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Three different types of atom accessibility descriptors are investigated in relation to site of metabolism predictions. To enable the integration of local accessibility we have constructed 2DSASA, a method for the calculation of the atomic solvent accessible surface area that is independent of 3D coordinates. The method was implemented in the SMARTCyp site of metabolism prediction models and improved the results by up to 4 percentage points for nine cytochrome P450 isoforms. The final models are made available at http://www.farma.ku.dk/smartcyp.

The mechanism of cytochrome P450(CYP)-catalyzed hydroxylation of primary amines is currently unclear, and is relevant to drug metabolism. Previous small model calculations have suggested two possible mechanisms: direct N-oxidation and H-abstraction/rebound. We have modeled the N-hydroxylation of (R)-mexiletine in CYP1A2 with hybrid quantum mechanics/molecular mechanics (QM/MM) methods, providing a more detailed and realistic model. Multiple reaction barriers have been calculated at the QM(B3LYP- D)/MM(CHARMM27) level for the direct N-oxidation and H-abstraction/rebound mechanisms. Our calculated barriers indicate that the direct N-oxidation mechanism is preferred and proceeds via the doublet spin state of Compound I. Molecular dynam- ics simulations indicate that the presence of an ordered water molecule in the active site assists in the binding of mexiletine in the active site, but is not a prerequisite for reaction via either mechanism. Several active site residues play a role in the binding of mexiletine in the active site, including Thr124 and Phe226. This work reveals key details in the N-hydroxylation of mexiletine and further demonstrates that mechanistic studies using QM/MM methods are useful for understanding drug metabolism. ispartof: Chemical Research in Toxicology vol:29 issue:6 pages:963-971 ispartof: location:United States status: published

The toxicity of phosphorodithioate pesticides is due to the formation of the active oxane product through desulfurization by cytochrome P450 enzymes, both in humans and insects. During this desulfurization, inhibition of cytochrome P450 and a loss of heme has been observed. Here, we study the mechanism of desulfurization and inhibition with density functional theory, using the B3LYP functional with and without dispersion correction. The results show that a reaction mechanism initiated by sulfur oxidation is most likely, with a reaction barrier of 47 kJ/mol. The sulfur oxidation is followed by a ring-closing mechanism with a barrier of 28 kJ/mol relative to the sulfur-oxidized intermediate. The enzymatic contribution to the ring-closing is very small. It is also shown that the apparent loss of heme might be due to the formation of a previously unknown inhibition complex, which changes the aromatic conjugation of the porphyrin ring. We also show that including dispersion correction has significant effects on a ring closure transition state (∼30 kJ/mol), whereas effects on the other steps in the reaction are relatively small (4-15 kJ/mol).

Three possible mechanisms have been suggested for the hydroxylation of primary and secondary amines by the cytochrome P450 enzyme family. We show that for the hydroxylation of primary alkyl amines, both the hydrogen abstraction and rebound mechanism and the direct oxygen transfer mechanism can contribute to the formation of the hydroxylated product. We also show that in the hydrogen abstraction and rebound mechanism the rebound step has higher activation energy than the hydrogen abstraction step, which is the opposite of the hydroxylation of aliphatic carbon atoms.

Cytochrome P450 enzymes (CYPs) metabolize alkyl- and arylamines, generating several different products. For the primary and secondary amines, some of these reactions result in hydroxylated amines, which may be toxic. Thus, when designing new drugs containing amine groups, it is important to be able to predict if a given compound will be a substrate for CYPs, in order to avoid toxic metabolites, and hence to understand the mechanism that is utilized by CYPs. Two possible mechanisms, for the N-hydroxylation of primary and secondary amines mediated by CYPs, are studied by density functional theory (DFT) for four different amines (aniline, N-methylaniline, propan-2-amine, and dimethylamine). The hydrogen abstraction and rebound mechanism is found to be preferred over a direct oxygen transfer mechanism for all four amines. However, in contrast to the same mechanism for the hydroxylation of aliphatic carbon atoms, the rebound step is shown to be rate-limiting in most cases.

