOR). ANF is generally known as a competitive inhibitor of the enzyme. However, in our tight-binding enzyme kinetics study, ANF acts as noncompetitive inhibitor in 7-ethoxycoumarin O-deethylation (ECOD) (K(i)=55.0 nM), but as competitive inhibitor in 7-ethoxyresorufin O-deethylation (EROD) (K(i)=1.4 nM). Based on homology modeling studies, ANF is positioned to bind to a hydrophobic cavity next to the active site where it may cause a direct effect on substrate binding. It is agreed with the predicted binding site of ANF in P450 3A4, in which ANF is rather known as a stimulating modulator. Our results suggest that ANF binds near the active site of P450 1A2 and exhibits differential inhibition mechanisms, possibly depending on the molecular structure of the substrate.     

Essential requirements for substrate binding affinity and selectivity toward human CYP2 family enzymes

A detailed analysis of substrate selectivity within the cytochrome P450 2 (CYP2) family is reported. From a consideration of specific interactions between drug substrates for human CYP2 family enzymes and the putative active sites of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1, it is likely that the number and disposition of hydrogen bond donor/acceptors and aromatic rings within the various P450 substrate molecules determines their enzyme selectivity and binding affinity, together with directing their preferred routes of metabolism by the CYP2 enzymes concerned. Although many aliphatic residues are present in most P450 active sites, it would appear that their main contribution centers around hydrophobic interactions and desolvation processes accompanying substrate binding. Molecular modeling studies based on the recent CYP2C5 crystal structure appear to show close agreement with site-directed mutagenesis experiments and with information on substrate metabolism and selectivity within the CYP2 family.     

Cholesterol Ester Oxidation by Mycobacterial Cytochrome P450

Mycobacteria share a common cholesterol degradation pathway initiated by oxidation of the alkyl side chain by enzymes of cytochrome P450 (CYP) families 125 and 142. Structural and sequence comparisons of the two enzyme families revealed two insertions into the N-terminal region of the CYP125 family (residues 58–67 and 100–109 in the CYP125A1 sequence) that could potentially sterically block the oxidation of the longer cholesterol ester molecules. Catalytic assays revealed that only CYP142 enzymes are able to oxidize cholesteryl propionate, and although CYP125 enzymes could oxidize cholesteryl sulfate, they were much less efficient at doing so than the CYP142 enzymes. The crystal structure of CYP142A2 in complex with cholesteryl sulfate revealed a substrate tightly fit into a smaller active site than was previously observed for the complex of CYP125A1 with 4-cholesten-3-one. We propose that the larger CYP125 active site allows for multiple binding modes of cholesteryl sulfate, the majority of which trigger the P450 catalytic cycle, but in an uncoupled mode rather than one that oxidizes the sterol. In contrast, the more unhindered and compact CYP142 structure enables enzymes of this family to readily oxidize cholesteryl esters, thus providing an additional source of carbon for mycobacterial growth.     

Differences in the cytochrome P-450 isoenzymes involved in the 2-hydroxylation of oestradiol and 17 alpha-ethinyloestradiol. Relative activities of rat and human liver enzymes.

The metabolism of oestradiol and 17 alpha-ethinyloestradiol to their 2-hydroxy derivatives is an important determinant in their biological effects. In this work, we have investigated which rat or human cytochrome P-450 isoenzymes are involved in catalysing these reactions. Oestradiol 2-hydroxylation was catalysed by a wide variety of rat cytochrome P-450s from gene families P450IA, P450IIB, P450IIC and P450IIIA. Interestingly, 17 alpha-ethinyloestradiol, which only differs structurally from oestradiol at a position distant from the site of oxidation, was metabolized predominantly by members of the P450IIC gene subfamily. In order to establish which enzymes are responsible for the oxidation of these substrates in man, antibodies to rat liver cytochrome P-450 isoenzymes were used to inhibit these reactions in a panel of human liver microsomal fractions. Also, possible correlations between the proteins recognized by the antibodies and the 2-hydroxylation rate were determined. These experiments provide evidence that 2-hydroxylation of 17 alpha-ethinyloestradiol in man is catalysed by cytochromes from the P450IIC, P450IIE and P450IIIA gene families. In contrast, the major proteins involved in oestradiol metabolism are from the P450IA gene family, although members of the P450IIC and P450IIE gene families may also play a role. These data demonstrate that the differences in the capacity of rat P-450s to metabolize these substrates are also present in the comparable enzymes involved in man, and that a variety of factors will determine the rate of disposition of these compounds in man.     

