Biomedical subjects

Danni L Harris

Publications and source records attributed to Danni L Harris.

8 recordsLinked to original sources

PubMed2006

Metabolism of bergamottin by cytochromes P450 2B6 and 3A5.

Cytochromes P450 (P450) 2B6 and 3A5 are inactivated by bergamottin (BG). P450 2B6 metabolized BG primarily to M3 and M4 and one minor metabolite (M1). The metabolites were analyzed, and the data indicated that M1 was bergaptol, M3 was 5'-OH-BG, and M4 was a mixture of 6'- and 7'-OH-BG. Because 6'- and 7'-OH-BG were the primary metabolites, it suggested that P450 2B6 preferentially oxidized the geranyloxy chain of BG. Metabolism of BG by P450 3A5 resulted in three major metabolites: [bergaptol, M3 (5'-OH-BG), and M5 (2'-OH-BG)], and two minor metabolites [M2 (6',7'-dihydroxy-BG) and M4 (6'- and 7'-OH-BG)]. Because bergaptol was the most abundant metabolite formed, it suggested that P450 3A5 metabolized BG mainly by cleaving the geranyl-oxy chain. Molecular modeling studies confirmed that docking of BG in the P450 2B6 active site favors oxidation in the terminal region of the geranyl-oxy chain, whereas positioning the 2'-carbon of BG nearest the heme iron is preferred by P450 3A5. Glutathione (GSH)-BG conjugates were formed by both P450. Each enzyme predominantly formed conjugates with m/z values of 662. Tandem mass spectrometry analysis of the GSH conjugates indicated that the oxidation forming a reactive intermediate occurred on the furan moiety of BG, presumably through the initial formation of an epoxide at the furan double bond. The data indicate that oxidation of the geranyl-oxy chain resulted in the formation of stable metabolites of BG, whereas oxidation of the furan ring produced reactive intermediates that may be responsible for binding to and inactivating P450 2B6 and 3A4.

Aryl Hydrocarbon Hydroxylases↗

PubMed2005

P450 active site architecture and reversibility: inactivation of cytochromes P450 2B4 and 2B4 T302A by tert-butyl acetylenes.

The inactivations of P450 2B4 and the T302A mutant of 2B4 by tert-butyl acetylene (tBA) and the inactivation of 2B4 T302A by tert-butyl 1-methyl-2-propynyl ether (tBMP) have been investigated. tBA and tBMP inactivated both enzymes in a mechanism-based manner with the losses in enzymatic activity corresponding closely to losses in P450 heme. HPLC and ESI-LC-MS analysis detected two different tBA- or tBMP-modified heme products with masses of 661 and 705 Da, respectively. Interestingly, the inactivations of the P450s 2B4 by tBA and tBMP were partially reversible by dialysis, and the tBA- or tBMP-modified heme products could only be observed with ESI-LC-MS/MS when the inactivated samples were acidified prior to analysis, indicating a requirement for protons in the formation of stable heme adducts in both the wild-type and mutant 2B4 enzymes. Results of studies using artificial oxidants to support enzyme inactivation suggest that the oxenoid-iron activated oxygen species is preferentially utilized during the inactivation of the P450s 2B4 by tBA. These results argue against the use of a peroxo-iron species by P450 2B4 T302A. Molecular dynamics studies of wild-type P450 2B4 reveal that contiguous hydrogen bond networks, including structural waters, link a conserved glutamate (E301) to the distal oxygen of the peroxo-heme species via threonine 302. Interestingly, models of 2B4 T302A reveal that a compensatory, ordered hydrogen bond network forms despite the removal of T302. These results indicate that while T302 may play a role in proton delivery in the formation of the oxenoid-iron complex and in the stabilization of acetylene heme adducts in 2B4, it is not essential for proton delivery given the presence of E301 in the binding site.

Alanine↗

PubMed2004

Antagonist analogue of 6-[3'-(1-adamantyl)-4'-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN) family of apoptosis inducers that effectively blocks AHPN-induced apoptosis but not cell-cycle arrest.

