Quantum Mechanics/Molecular Mechanics Modeling of Drug Metabolism: Mexiletine N-Hydroxylation by Cytochrome P450 1A2.

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 dynamics 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 this 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 of the N-hydroxylation of mexiletine and further demonstrates that mechanistic studies using QM/MM methods are useful for understanding drug metabolism.

Mechanism of the N-hydroxylation of primary and secondary amines by cytochrome P450.

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.

Density functional theory study on the formation of reactive benzoquinone imines by hydrogen abstraction.

Many drug compounds are oxidized by cytochrome P450 (CYP) enzymes to form reactive metabolites. This study presents density functional theory calculations of the CYP-mediated metabolism of acetaminophen and a series of related compounds that can form reactive metabolites by hydrogen abstraction. The substitution pattern affects the activation barrier for hydrogen abstraction by up to 30 kJ/mol. A correlation (R(2) = 0.72) between the transition-state energies and the corresponding substrate radical energies has been established. Using this correlation is significantly less time-demanding than using the porphyrin model to determine the activation energies. We have used this correlation on monosubstituted phenols to rationalize the effect of the various substituents in the drug compounds. In addition to facilitating a chemical interpretation, the approach is sufficiently fast and reliable to be used as an in silico method in the design of new compounds with improved metabolic stability.

WhichCyp: prediction of cytochromes P450 inhibition.

SUMMARY: In this work we present WhichCyp, a tool for prediction of which cytochromes P450 isoforms (among 1A2, 2C9, 2C19, 2D6 and 3A4) a given molecule is likely to inhibit. The models are built from experimental high-throughput data using support vector machines and molecular signatures. AVAILABILITY: The WhichCyp server is freely available for use on the web at http://drug.ku.dk/whichcyp, where the WhichCyp Java program and source code is also available for download.

RS-WebPredictor: a server for predicting CYP-mediated sites of metabolism on drug-like molecules.

SUMMARY: Regioselectivity-WebPredictor (RS-WebPredictor) is a server that predicts isozyme-specific cytochrome P450 (CYP)-mediated sites of metabolism (SOMs) on drug-like molecules. Predictions may be made for the promiscuous 2C9, 2D6 and 3A4 CYP isozymes, as well as CYPs 1A2, 2A6, 2B6, 2C8, 2C19 and 2E1. RS-WebPredictor is the first freely accessible server that predicts the regioselectivity of the last six isozymes. Server execution time is fast, taking on average 2s to encode a submitted molecule and 1s to apply a given model, allowing for high-throughput use in lead optimization projects. AVAILABILITY: RS-WebPredictor is accessible for free use at http://reccr.chem.rpi.edu/Software/RS-WebPredictor/

The contribution of atom accessibility to site of metabolism models for cytochromes P450.

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.

FAst MEtabolizer (FAME): A rapid and accurate predictor of sites of metabolism in multiple species by endogenous enzymes.

FAst MEtabolizer (FAME) is a fast and accurate predictor of sites of metabolism (SoMs). It is based on a collection of random forest models trained on diverse chemical data sets of more than 20 000 molecules annotated with their experimentally determined SoMs. Using a comprehensive set of available data, FAME aims to assess metabolic processes from a holistic point of view. It is not limited to a specific enzyme family or species. Besides a global model, dedicated models are available for human, rat, and dog metabolism; specific prediction of phase I and II metabolism is also supported. FAME is able to identify at least one known SoM among the top-1, top-2, and top-3 highest ranked atom positions in up to 71%, 81%, and 87% of all cases tested, respectively. These prediction rates are comparable to or better than SoM predictors focused on specific enzyme families (such as cytochrome P450s), despite the fact that FAME uses only seven chemical descriptors. FAME covers a very broad chemical space, which together with its inter- and extrapolation power makes it applicable to a wide range of chemicals. Predictions take less than 2.5 s per molecule in batch mode on an Ultrabook. Results are visualized using Jmol, with the most likely SoMs highlighted.

Structure-activity relationships and identification of optmized CC-chemokine receptor CCR1, 5, and 8 metal-ion chelators.

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.