Structure of human microsomal cytochrome P450 2C8. Evidence for a peripheral fatty acid binding site

A 2.7-Angstrom molecular structure of human microsomal cytochrome P450 2C8 (CYP2C8) was determined by x-ray crystallography. The membrane protein was modified for crystallization by replacement of the hydrophobic N-terminal transmembrane domain with a short hydrophilic sequence before residue 28. The structure of the native sequence is complete from residue 28 to the beginning of a C-terminal histidine tag used for purification. CYP2C8 is one of the principal hepatic drug-metabolizing enzymes that oxidizes therapeutic drugs such as taxol and cerivastatin and endobiotics such as retinoic acid and arachidonic acid. Consistent with the relatively large size of its preferred substrates, the active site volume is twice that observed for the structure of CYP2C5. The extended active site cavity is bounded by the beta1 sheet and helix F' that have not previously been implicated in substrate recognition by mammalian P450s. CYP2C8 crystallized as a symmetric dimer formed by the interaction of helices F, F', G', and G. Two molecules of palmitic acid are bound in the dimer interface. The dimer is observed in solution, and mass spectrometry confirmed the association of palmitic acid with the enzyme. This novel finding identifies a peripheral binding site in P450s that may contribute to drug-drug interactions in P450 metabolism.     

Enhancement of 7-methoxyresorufin O-demethylation activity of human cytochrome P450 1A2 by molecular breeding

Alkylresorufins are model substrates for cytochrome P450 (P450) 1A2. The ability of human P450 1A2 to catalyze 7-methoxyresorufin O-demethylation was improved by screening of random mutant libraries (expressed in Escherichia coli) on the basis of 7-methoxyresorufin O-demethylation. After three rounds of mutagenesis and screening, the triple mutant E163K/V193M/K170Q yielded a kcat > five times faster than wild type P450 1A2 in steady-state kinetic analysis using either isolated membrane fractions or purified, reconstituted enzymes. The enhanced catalytic activity was not attributed to changes in substrate affinity. The kinetic hydrogen isotope effect of the triple mutant did not change from wild type enzyme and suggests that C-H bond cleavage is rate-limiting in both enzymes. Homology modeling, based on an X-ray structure of rabbit P450 2C5, suggests that the locations of mutated residues are not close to the substrate binding site and therefore that structural elements outside of this site play roles in changing the catalytic activity. This approach has potential value in understanding P450 1A2 and generating engineered enzymes with enhanced catalytic activity.     

A predicted three-dimensional structure of human cytochrome P450: implications for substrate specificity

A three-dimensional structure for human cytochrome P450IA1 was predicted based on the crystal coordinates of cytochrome P450cam from Pseudomonas putida. As there was only 15% residue identity between the two enzymes, additional information was used to establish an accurate sequence alignment that is a prerequisite for model building. Twelve representative eukaryotic sequences were aligned and a net prediction of secondary structure was matched against the known alpha-helices and beta-sheets of P450cam. The cam secondary structure provided a fixed main-chain framework onto which loops of appropriate length from the human P450IA1 structure were added. The model-built structure of the human cytochrome conformed to the requirements for the segregation of polar and nonpolar residues between the core and the surface. The first 44 residues of human cytochrome P450 could not be built into the model and sequence analysis suggested that residues 1-26 formed a single membrane-spanning segment. Examination of the sequences of cytochrome P450s from distinct gene families suggested specific residues that could account for the differences in substrate specificity. A major substrate for P450IA1, 3-methyl-cholanthrene, was fitted into the proposed active site and this planar aromatic molecule could be accommodated into the available cavity. Residues that are likely to interact with the haem were identified. The sequence similarity between 59 eukaryotic enzymes was represented as a dendrogram that in general clustered according to gene family. Until a crystallographic structure is available, this model-building study identifies potential residues in cytochrome P450s important in the function of these enzymes and these residues are candidates for site-directed mutagenesis.     