The retinoid 6-[3'-(1-adamantyl)-4'-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN) and its active analogues induce cell-cycle arrest and programmed cell death (apoptosis) in cancer cells independently of retinoic acid receptor (RAR) interaction. Its analogue, (E)-4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-(3'-acetamidopropyloxy)cinnamic acid (3-A-AHPC) selectively antagonized cell apoptotic events (TR3/nur77/NGFI-B expression and nuclear-to-mitochondrial translocation) but not the proliferative events (cell-cycle arrest and p21(WAF1/CIP1) expression) induced by proapoptotic AHPN and its analogues. The syntheses of 3-A-AHPC and proapoptotic (E)-6-[3'-(1-adamantyl)-4'-hydroxyphenyl]-5-chloronaphthalenecarboxylic acid (5-Cl-AHPN) are described. Computational studies on AHPN, AHPC, and three substituted analogues (5-Cl-AHPN, 3-Cl-AHPC, and 3-A-AHPC) suggested reasons for their diametric effects on RAR activation. Density functional theory studies indicated that the 1-adamantyl (1-Ad) groups of the AHPN and AHPC configurations assumed positions that were nearly planar with the aromatic rings of their polar termini. In contrast, in the configurations of the substituted analogues having chloro and 3-acetamidopropyloxy groups, rather than a hydrogen, ortho to the diaryl bonds, the diaryl bond torsion angles increased so that the 1-Ad groups were oriented out of this plane. Docking and molecular dynamics of AHPN, AHPC, and these substituted analogues in the RARgamma ligand-binding domain illustrated how specific substituents on the AHPN and AHPC scaffolds modulated the positions and dynamics of the 1-Ad groups. As a result, the position of RARgamma helix H12 in forming the coactivator-binding site was impacted in a manner consistent with the experimental effect of each analogue on RARgamma transcriptional activation.

Adamantane↗

PubMed2004

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.

Amino Acid Sequence↗

PubMed2004

In silico predictive metabolism: a structural/electronic filter method.

This article reviews recent progress in our current fundamental understanding of structure, mechanism and metabolism of cytochrome P450s, the principal enzymatic system involved in phase I metabolism, with the aim of critically appraising the potential of in silico predictive metabolism based on CYP450 structure, CYP450-drug configuration sampling, and accurate quantum chemical determinants of the rate limiting steps of drug metabolism. Such an approach does not rely on training sets or multivariate statistical approaches. The latter approaches have been comprehensively described in a review by Wilson et al (Curr Opin Drug Discovery Dev (2003) 6(1):123-128).

Animals↗

PubMed2003

Determination of the rate of reduction of oxyferrous cytochrome P450 2B4 by 5-deazariboflavin adenine dinucleotide T491V cytochrome P450 reductase.

The use of 5-deazaFAD T491V cytochrome P450 reductase has made it possible to directly measure the rate of electron transfer to microsomal oxyferrous cytochrome (cyt) P450 2B4. In this reductase the FMN moiety can be reduced to the hydroquinone, FMNH(2), while the 5-deazaFAD moiety remains oxidized [Zhang, H., et al. (2003) Biochemistry 42, 6804-6813]. The rate of electron transfer from 5-deazaFAD cyt P450 reductase to oxyferrous cyt P450 was determined by rapidly mixing the ferrous cyt P450-2-electron-reduced 5-deazaFAD T491V reductase complex with oxygen in the presence of substrate. The 5-deazaFAD T491V reductase which can only donate a single electron reduces the oxyferrous cyt P450 and oxidizes to the air-stable semiquinone, with rate constants of 8.4 and 0.37 s(-1) at 15 degrees C. Surprisingly, oxyferrous cyt P450 turns over more slowly with a rate constant of 0.09 s(-1), which is the rate of catalysis under steady-state conditions at 15 degrees C (k(cat) = 0.08 s(-1)). In contrast, the rate constant for electron transfer from ferrous cyt b(5) to oxyferrous cyt P450 is 10 s(-1) with oxyferrous cyt P450 and cyt b(5) simultaneously undergoing spectral changes. Quantitative analyses by LC-MS/MS revealed that the product, norbenzphetamine, was formed with a coupling efficiency of 52% with cyt b(5) and 32% with 5-deazaFAD T491V reductase. Collectively, these results suggest that during catalysis a relatively stable reduced oxyferrous intermediate of cyt P450 is formed in the presence of cyt P450 reductase but not cyt b(5) and that the rate-limiting step in catalysis follows introduction of the second electron.

