Heterobifunctional photoaffinity probes for cytochrome P450 2B.

Three heterobifunctional photoaffinity probes, N-(p-azidobenzyl)-N-methyl-p-aminobenzylamine (I), N-(p-azidobenzyl)-N-methyl-p-aminophenethylamine (II), and N-(p-azidophenethyl)-N-methyl-p-aminophenethylamine (III), were synthesized and characterized. These probes, containing a photolabile azido-group and an amino-group on opposite sides of the molecule, were designed for photoaffinty labeling of the cytochrome P450 (CYP) 2B active site cavity differing in distance from the heme iron. Spectroscopic studies proved that probes I and II coordinated with the heme iron via their amino-group in the enzyme active center, whereas probe III did not. This result in conjunction with data from kinetic studies suggests probes I and II are appropriate for photoaffinity labeling of the CYP 2B active center. Thus, probe II was used to identify amino acid residues within a distance of the probe length (about 16.5 A) from the heme. Analysis of a Lys-C digest of the probe II-labeled CYP 2B4 revealed a single labeled hexapeptide corresponding to position 192-197 of the CYP 2B4 sequence. Using postsource decay/matrix-assisted laser desorption ionization-time of flight, Arg197 was identified as a probe II target. The location of the labeled site in three-dimensional structures of bacterial CYPs and in CYP 2B homology models is discussed.     

Mapping of cytochrome P450 2B4 substrate binding sites by photolabile probe 3-azidiamantane: identification of putative substrate access regions

To investigate structure-function relationships of cytochromes P450 (CYP), 3-azidiamantane was employed for photoaffinity labeling of rabbit microsomal CYP2B4. Four diamantane labeled tryptic fragments were identified by mass spectrometry and sequencing: peptide I (Leu³⁵⁹-Lys³⁷³), peptide II (Leu³⁰-Arg⁴⁸), peptide III (Phe¹²⁷-Arg¹⁴⁰), and peptide IV (Arg⁴³⁴-Arg⁴⁴³). Their positions were projected into CYP2B4 model structures and compared with substrate binding sites, proposed by docking of diamantane. We identified novel binding regions outside the active site of CYP2B4. One of them, defined with diamantane modified Arg¹³³, marks a possible entrance to the active site from the heme proximal face. In addition to crystal structures of CYP2B4 chimeras and molecular dynamics simulations, our data of photoaffinity labeling of the full CYP2B4 molecule provide further insight into functional and structural aspects of substrate binding.     

Structural requirements for inhibitors of cytochromes P450 2B: assessment of the enzyme interaction with diamondoids

The series of diamondoids: adamantane, diamantane, triamantane, 2-isopropenyl-2-methyladamantane and 3-isopropenyl-3-methyldiamantane (3-IPMDIA), were employed to elucidate the molecular basis of their interaction with the active site of cytochromes P450 (CYP) of a 2B subfamily. These potent inhibitors of CYP2B enzymes were docked into the homology model of CYP2B4. Apparent dissociation constants calculated for the complexes of CYP2B4 with docked diamandoids agreed closely with the experimental data showing inhibition potency of the compounds and their binding affinity to CYP2B4. Superimposed structures of docked diamondoids mapped binding site residues. As they are mainly non-polar residues, the hydrophobicity plays the major role in the binding of diamondoids. Overlapping structure of diamondoids defined an elliptical binding cavity (5.9A inner diameter, 7.9A length) forming an angle of approximately 43 degrees with the heme plane. CYP2B specific diamondoids, namely 3-IPMDIA, showing the highest binding affinity, should be considered for a potential clinical use.     