The structure of human microsomal cytochrome P450 3A4 determined by X-ray crystallography to 2.05-A resolution

The structure of P450 3A4 was determined by x-ray crystallography to 2.05-A resolution. P450 3A4 catalyzes the metabolic clearance of a large number of clinically used drugs, and a number of adverse drug-drug interactions reflect the inhibition or induction of the enzyme. P450 3A4 exhibits a relatively large substrate-binding cavity that is consistent with its capacity to oxidize bulky substrates such as cyclosporin, statins, taxanes, and macrolide antibiotics. Family 3A P450s also exhibit unusual kinetic characteristics that suggest simultaneous occupancy by smaller substrates. Although the active site volume is similar to that of P450 2C8 (PDB code: 1PQ2), the shape of the active site cavity differs considerably due to differences in the folding and packing of portions of the protein that form the cavity. Compared with P450 2C8, the active site cavity of 3A4 is much larger near the heme iron. The lower constraints on the motions of small substrates near the site of oxygen activation may diminish the efficiency of substrate oxidation, which may, in turn, be improved by space restrictions imposed by the presence of a second substrate molecule. The structure of P450 3A4 should facilitate a better understanding of the substrate selectivity of the enzyme.     

Magnetic resonance study of the structure and functions of cytochrome P450

Cytochrome P450 is a membrane-bound enzyme providing oxidation of numerous organic compounds in organisms. The objective of this review is to show the wide possibilities that are provided by Electron Spin Resonance (ESR) and Nuclear Magnetic Resonance (NMR) techniques to the study of the structure and functions of this unique enzyme. High sensitivity of ESR spectra of cytochrome P450 to its functional state and interaction with substrates and inhibitors is illustrated. NMR and proton relaxation make it possible to obtain unique information about the structure of the active center of cytochrome P450 under physiological conditions. ESR and NMR methods allow one to obtain structural data on location of substrates, inhibitors, and their spin-labeled analogs with respect to Fe3+ ions in the enzyme-active center. Of special interest seems to be coupling of ESR with the affinity modification method. For this purpose, the spin-labeled analogs of cytochrome P450 substrates containing alkylating groups were used. As a result, an important datum has been obtained on the structure of active centers of cytochrome P450 in microsomes and in a highly purified state. In conclusion, the problems of the structure and functions of cytochrome P450, which can be most efficiently resolved with the use of magnetic resonance methods, are discussed.     

Role of protein and substrate dynamics in catalysis by Pseudomonas putida cytochrome P450cam