We have estimated the activation energy for hydrogen abstraction by compound I in cytochrome P450 for a diverse set of 24 small organic substrates using state-of-the-art density functional theory (B3LYP). We then show that these results can be reproduced by computationally less demanding methods, for example, by using small organic mimics of compound I with both B3LYP and the semiempirical AM1 method (mean absolute error of 3-4 kJ/mol) or by calculating the bond dissociation energy, without relaxation of the radical (B3LYP) or estimated from three-point fit to a Morse potential (AM1; errors of 4 and 5 kJ/mol, respectively). We can assign activation energies of 74, 61, 53, 47, and 30 kJ/mol to primary carbons, secondary/tertiary carbons, carbons with adjacent sp(2) or aromatic groups, ethers/thioethers, and amines, respectively, which gives a very simple and predictive model. Finally, some of the less demanding methods are applied to study the CYP3A4 metabolism of progesterone and dextromethorphan.

Chemokine receptors are involved in trafficking of leukocytes and represent targets for autoimmune conditions, inflammatory diseases, viral infections, and cancer. We recently published CCR1, CCR8, and CCR5 agonists and positive modulators based on a three metal-ion chelator series: 2,2'-bipyridine, 1,10-phenanthroline, and 2,2';6',2″-terpyridine. Here, we have performed an in-depth structure-activity relationship study and tested eight new optimized analogs. Using density functional theory calculations we demonstrate that the chelator zinc affinities depend on how electron-donating and -withdrawing substituents modulate the partial charges of chelating nitrogens. The zinc affinity was found to constitute the major factor for receptor potency, although the activity of some chelators deviate suggesting favorable or unfavorable interactions. Hydrophobic and halogen substituents are generally better accommodated in the receptors than polar groups. The new analog brominated terpyridine (29) resulted in the highest chelator potencies observed so far CCR1 (EC50: 0.49 μM) and CCR8 (EC50: 0.28 μM). Furthermore, we identified the first selective CCR5 agonist chelator, meta dithiomethylated bipyridine (23). The structure-activity relationships contribute to small-molecule drug development, and the novel chelators constitute valuable tools for studies of structural mechanisms for chemokine receptor activation.

To enhance the discrimination rate for methods applying structural alerts and biotransformation rules in the prediction of toxicity and drug metabolism we have developed a set of novel fragment based atomic descriptors. These atomic descriptors encode the properties of the fragments separating an atom from the closest end of a branch or the molecule. The end of a branch and the end of a molecule, as well as the selection of the fragments, are made by an algorithm that uses only the distance matrix of the molecule. The novel descriptors are applied to a small set of biotransformation rules and are shown to be able to reduce the number of unconfirmed positives by up to 58 %.

A ligand-based method based on the SMARTCyp approach that predicts the sites of cytochrome P450 2D6-mediated metabolism of druglike molecules has been developed. The method uses only two descriptors besides the reactivity from SMARTCyp: the distance to a protonated nitrogen atom and the distance to the end of the molecule. Hence, the site of metabolism is predicted directly from the 2D structure of a molecule, without requiring calculation of electronic properties or generation of 3D structures. Testing on an independent test set gives an area under the curve value of 0.94, and a site of metabolism is found among the top two ranked atoms for 91% of the compounds.

This article describes RegioSelectivity-Predictor (RS-Predictor), a new in silico method for generating predictive models of P450-mediated metabolism for drug-like compounds. Within this method, potential sites of metabolism (SOMs) are represented as "metabolophores": A concept that describes the hierarchical combination of topological and quantum chemical descriptors needed to represent the reactivity of potential metabolic reaction sites. RS-Predictor modeling involves the use of metabolophore descriptors together with multiple-instance ranking (MIRank) to generate an optimized descriptor weight vector that encodes regioselectivity trends across all cases in a training set. The resulting pathway-independent (O-dealkylation vs N-oxidation vs Csp(3) hydroxylation, etc.), isozyme-specific regioselectivity model may be used to predict potential metabolic liabilities. In the present work, cross-validated RS-Predictor models were generated for a set of 394 substrates of CYP 3A4 as a proof-of-principle for the method. Rank aggregation was then employed to merge independently generated predictions for each substrate into a single consensus prediction. The resulting consensus RS-Predictor models were shown to reliably identify at least one observed site of metabolism in the top two rank-positions on 78% of the substrates. Comparisons between RS-Predictor and previously described regioselectivity prediction methods reveal new insights into how in silico metabolite prediction methods should be compared.