RS-Predictor models augmented with SMARTCyp reactivities: robust metabolic regioselectivity predictions for nine CYP isozymes.

RS-Predictor is a tool for creating pathway-independent, isozyme-specific, site of metabolism (SOM) prediction models using any set of known cytochrome P450 (CYP) substrates and metabolites. Until now, the RS-Predictor method was only trained and validated on CYP 3A4 data, but in the present study, we report on the versatility the RS-Predictor modeling paradigm by creating and testing regioselectivity models for substrates of the nine most important CYP isozymes. Through curation of source literature, we have assembled 680 substrates distributed among CYPs 1A2, 2A6, 2B6, 2C19, 2C8, 2C9, 2D6, 2E1, and 3A4, the largest publicly accessible collection of P450 ligands and metabolites released to date. A comprehensive investigation into the importance of different descriptor classes for identifying the regioselectivity mediated by each isozyme is made through the generation of multiple independent RS-Predictor models for each set of isozyme substrates. Two of these models include a density functional theory (DFT) reactivity descriptor derived from SMARTCyp. Optimal combinations of RS-Predictor and SMARTCyp are shown to have stronger performance than either method alone, while also exceeding the accuracy of the commercial regioselectivity prediction methods distributed by Optibrium and Schrödinger, correctly identifying a large proportion of the metabolites in each substrate set within the top two rank-positions: 1A2 (83.0%), 2A6 (85.7%), 2B6 (82.1%), 2C19 (86.2%), 2C8 (83.8%), 2C9 (84.5%), 2D6 (85.9%), 2E1 (82.8%), 3A4 (82.3%), and merged (86.0%). Comprehensive datamining of each substrate set and careful statistical analyses of the predictions made by the different models revealed new insights into molecular features that control metabolic regioselectivity and enable accurate prospective prediction of likely SOMs.

Binding affinities in the SAMPL3 trypsin and host-guest blind tests estimated with the MM/PBSA and LIE methods.

We have estimated affinities for the binding of 34 ligands to trypsin and nine guest molecules to three different hosts in the SAMPL3 blind challenge, using the MM/PBSA, MM/GBSA, LIE, continuum LIE, and Glide score methods. For the trypsin challenge, none of the methods were able to accurately predict the experimental results. For the MM/GB(PB)SA and LIE methods, the rankings were essentially random and the mean absolute deviations were much worse than a null hypothesis giving the same affinity to all ligand. Glide scoring gave a Kendall's τ index better than random, but the ranking is still only mediocre, τ = 0.2. However, the range of affinities is small and most of the pairs of ligands have an experimental affinity difference that is not statistically significant. Removing those pairs improves the ranking metric to 0.4-1.0 for all methods except CLIE. Half of the trypsin ligands were non-binders according to the binding assay. The LIE methods could not separate the inactive ligands from the active ones better than a random guess, whereas MM/GBSA and MM/PBSA were slightly better than random (area under the receiver-operating-characteristic curve, AUC = 0.65-0.68), and Glide scoring was even better (AUC = 0.79). For the first host, MM/GBSA and MM/PBSA reproduce the experimental ranking fairly good, with τ = 0.6 and 0.5, respectively, whereas the Glide scoring was considerably worse, with a τ = 0.4, highlighting that the success of the methods is system-dependent.

The SMARTCyp cytochrome P450 metabolism prediction server.

SUMMARY: The SMARTCyp server is the first web application for site of metabolism prediction of cytochrome P450-mediated drug metabolism. AVAILABILITY: The SMARTCyp server is freely available for use on the web at www.farma.ku.dk/smartcyp where the SMARTCyp Java program and source code is also available for download. CONTACT: smartcyp@farma.ku.dk; lo@farma.ku.dk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

RS-predictor: a new tool for predicting sites of cytochrome P450-mediated metabolism applied to CYP 3A4.

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.

Branched nanotrees with immobilized acetylcholine esterase for nanobiosensor applications.

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.

The accuracy of geometries for iron porphyrin complexes from density functional theory.