The role of protein structural flexibility and substrate dynamics in catalysis by cytochrome P450 enzymes is an area of current interest. We have addressed these in cytochrome P450(cam) (P450(cam)) and its Y96A mutant with camphor and its related compounds using fluorescence spectroscopy. Previously [Prasad et al. (2000) FEBS Lett. 477, 157-160], we provided experimental support to dynamic fluctuations in P450(cam), and substrate access into the active site region via the channel next to the flexible F-G helix-loop-helix segment. In the investigation described here, we show that the dynamic fluctuations in the enzyme are substrate dependent as reflected by tryptophan fluorescence quenching experiments. The orientation of tryptophan relative to heme (kappa(2)) for W42 obtained from time-resolved tryptophan fluorescence measurements show variation with type of substrate bound to P450(cam) suggesting regions distant from heme-binding site are affected by physicochemical and steric characteristics/protein-substrate interactions of P450(cam) active site. We monitored substrate dynamics in the active site region of P450(cam) by time-resolved substrate anisotropy measurements. The anisotropy decay of substrates bound to P450(cam) indicate that mobility of substrates is modulated by physicochemical and steric characteristics/protein-substrate interactions of local active site structure, and provides an understanding of factors controlling observed hydroxylated products for substrate bound P450(cam) complexes. The present study shows that P450(cam) local and peripheral structural flexibility and heterogeneity along with substrate mobility play an important role in regulating substrate binding orientation during catalysis and accommodating diverse range of substrates within P450(cam) heme pocket.     

Molecular Lego: design of molecular assemblies of P450 enzymes for nanobiotechnology.

This paper reports on the application of the molecular Lego approach to P450 enzymes. Protein domains are used as catalytic (P450 BM3 haem domain and human P450 2E1) or electron transfer (flavodoxin and P450 BM3 reductase) modules. The objectives are to build assemblies with improved electrochemical properties, to construct soluble human P450 enzymes, and to generate libraries of new P450 catalytic modules based on P450 BM3. A rationally designed, gene-fused assembly (BMP-FLD) was obtained from the soluble haem domain of cytochrome P450 BM3 from Bacillus megaterium (BMP) and flavodoxin from Desulfovibrio vulgaris (FLD). The assembly was expressed successfully and characterised in its active form, displaying improved electrochemical properties. Solubilisation of the human, membrane-bound P450 2E1 (2E1) was achieved by fusing key elements of the 2E1 enzyme with selected parts of P450 BM3. An assembly containing the first 54 residues of P450 BM3, the whole sequence of P450 2E1 from residue 81 and the reductase domain of P450 BM3 was constructed. The 2E1-BM3 assembly was successfully expressed in the cytosol of Escherichia coli. The soluble form of 2E1-BM3 was reduced in carbon monoxide atmosphere and displayed the typical absorption peak at 450 nm, characteristic of a folded and active P450 enzyme. Finally, the alkali method previously developed in this laboratory was used to screen for P450 activity within a library of random mutants of P450 BM3. A number of variants active towards non-physiological substrates, such as pesticides and polyaromatic hydrocarbons were identified, providing new P450 catalytic modules. The combination of these three areas of research provide interesting tools for exploitation in nanobiotechnology.     

Catalysis of the oxidation of steroid and stilbene estrogens to estrogen quinone metabolites by the beta-naphthoflavone-inducible cytochrome P450 IA family.

Diethylstilbestrol (DES) or catecholestrogens are metabolized by microsomal enzymes to quinones, DES Q or catecholestrogen quinones, respectively, which have been shown to bind covalently to DNA and to undergo redox cycling. The isoforms of cytochrome P450 catalyzing this oxidation of estrogens to genotoxic intermediates were not known and have been identified in this study by (a) using microsomes of rats treated with various inducers of cytochrome P450; (b) using purified cytochrome P450 isoforms; and (c) examining the peroxide cofactor concentrations necessary for this oxidation by microsomes or pure isoenzymes. The highest rate of oxidation of DES to DES Q was obtained using beta-naphthoflavone-induced microsomes (14.0 nmol DES Q/mg protein/min) or cytochrome P450 IA1 (6.4 pmol DES Q/min/pmol P450). Isosafrole-induced microsomes or cytochrome P450 IA2 oxidized DES to quinone at one-third or one-fifth of that rate, respectively. Low or negligible rates of oxidation were measured when oxidations were catalyzed by microsomal rat liver enzymes induced by phenobarbital, ethanol, or pregnenolone-16 alpha-carbonitrile or by pure cytochromes P450 IIB1, IIB4, IIC3, IIC6, IIE1, IIE2, IIG1, or IIIA6. Cytochrome P450 IA1 also catalyzed the oxidation of 2- or 4-hydroxyestradiol to their corresponding quinones. The beta-naphthoflavone-induced microsomes and cytochrome P450 IA1 had the highest "affinity" for cumene hydroperoxide cofactor (Km = 77 microM). Cofactor concentrations above 250 microM resulted in decreased rates of oxidation. The other cytochrome P450 isoforms required much higher cofactor concentrations and were not inactivated at high cofactor concentrations. The data demonstrate that beta-naphthoflavone-inducible cytochrome P450 IA family enzymes catalyze most efficiently the oxidation of estrogenic hydroquinones to corresponding quinones. This oxidation may represent a detoxification pathway to keep organic hydroperoxides at minimal concentrations. The resulting quinone metabolites may be detoxified by other pathways. However, in cells with decreased detoxifying enzyme activities, quinones metabolites may accumulate and initiate carcinogenesis or cell death by covalent arylation of DNA or proteins.     