Summary The ectodomain of olfactory cell adhesion molecule (OCAM/NCAM2/RNCAM) consists of five immunoglobulin (Ig) domains (IgI–V), followed by two fibronectin-type 3 (Fn3) domains (Fn3I–II). A complete structural model of the entire ectodomain of human OCAM has been assembled from crystal structures of six recombinant proteins corresponding to different regions of the ectodomain. The model is the longest experimentally based composite structural model of an entire IgCAM ectodomain. It displays an essentially linear arrangement of IgI–V, followed by bends between IgV and Fn3I and between Fn3I and Fn3II. Proteins containing IgI–IgII domains formed stable homodimers in solution and in crystals. Dimerization could be disrupted in vitro by mutations in the dimer interface region. In conjunction with the bent ectodomain conformation, which can position IgI–V parallel with the cell surface, the IgI–IgII dimerization enables OCAM-mediated trans- interactions with an intercellular distance of about 20 nm, which is consistent with that observed in synapses.

Cytochrome P450 mediated metabolism of drugs is one of the major determinants of their kinetic profile, and prediction of this metabolism is therefore highly relevant during the drug discovery and development process. A new rule-based method, based on results from density functional theory calculations, for predicting activation energies for aliphatic and aromatic oxidations by cytochromes P450 is developed and compared with several other methods. Although the applicability of the method is currently limited to a subset of P450 reactions, these reactions describe more than 90% of the metabolites. The rules employed are relatively few and general, and when combined with solvent-accessible surface area calculations to account for steric accessibility, the method gives a major P450 metabolite as first-ranked position for 75% of the substrates, and ranked in the top three for 90% of substrates for a set of 20 substrates. In combination with docking, it can predict isoform-specific metabolism, and we apply this on CYP1A2 with very good results on 81 substrates, for which we find a major metabolite ranked in the top three for 90% of the substrates (100% in the training set and 87% in the larger test set). © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.

This chapter illustrates how quantum chemical calculations can be used to elucidate structural and functional aspects of tetrapyrrole cofactors, focusing on porphyrins, cobalamins, coenzyme F430, and chlorophyll. A particular emphasis is put on the biochemical significance of axial ligands, which can tune the function of the tetrapyrroles. With the use of quantum chemical calculations, it is possible to draw important conclusions regarding aspects of tetrapyrroles that could not otherwise be accessed. The results show that the general reactivity is mainly determined by the metal and the tetrapyrrole ring system, whereas the electronic structure and reactivity are tuned by the choice of axial ligands, providing a unique insight into the design of cofactors in nature. (Less)

Peroxidases, oxygenases and catalases have similar high-valent metal-ion intermediates in their respective reaction cycles. In this review, haem-based examples will be discussed. The intermediates of the haem-containing enzymes have been extensively studied for many years by different spectroscopic methods like UV-Vis, EPR (electron paramagnetic resonance), resonance Raman, Mossbauer and MCD (magnetic circular dichroism). The first crystal structure of one of these high-valent intermediates was on cytochrome c peroxidase in 1987. Since then, structures have appeared for catalases in 1996, 2002, 2003, putatively for cytochrome P450 in 2000, for myoglobin in 2002, for horseradish peroxidase in 2002 and for cytochrome c peroxidase again in 1994 and 2003. This review will focus on the most recent structural investigations for the different intermediates of these proteins. The structures of these intermediates will also be viewed in light of quantum mechanical (QM) calculations on haem models. In particular quantum refinement, which is a combination of QM calculations and crystallography, will be discussed. Only small structural changes accompany the generation of these intermediates. The crystal structures show that the compound I state, with a so called pi-cation radical on the haem group, has a relatively short iron-oxygen bond (1.67-1.76A) in agreement with a double-bond character, while the compound II state or the compound I state with a radical on an amino acid residue have a relatively long iron-oxygen bond (1.86-1.92A) in agreement with a single-bond character where the oxygen-atom is protonated.