CYP2E1 active site residues in substrate recognition sequence 5 identified by photoaffinity labeling and homology modeling

Despite its biological importance, our knowledge of active site structure and relevance of critical amino acids in CYP2E1 catalytic processes remain limited. In this study, we identified CYP2E1 active site residues using photoaffinity labeling with 7-azido-4-methylcoumarin (AzMC) coupled with a CYP2E1 homology model. In the absence of light, AzMC was an effective competitor against substrate p-nitrophenol oxidation by CYP2E1. Photoactivation of AzMC led to a concentration-dependent loss in CYP2E1 activity and structural integrity resulting from the modification of both heme and protein. The photo-labeling reaction degraded heme and produced a possible heme adduct. Probe incorporation into the protein occurred at multiple sites within substrate recognition sequence 5 (SRS-5). Based on a CYP2E1 homology model, we hypothesize AzMC labels SRS-5 residues, Leu363, Val364, and Leu368, in the active site. In addition, we propose a series of phenylalanines, especially Phe106, mediate contacts with the coumarin.     

Heterologous expression of CYP102A5 variant from Bacillus cereus CYPPB-1: Validation of model for predicting drug metabolism of human P450 probe substrates

CYP102A5 variant (ADL27534) from isolated Bacillus cereus CYPPB-1 was heterologously expressed in Escherichia coli Top 10 cells. Comparative sequence analysis of purified CYP102A5 variant with respect to reported CYP102A5 (AAP10153) from Bacillus cereus ATCC 14579 revealed amino acid sequence changes at positions P245S and M318I of heme domain. The binding affinities of 15 selected human P450 probe substrates towards isolated CYP102A5 were analyzed in silico using a homology model together with molecular docking techniques to predict the human drug metabolism. In vitro analysis suggested that the purified CYP102A5 metabolizes typical substrates of human CYP2C9, CYP2D6, CYP2E1, and CYP3A4, such as coumarin, propranolol, aniline, chlorzoxazone, p-nitrophenol, and nifedipine. The calculated K _M values for propranolol, chloroxazone, coumarin, aniline, and 4-nitrophenol were calculated to be 0.962?±?0.041, 1.254?±?0.057, 2.859?±?0.083, 2.732?±?0.106, and 2.528?±?0.11 mM, respectively. Importantly, taking a ChemScore cutoff value of ?31 kJ/mol, substrate binding at active site and in vitro activity as the distinguishing lines between “substrates” and “nonsubstrates” revealed one false-positive and one false-negative results out of the 15 compounds examined. This is the first report on validation of CYP102A family homology model for in silico prediction of human drug metabolism.     

Comparing the electronic properties and docking calculations of heme derivatives on CYP2B4

Cytochrome P-450 is a group of enzymes involved in the biotransformation of many substances, including drugs. These enzymes possess a heme group (1) that when it is properly modified induces several important physicochemical changes that affect their enzymatic activity. In this work, the five structurally modified heme derivatives 2–6 and the native heme 1 were docked on CYP2B4, (an isoform of P450), in order to determine whether such modifications alter their binding form and binding affinity for CYP2B4 apoprotein. In addition, docking calculations were used to evaluate the affinity of CYP2B4 apoprotein-heme complexes for aniline (A) and N-methyl-aniline (NMA). Results showing the CYP2B4 heme 4- and heme 6-apoprotein complexes to be most energetically stable indicate that either hindrance effects or electronic properties are the most important factors with respect to the binding of heme derivatives at the heme-binding site. Furthermore, although all heme-apoprotein complexes demonstrated high affinity for both A and NMA, the CYP2B4 apoprotein-5 complex had higher affinity for A, and the heme 6 complex had higher affinity for NMA. Finally, surface electronic properties (SEP) were calculated in order to explain why certain arginine residues of CYP2B4 apoprotein interact with polarizable functionalities, such as ester groups or sp ² carbons, present in some heme derivates. The main physicochemical parameter involved in the recognition process of the heme derivatives, the CYP2B4 apoprotein and A or NMA, are reported. Figure Scheme of steps to be followed for obtaining five new CYP2B4 apoprotein-heme complexes by docking     

Analysis of clinically relevant substrates of CYP2B6 enzyme by computational methods