Structural insight into the altered substrate specificity of human cytochrome P450 2A6 mutants

Human P450 2A6 displays a small active site that is well adapted for the oxidation of small planar substrates. Mutagenesis of CYP2A6 resulted in an increased catalytic efficiency for indole biotransformation to pigments and conferred a capacity to oxidize substituted indoles (Wu, Z.-L., Podust, L.M., Guengerich, F.P. J. Biol. Chem. 49 (2005) 41090-41100.). Here, we describe the structural basis that underlies the altered metabolic profile of three mutant enzymes, P450 2A6 N297Q, L240C/N297Q and N297Q/I300V. The Asn297 substitution abolishes a potential hydrogen bonding interaction with substrates in the active site, and replaces a structural water molecule between the helix B'-C region and helix I while maintaining structural hydrogen bonding interactions. The structures of the P450 2A6 N297Q/L240C and N297Q/I300V mutants provide clues as to how the protein can adapt to fit the larger substituted indoles in the active site, and enable a comparison with other P450 family 2 enzymes for which the residue at the equivalent position was seen to function in isozyme specificity, structural integrity and protein flexibility.     

Modelling human cytochromes P450 involved in drug metabolism from the CYP2C5 crystallographic template

A historical background to homology modelling of human P450s involved in drug metabolism is outlined, showing that the progress in crystallographic studies of bacterial forms of enzyme and, latterly, determination of a mammalian P450 crystal structure, has enabled the production of increasingly satisfactory models of human P450 enzymes. The methodology for the generation of P450 models by homology with crystallographic template structures is summarized, and recent results of CYP2C5-constructed models of P450s are described. These indicate that selective substrates are able to fit within the putative active sites of each enzyme, where key contacts with complementary amino acid residues are largely consistent with the results of site-directed mutagenesis experiments and metabolic studies. Consequently, the CYP2C5 crystal structure can be regarded at the current paradigm for homology modelling of the drug metabolizing P450s, especially those from the CYP2 family.     

Molecular Modeling of Cytochrome P450 2B1: Mode of Membrane Insertion and Substrate Specificity

A molecular model of a mammalian membrane-bound cytochrome P450, rat P450 2B1, was constructed in order to elucidate its mode of attachment to the endoplasmic reticulum and the structural basis of substrate specificity. The model was primarily derived from the structure of P450BM-3, which as a class II P450 is the most functionally similar P450 of known structure. However, model development was also guided by the conserved core regions of P450cam and P450terp. To optimally align the P450 2B1 and P450BM-3 sequences, multiple alignment was performed using sequences of five P450s in the II family, followed by minor adjustments on the basis of secondary structure predictions. The resulting P450 2B1 homology model structure was refined by molecular dynamics heating, equilibration, simulation, and energy minimization. The model suggests that the F–G loop serves as both a hydrophobic membrane anchor and entrance channel for hydrophobic substrates from the membrane to the P450 active site. To assess the mode of substrate binding, benzphetamine, testosterone, and benzo[a]pyrene were docked into the active site. The hydrophobic substrate-binding pocket is consistent with the preferences of this P450 toward hydrophobic substrates, while the presence of an acidic Glu-105 in this pocket is consistent with the preference of this P450 for the cationic substrate benzphetamine. This model is thus consistent with several known experimental properties of this P450, such as membrane attachment and substrate selectivity.     