Three microperoxidases--hemin-6(7)-gly-gly-his methyl ester (HGGH), mesohemin-6(7)-gly-gly-his methyl ester (MGGH) and deuterohemin-6(7)-gly-gly-his methyl ester (DGGH)--have been prepared as models for heme-containing peroxidases by condensation of glycyl-glycyl-L-histidine methyl ester with the propionic side chains of hemin, mesohemin and deuterohemin, respectively. The three microperoxidases differ in two substituents, R, of the protoporphyrin IX framework (HGGH: R=vinyl, MGGH: R=ethyl, DGGH: R=H). X-band and high field EPR spectra show that the microperoxidases exhibit spectroscopic properties similar to those of metmyoglobin, i.e. a high spin ferric S=5/2 signal at g(perpendicular)=6 and g parallel)=2 and an estimated D value of 7.5+/-1cm(-1). The catalytic activities of the microperoxidases towards K4[Fe(CN)6], L-tyrosine methyl ester and 2,2'-azino(bis(3-ethylbenzothiazoline-6-sulfonic acid)) (ABTS) have been investigated. It was found that all three microperoxidases exhibit peroxidase activity and that the reactions follow the generally accepted peroxidase reaction scheme [Biochem. J. 145 (1975) 93-103] with the exception that the initial formation of a Compound I analogue is the rate-limiting step for the whole process. The general activity trend was found to be MGGH approximately DGGH>HGGH. For each microperoxidase, DFT calculations (B3LYP) were made on the reactions of compounds 0, I and II with H+, e- and H+ + e-, respectively, in order to probe the possible relationship between the nature of the 2- and 4-substituents of the hemin and the observed reactivity. The computational modeling indicates that the relative energy differences are very small; solvation and electrostatic effects may be factors that decide the relative activities of the microperoxidases.

We present a systematic investigation of how the axial ligand in heme proteins influences the geometry, electronic structure, and spin states of the active site, and the energies of the reaction cycles. Using the density functional B3LYP method and medium-sized basis sets, we have compared models with His, His+Asp, Cys, Tyr, and Tyr+Arg as found in myoglobin and hemoglobin, peroxidases, cytochrome P450, and heme catalases, respectively. We have studied 12 reactants and intermediates of the reaction cycles of these enzymes, including complexes with H(2)O, OH(-), O(2-), CH(3)OH, O(2), H(2)O(2), and HO(2)(-) in various formal oxidation states of the iron ion (II to V). The results show that His gives ~0.6 V higher reduction potentials than the other ligands. In particular, it is harder to reduce and protonate the O(2) complex with His than with the other ligands, in accordance with the O(2) carrier function of globins and the oxidative chemistry of the other proteins. For most properties, the trend Cys

A novel lab-on-a-chip nanotree enzyme reactor is demonstrated for the detection of acetylcholine. The reactors are intended for use in the RISFET (regional ion sensitive field effect transistor) nanosensor, and are constructed from gold-tipped branched nanorod structures grown on SiN(x)-covered wafers. Two different reactors are shown: one with simple, one-dimensional nanorods and one with branched nanorod structures (nanotrees). Significantly higher enzymatic activity is found for the nanotree reactors than for the nanorod reactors, most likely due to the increased gold surface area and thereby higher enzyme binding capacity. A theoretical calculation is included to show how the enzyme kinetics and hence the sensitivity can be influenced and increased by the control of electrical fields in relation to the active sites of enzymes in an electronic biosensor. The possible effects of electrical fields employed in the RISFET on the function of acetylcholine esterase is investigated using quantum chemical methods, which show that the small electric field strengths used are unlikely to affect enzyme kinetics. Acetylcholine esterase activity is determined using choline oxidase and peroxidase by measuring the amount of choline formed using the chemiluminescent luminol reaction.