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

Unusual regioselectivity and active site topology of human cytochrome P450 2J2

The oxidation of six derivatives of terfenadone by recombinant human CYP2J2 (CYP = cytochrome P450) was studied by high-performance liquid chromatography coupled to mass spectrometry (MS) using tandem MS techniques and by 1H NMR spectroscopy. CYP2J2 exhibited a surprising regioselectivity in favor of the hydroxylation of the substrate terminal chain at the weakly reactive homobenzylic position. In contrast, hydroxylation of the same substrates by CYP3A4 mainly occurred on the most chemically reactive sites of the substrates (N-oxidation and benzylic hydroxylation). A 3D homology model of CYP2J2 was constructed using recently published structures of CYP2A6, CYP2B4, CYP2C8, CYP2C9, and CYP2D6 as templates. In contrast with other CYP2 structures, it revealed an active site cavity with a severely restricted access of substrates to the heme through a narrow hydrophobic channel. Dynamic docking of terfenadone derivatives in the CYP2J2 active site allowed one to interpret the unexpected regioselectivity of the hydroxylation of these substrates by CYP2J2, which is mainly based on this restricted access to the iron. The structural features that have been found to be important for recognition of substrates or inhibitors by CYP2J2 were also interpreted on the basis of CYP2J2-substrate interactions in this model.     

CYP2E1 substrate inhibition. Mechanistic interpretation through an effector site for monocyclic compounds

In this study we offer a mechanistic interpretation of the previously known but unexplained substrate inhibition observed for CYP2E1. At low substrate concentrations, p-nitrophenol (pNP) was rapidly turned over (47 min(-1)) with relatively low K(m) (24 microM); nevertheless, at concentrations of >100 microM, the rate of pNP oxidation gradually decreased as a second molecule bound to CYP2E1 through an effector site (K(ss) = 260 microm), which inhibited activity at the catalytic site. 4-Methylpyrazole (4MP) was a potent inhibitor for both sites through a mixed inhibition mechanism. The K(i) for the catalytic site was 2.0 microM. Although we were unable to discriminate whether an EIS or ESI complex formed, the respective inhibition constants were far lower than K(ss). Bicyclic indazole (IND) inhibited catalysis through a single CYP2E1 site (K(i) = 0.12 microM). Similarly, 4MP and IND yielded type II binding spectra that reflected the association of either two 4MP or one IND molecule(s) to CYP2E1, respectively. Based on computational docking studies with a homology model for CYP2E1, the two sites for monocyclic molecules, pNP and 4MP, exist within a narrow channel connecting the active site to the surface of the enzyme. Because of the presence of the heme iron, one site supports catalysis, whereas the other more distal effector site binds molecules that can influence the binding orientation and egress of molecules for the catalytic site. Although IND did not bind these sites simultaneously, the presence of IND at the catalytic site blocked binding at the effector site.     

Rapid P450 heme iron reduction by laser photoexcitation of Mycobacterium tuberculosis CYP121 and CYP51B1. Analysis of CO complexation reactions and reversibility of the P450/P420 equilibrium

We demonstrate that photoexcitation of NAD(P)H reduces heme iron of Mycobacterium tuberculosis P450s CYP121 and CYP51B1 on the microsecond time scale. Rates of formation for the ferrous-carbonmonoxy (Fe(II)-CO) complex were determined across a range of coenzyme/CO concentrations. CYP121 reaction transients were biphasic. A hyperbolic dependence on CO concentration was observed, consistent with the presence of a CO binding site in ferric CYP121. CYP51B1 absorption transients for Fe(II)-CO complex formation were monophasic. The reaction rate was second order with respect to [CO], suggesting the absence of a CO-binding site in ferric CYP51B1. In the absence of CO, heme iron reduction by photoexcited NAD(P)H is fast ( approximately 10,000-11,000 s(-1)) with both P450s. For CYP121, transients revealed initial production of the thiolate-coordinated (P450) complex (absorbance maximum at 448 nm), followed by a slower phase reporting partial conversion to the thiol-coordinated P420 species (at 420 nm). The slow phase amplitude increased at lower pH values, consistent with heme cysteinate protonation underlying the transition. Thus, CO binding occurs to the thiolate-coordinated ferrous form prior to cysteinate protonation. For CYP51B1, slow conversions of both the ferrous/Fe(II)-CO forms to species with spectral maxima at 423/421.5 nm occurred following photoexcitation in the absence/presence of CO. This reflected conversion from ferrous thiolate- to thiol-coordinated forms in both cases, indicating instability of the thiolate-coordinated ferrous CYP51B1. CYP121 Fe(II)-CO complex pH titrations revealed reversible spectral transitions between P450 and P420 forms. Our data provide strong evidence for P420 formation linked to reversible heme thiolate protonation, and demonstrate key differences in heme chemistry and CO binding for CYP121 and CYP51B1.     