A molecular model for the enzyme cytochrome P450(17 alpha), a major target for the chemotherapy of prostatic cancer

The enzyme cytochrome P450(17 alpha) catalyses two key steps in the biosynthesis of the androgens from pregnanes: the 17 alpha hydroxylation step and the subsequent 17-20 lyase reaction. Using a variety of techniques, including sequence alignment, secondary structure prediction, molecular mechanics and molecular dynamics, we have constructed a model for the three-dimensional structure of P450(17 alpha) based on that of P450cam, the only cytochrome P450 enzyme for which the crystal structure is known. The model suggests the possibility of two modes of binding of steroid substrates at the active site, perhaps reflecting the dual functionality of the enzyme.     

Use of kinetic isotope effects to delineate the role of phenylalanine 87 in P450(BM-3)

The substrate oxidation rates of P450(BM-3) are unparalleled in the cytochrome P450 (CYP) superfamily of enzymes. Furthermore, the bacterial enzyme, originating from Bacillus megaterium, has been used repeatedly as a model to study the metabolism of mammalian P450s. A specific example is presented where studying P450(BM-3) substrate dynamics can define important enzyme-substrate characteristics, which may be useful in modeling omega-hydroxylation seen in mammalian P450s. In addition, if the reactive species responsible for metabolism can be controlled to produce specific products this enzyme could be a useful biocatalyst. Based on crystal structures and the fact that the P450(BM-3) F87A mutant produces a large isotope in contrast to the native enzyme, we propose that phenylalanine 87 is responsible for hindering substrate access to the active oxygen species for nonnative substrates. Using kinetic isotopes and two aromatic substrates, p-xylene and 4,4'-dimethylbiphenyl, the role phenylalanine 87 plays in active-site dynamics is characterized. The intrinsic KIE is 7.3 +/- 2 for wtP450(BM-3) metabolism of p-xylene. In addition, stoichiometry differences were measured with the native and mutant enzyme and 4,4'-dimethylbiphenyl. The results show a more highly coupled substrate/NADPH ratio in the mutant than in the wtP450(BM-3).     

Interactions of microsomal cytochrome P-450 with spin-labeled substrate analogs

The iodine-containing stable iminoxyl radicals with various distances between the N-O-group and the iodine atom are proposed to be used to study the structure of the active center of the microsomal cytochrome P-450. The radicals used induce changes in the optical spectra of the Fe3+ ion located in the active center of the enzyme, as in the case of type 1 substrates and inhibit essentially the microsomal oxidation of cytochrome P-450 substrates of type 1 and 2. This inhibition is neither due to suppression of the NADPH-cytochrome c reductase activity nor to cytochrome P-450 conversion to cytochrome P-420. Cytochrome P-450 substrates (aminopyrine) protect the enzyme against the radical-induced inactivation. The iodine-containing radicals are covalently bound to cytochrome P-450 in the vicinity of active center. The values of dissociation constants for the reversible enzyme-radical constants and the rate constants for the monomolecular transformation in the complex, k, were determined. The EPR method was used to detect the coupling between Fe3+ and the radical located in the active center of cytochrome P-450. The saturation curves of radical SPR spectra at 77 degrees K were employed to determine the contribution of Fe3+ to the relaxation time, T1, of the radicals covalently bound to cytochrome P-450 and to estimate the distances between the Fe3+ ion and the N-O-group of these radicals in the enzyme active center.