Aryl Hydrocarbon Hydroxylases↗

PubMed2002

Oxidation and electronic state dependence of proton transfer in the enzymatic cycle of cytochrome P450eryF.

Hydrogen bond networks, consisting of hydrogen bonded waters anchored by polar/acidic amino acid sidechains, are often present in the vicinity of the oxygen binding clefts of P450s. Density functional and quantum dynamics calculations of a O(2) binding cleft network model of cytochrome P450eryF(CYP107A1) indicate that such structural motifs facilitate ultrafast proton transfer from network waters to the dioxygen of the reduced oxyferrous species via a multiple proton translocation mechanism with barriers of 7-10 kcal/mol on its doublet ground state, and that the energies of the proton transfer reactant and constrained proton transfer products have an electronic and oxidation state dependence [J. Am. Chem. Soc. 124 (2002) 1430]. In the present study, the origin of the oxidation state dependence is shown to have its roots in differential proton affinities while the electronic state dependence of the reduced oxyferrous heme has its origins in subtle differences in network topologies near the transition state of the initial proton transfer event. Relaxed potential surface scans and unconstrained proton transfer product optimizations indicate that the proton transfer product in both the singlet oxyferrous heme and the reduced oxyferrous heme species in a quartet state are not viable stable (bound) states relative to the reactant form. While the proton affinity of H(3)O(+) is sufficient for it to protonate both the oxyferrous and the reduced oxyferrous heme species, hydrogen bond network stabilized water is only capable of protonating the reduced oxyferrous form. This interpretation is substantiated by study of the NO bound reduced ferrous heme of P450nor, which is isoelectronic with the oxyferrous heme and has a similar proton affinity. Density functional calculations on a more extensive O(2) binding cleft model support the multiple proton translocation mechanism of transfer but indicates that the significant negative charge density on the bound dioxygen of the reduced oxyferrous heme species, in its doublet ground state, polarizes the associated hydrogen bond network sufficiently so as to result in short, strong, low-barrier hydrogen bonds. The computed O-H-O bond distances are less than 2.55 A and have a near degeneracy of the proton transfer reactant and initial (sudden) proton transfer products. These low-barrier hydrogen bond features, in addition to the finding of a (zero point uncorrected) barrier of 1.3 kcal/mol, indicate that proton transfer from water to the distal oxygen should be rapid, facile and may not require large curvature tunneling as originally suggested by use of a smaller model. An initial assessment of protonation of the reduced oxyferrous heme distal oxygen by a model of 6-deoxyerythronolide B (6-DEB) indicates it to be low barrier (3.8 kcal/mol) and exothermic (-2.9 kcal/mol). The combined results indicate the plausibility of simultaneous diprotonation of the distal oxygen of the reduced oxyferrous heme, leading to O-O bond scission, using the combined water network and 6-DEB substrate protonation agents.

Bacterial Proteins↗

PubMed2002

Proton-transfer dynamics in the activation of cytochrome P450eryF.

Molecular dynamics simulations are combined with quantum chemistry calculations of instantaneous proton-transfer energy profiles to investigate proton-transfer events in the transient pathway of cytochrome P450eryF (6-deoxyerythronolide B hydroxylase; CYP107A1), from the oxyferrous species to the catalytically active ferryl oxygen species (compound I). This reaction is one of the most fundamental unresolved aspects in the mechanism of oxidation that is common to all cytochrome P450s. We find that this process involves an ultrafast proton transfer from the crystallographic water molecule W519 to the distal oxygen bound to the heme group, and a subsequent proton-transfer event from W564 to W519. Both proton-transfer events are found to be endothermic in the oxyferrous state, suggesting that the oxyferrous reduction is mechanistically linked to the proton-transfer dynamics. These findings indicate that the hydrogen bond network, proximate to the O(2)-binding cleft, plays a crucial functional role in the enzymatic activation of P450s. Our results are consistent with the effect of mutations on the enzymatic efficacy.

Bacterial Proteins↗