Effects of pH and temperature on photoaffinity labeling of Family B G protein-coupled receptors

The efficiency of covalent labeling of a receptor by a photolabile analogue of its natural ligand is dependent on the spatial approximation of the probe and its target. Systematic application of intrinsic photoaffinity labeling to the secretin receptor, a prototypic Family B G protein-coupled receptor, demonstrated reduced efficiency of labeling for amino-terminal and mid-region sites of labeling relative to carboxyl-terminal sites. Reduction of pH from 7.4 to 5.5 and reduction of temperature from 25 °C to 4 °C improved the efficiency of covalent labeling of the receptor with these probes. This correlated with sites of labeling at the interface between the receptor amino terminus and the receptor core, a region containing histidine residues that have their ionization affected in this pH range. Application to the calcitonin receptor, another Family B G protein-coupled receptor, yielded analogous results. These results support the consistent mode of docking peptide ligands to this group of receptors.     

Nile Red is a fluorescent allosteric substrate of cytochrome P450 3A4

Cytochrome P450 3A4 (CYP3A4) plays a critical role in the metabolism of many drugs. CYP3A4 exhibits extraordinary substrate promiscuity and unusual allosteric kinetics. In addition, many CYPs catalyze sequential oxidations on a single substrate, but in most cases, mechanistic details of these processes are not well-established. As a result, in vivo clearance of many drugs and their metabolites is difficult to predict on the basis of the complex in vitro kinetics, and new in vitro probes are required to understand these behaviors. The near-IR fluorescent probe Nile Red, which has strong solvatochromic behavior, was investigated as a probe of allostery and sequential metabolism with CYP3A4. Nile Red binds with apparent Kd values of 0.05 and 2.3 muM, based on a sigmoidal dependence of heme spin state on Nile Red concentration, where the first equivalent of Nile Red increased the high-spin fraction by only 13% of the total change at saturation. Mass spectrometry analysis indicates that Nile Red is metabolized sequentially by CYP3A4 to the N-monoethyl and N-desethyl products, confirming that the immediate vicinity of the heme iron is one binding site. In the presence of CYP3A4, steady-state fluorescence emission and excitation spectra, as well as excited-state lifetimes at varying Nile Red concentrations, indicate a high-affinity site that modulates the fluorescent properties of Nile Red. The Nile Red binding site is competitively eliminated by itraconazole, which is a high-affinity ligand known to coordinate to the heme iron. Together, the data suggest that Nile Red binds to the active site with high affinity ( approximately 50 nM), where it is desolvated in a low-dielectric environment. In addition, Nile Red is sequentially oxidized at rates comparable to or faster than those of other in vitro probes, which emphasizes its utility in the further examination of this important kinetic phenomenon in vitro.     

Design, synthesis and evaluation of the inhibitory selectivity of novel trans-resveratrol analogues on human recombinant CYP1A1, CYP1A2 and CYP1B1

A series of trans-stilbene derivatives containing 4'-methylthio substituent were synthesized and evaluated for inhibitory activities on human recombinant cytochrome P450(s): CYP1A1, CYP1A2, and CYP1B1. CYP1A2-related metabolism of stilbene derivatives was estimated by using NADPH oxidation assay. Additionally, for CYP1A2 and CYP1B1 molecular docking analysis was carried out to provide information on enzyme-ligand interactions and putative site of metabolism. 3,4,5-Trimethoxy-4'-methylthio-trans-stilbene, an analogue of DMU-212 (3,4,5,4'-tetramethoxy-trans-stilbene) was an effective inhibitor of all CYP1 enzymes. On the other hand, 2,3,4-trimethoxy-4'-methylthio-trans-stilbene, appeared to be the most selective inhibitor of the isozymes CYP1A1 and CYP1B1, displaying extremely low affinity towards CYP1A2. Molecular modeling suggested that the most probable binding poses of the methylthiostilbene derivatives in CYP1A2 active sites are those with the methylthio substituent directed towards the heme iron. Products of CYP1A2-catalyzed oxidation of 2,4,5-trimethoxy-4'-methylthiostilbene and 3,4,5-trimethoxy-4'-methylthiostilbene were identified as monohydroxylated compounds. Other studied derivatives appeared to be poor substrates of CYP1A2. Structure-activity relationship analysis rendered better understanding of the mechanism of action of xenobiotic-metabolizing enzymes crucial at the early stage of carcinogenesis.     

Theoretical study of the ligand-CYP2B4 complexes: effect of structure on binding free energies and heme spin state

The molecular origins of temperature-dependent ligand-binding affinities and ligand-induced heme spin state conversion have been investigated using free energy analysis and DFT calculations for substrates and inhibitors of cytochrome P450 2B4 (CYP2B4), employing models of CYP2B4 based on CYP2C5(3LVdH)/CYP2C9 crystal structures, and the results compared with experiment. DFT calculations indicate that large heme-ligand interactions (ca. -15 kcal/mol) are required for inducing a high to low spin heme transition, which is correlated with large molecular electrostatic potentials (approximately -45 kcal/mol) at the ligand heteroatom. While type II ligands often contain oxygen and nitrogen heteroatoms that ligate heme iron, DFT results indicate that BP and MF heme complexes, with weak substrate-heme interactions (ca. -2 kcal/mol), and modest MEPS minima (>-35 kcal/mol) are high spin. In contrast, heme complexes of the CYP2B4 inhibitor, 4PI, the product of benzphetamine metabolism, DMBP, and water are low spin, have substantial heme-ligand interaction energies (<-15 kcal/mol) and deep MEPS minima (<-45 kcal/mol) near their heteroatoms. MMPBSA analysis of MD trajectories were made to estimate binding free energies of these ligands at the heme binding site of CYP2B4. In order to initially assess the realism of this approach, the binding free energy of 4PI inhibitor was computed and found to be a reasonable agreement with experiment: -7.7 kcal/mol [-7.2 kcal/mol (experiment)]. BP was determined to be a good substrate [-6.3 kcal/mol (with heme-ligand water), -7.3 kcal/mol (without ligand water)/-5.8 kcal/mol (experiment)], whereas the binding of MF was negligible, with only marginal binding binding free energy of -1.7 kcal/mol with 2-MF bound [-3.8 kcal/mol (experiment)], both with and without retained heme-ligand water. Analysis of the free energy components reveal that hydrophobic/nonpolar contributions account for approximately 90% of the total binding free energy of these substrates and are the source of their differential and temperature-dependent CYP2B4 binding. The results indicate the underlying origins of the experimentally observed differential binding affinities of BP and MF, and indicate the plausibility of the use of models derived from moderate sequence identity templates in conjunction with approximate free energy methods in the estimation of ligand-P450 binding affinities.     

Differential mechanisms for the inhibition of human cytochrome P450 1A2 by apigenin and genistein

The inhibitory effects of flavonoids on the human cytochrome P450 1A2 (CYP1A2) were examined. Among flavonoids tested, galangin, kaempferol, chrysin, and apigenin were potent inhibitors. Although apigenin belonging to flavones and genistein belonging to isoflavones are similar in the chemical structures, the inhibitory potencies for CYP1A2 were distinguished markedly between these two flavonoids. In computer‐docking simulation, apigenin interacted with the same mode of cocrystallized α‐naphthoflavone in the active site of CYP1A2, and then the B ring of apigenin was placed close to the heme iron of the enzyme with a single orientation. In contrast, the docked genistein conformation showed two different binding modes, and the A ring of genistein was oriented to the heme iron of CYP1A2. Furthermore, the binding free energy of apigenin was lower than that of genistein. These results demonstrate a possible mechanism that causes the differential inhibitory potencies of apigenin and genistein for CYP1A2. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:230–234, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20328     

Evaluation of 3-azidiamantane as photoaffinity probe of cytochrome P450.

3-azidiamantane (DIA-N2) has been shown to be a photolabile carbene-generating probe interacting specifically with cytochrome P450 (P450) active centre. To evaluate the modification of P450 by the probe, radiolabelled [9-3H]-3-azidiamantane was prepared by reductive dehalogenation of its precursor, 3-oxo-9-bromodiamantane ethylene ketal. The synthesis was optimized as the proper precursor and reaction conditions were concerned to produce 96% pure product (overall yield 59%). An incorporation efficacy of the probe photoactivated at 366 nm was examined with two different proteins, BSA and rat phenobarbital-inducible P450 2B1, both having hydrophobic binding sites. Under photolysis the photoaffinity probe generated short-lived (> 90%) intermediates binding immediately to the protein. The yield of photoactivated DIA-N2 incorporation was 12% and 11% for BSA and P450, respectively. The presence of reduced glutathione, a scavenger of reactive intermediates, did not affect the probe incorporation markedly. On the other hand, scavengers entering the P450 active centre, methanol and dithiothreitol, reduced the protein labelling by 36% and 42%, respectively. Similarly, at DIA-N2, aminopyrine (substrates), and metyrapone (inhibitor) 50 times molar excess over the probe, prevented its binding by about 40%. In addition, when photoaffinity labelling was carried out with microsomal preparation, the substrate with a high affinity for the P450 2B1, diamantane, (at 20 times molar excess to the probe) caused 47% inhibition of the P450 covalent labelling. These results, suggesting a high specificity of the probe binding, show that it can be applied as a photoaffinity probe for cytochrome P450 2B1 active centre studies.     

Identification of variable amino acids in the SRS1 region of CYP6B1 modulating furanocoumarin metabolism

The homology model of Papilio polyxenes CYP6B1 places Ile115, one of two variable amino acids, in the SRS1 of various CYP6B subfamily proteins in close proximity to the heme and Ala113, another variable amino acid, in a more distal position. We have constructed mutant CYP6B1 proteins altered at either of these positions and homology models of each based on multiple alignments with crystallized P450 proteins. The homology models suggest the existence of significant structural diversity in the hydrogen bond network surrounding the heme as a result of single point mutations in SRS1. Mutagenesis of Ile115 or Ala113 to other residues present in the insect CYP6B subfamily indicates that these amino acids control the spin state of the heme and, as a result, the catalytic activity of this monooxygenase. In particular, the I115L mutation significantly increases the spin state of the heme coordinately with 2- to 4-fold increases in its turnover of linear furanocoumarins. Other A113V, A113L, A113Q, and A113E mutations display more variation in their effects but, in each case, strong correlations exist between furanocoumarin turnover and heme spin state. These data demonstrate that variable amino acids in SRS1 of the insect CYP6B subfamily exert dramatic effects on the range of furanocoumarins metabolized, even when they occur in positions potentially distal from the substrate. These effects are possibly mediated through rearrangement of the local hydrogen bond network.     

Sites of covalent attachment of CYP4 enzymes to heme: evidence for microheterogeneity of P450 heme orientation

Typical cytochrome P450s secure the heme prosthetic group with a cysteine thiolate ligand bound to the iron, electrostatic interactions with the heme propionate carboxylates, and hydrophobic interactions with the heme periphery. In addition to these interactions, CYP4B1 covalently binds heme through a monoester link furnished, in part, by a conserved I-helix acid, Glu310. Chromatography, mass spectrometry, and NMR have now been utilized to identify the site of attachment on the heme. Native CYP4B1 covalently binds heme solely at the C-5 methyl position. Unexpectedly, recombinant CYP4B1 from insect cells and Escherichia coli also bound their heme covalently at the C-8 methyl position. Structural heterogeneity may be common among recombinant CYP4 proteins because CYP4A3 exhibited this duality. Attempts to evaluate functional heterogeneity were complicated by the complexity of the system. The phenomenon of covalent heme binding to P450 provides a novel method for assessing microheterogeneity in heme orientation and raises questions about the fidelity of heme incorporation in recombinant systems.     

Photoaffinity labeling of P450Cam by an imidazole-tethered benzophenone probe

[(3)H]4-Benzoyl-N-[2-(imidazole-4-yl)ethyl]benzamide ([(3)H]HBP) was synthesized and used to photoaffinity label P450(Cam). The imidazole moiety of HBP anchors the compound in the P450(Cam) active site by coordination of the heme iron, thereby insuring that covalent modification occurs in the active site. Additionally, the imidazole anchor provides a known binding orientation of HBP to P450(Cam) from which conclusions about enzyme structure can be drawn based upon the locations of photoadducted residues. Two sites of adduction were identified by MS analysis of digested, photoaffinity labeled P450(Cam). Photoaffinity labeling experiments in the presence of the type II competitive inhibitor, 1-phenylimidazole, were used to assess the specificity of the photoadducts characterized. One adduct was located at Met103 on the flexible B'/C loop region of P450(Cam). The other adduct was localized on the C-helix at Met121. The implications of these data are discussed.     

1,2,3-triazole-heme interactions in cytochrome p450: functionally competent triazole-water-heme complexes

In comparison to imidazole (IMZ) and 1,2,4-triazole (1,2,4-TRZ), the isosteric 1,2,3-triazole (1,2,3-TRZ) is unrepresented among cytochrome P450 (CYP) inhibitors. This is surprising because 1,2,3-TRZs are easily obtained via "click" chemistry. To understand this underrepresentation of 1,2,3-TRZs among CYP inhibitors, thermodynamic and density functional theory computational studies were performed with unsubstituted IMZ, 1,2,4-TRZ, and 1,2,3-TRZ. The results indicate that the lower affinity of 1,2,3-TRZ for the heme iron includes a large unfavorable entropy term likely originating in solvent-1,2,3-TRZ interactions; the difference is not solely due to differences in the enthalpy of heme-ligand interactions. In addition, the 1,2,3-TRZ fragment was incorporated into a well-established CYP3A4 substrate and mechanism-based inactivator, 17-α-ethynylestradiol (17EE), via click chemistry. This derivative, 17-click, yielded optical spectra consistent with low-spin ferric heme iron (type II) in contrast to 17EE, which yields a high-spin complex (type I). Furthermore, the rate of CYP3A4-mediated metabolism of 17-click was comparable to that of 17EE, with a different regioselectivity. Surprisingly, continuous-wave electron paramagnetic resonance (EPR) and HYSCORE EPR spectroscopy indicate that 17-click does not displace water from the sixth axial ligand position of CYP3A4 as expected for a type II ligand. We propose a binding model in which 17-click pendant 1,2,3-TRZ hydrogen bonds with the sixth axial water ligand. The results demonstrate the potential for 1,2,3-TRZ to form metabolically labile water-bridged low-spin heme complexes, consistent with recent evidence that nitrogenous type II ligands of CYPs can be efficiently metabolized. The specific case of [CYP3A4·17-click] highlights the risk of interpreting CYP-ligand complex structure on the basis of optical spectra.