Iron porphyrin complexes are cofactors in many important proteins such as cytochromes P450, hemoglobin, heme peroxidases, etc. Many computational studies on these systems have been done over the past decade. In this study, the performance of some of the most commonly used density functional theory functionals is evaluated with regard to how they reproduce experimental structures. Seven different functionals (BP86, PBE, PBE0, TPSS, TPSSH, B3LYP, and B97-D) are used to study eight different iron porphyrin complexes. The results show that the TPSSH, PBE0, and TPSS functionals give the best results (absolute bond distance deviations of 0.015-0.016 A), but the geometries are well-reproduced by all functionals except B3LYP. We also test four different basis sets of double-zeta quality, and we find that a combination of double-zeta basis set of Schafer et al. on the iron atom and the 6-31G* basis set on the other atoms performs best. Finally, we remove the porphyrin side chains and increase the basis set size systematically to see if this affects the results. We show that basis sets larger than double-zeta quality are not necessary to get accurate geometries, and nonaromatic side chains do not affect the geometries.

Protonation of the proximal histidine ligand in heme peroxidases.

The heme peroxidases have a histidine group as the axial ligand of iron. This ligand forms a hydrogen bond to an aspartate carboxylate group by the other nitrogen atom in the side chain. The aspartate is not present in the globins and it has been suggested that it gives an imidazolate character to the histidine ligand. Quantum chemical calculations have indicated that the properties of the heme site strongly depend on the position of the proton in this hydrogen bond. Therefore, we have studied the location of this proton in all intermediates in the reaction mechanism, using a set of different quantum mechanical and combined experimental and computational methods. Quantum refinements of a crystal structure of the resting FeIII state in yeast cytochrome c peroxidase show that the geometric differences of the two states are so small that it cannot be unambiguously decided where the proton is in the crystal structure. Vacuum calculations indicate that the position of the proton is sensitive to the surroundings and to the side chains of the porphyrin ring. Combined quantum and molecular mechanics (QM/MM) calculations indicate that the proton prefers to reside on the His ligand in all states in the reaction mechanism of the peroxidases. QM/MM free energy perturbations confirm these results, but reduce the energy difference between the two states to 12-44 kJ/mol.

Prediction of activation energies for aromatic oxidation by cytochrome P450.

We have estimated the activation energy for aromatic oxidation by compound I in cytochrome P450 for a diverse set of 17 substrates using state-of-the-art density functional theory (B3LYP) with large basis sets. The activation energies vary from 60 to 87 kJ/mol. We then test if these results can be reproduced by computationally less demanding methods. The best methods (a B3LYP calculation of the activation energy of a methoxy-radical model or a partial least-squares model of the semiempirical AM1 bond dissociation energies and spin densities of the tetrahedral intermediate for both a hydroxyl-cation and a hydroxyl-radical model) give correlations with r(2) of 0.8 and mean absolute deviations of 3 kJ/mol. Finally, we apply these simpler methods on several sets of reactions for which experimental data are available and show that we can predict the reactive sites by combining calculations of the activation energies with the solvent-accessible surface area of each site.

Dynamics of water molecules in the active-site cavity of human cytochromes P450.

We have studied the dynamics of water molecules in six crystal structures of four human cytochromes P450, 2A6, 2C8, 2C9, and 3A4, with molecular dynamics simulations. In the crystal structures, only a few water molecules are seen and the reported sizes of the active-site cavity vary a lot. In the simulations, the cavities are completely filled with water molecules, although with approximately 20% lower density than in bulk water. The 2A6 protein differs from the other three in that it has a very small cavity with only two water molecules and no exchange with the surroundings. The other three proteins have quite big cavities, with 41 water molecules on average in 2C8 and 54-58 in 2C9 and 3A4, giving a water volume of 1500-2100 A3. The two crystal structures of 2C9 differ quite appreciably, whereas those of 3A4 are quite similar. The active-site cavity is connected to the surroundings by three to six channels, through which there is a quite frequent exchange of water molecules (one molecule is exchanged every 30-200 ps), except in 2A6. Most of the channels are observed also in the crystal structures, but two to three channels in each protein open only during the simulations. There are no water molecules close to the heme iron ion in these simulations of the high-spin ferric state (the average distance to the closest water molecule is 3.3-5 A), and there are few ordered water molecules in the active sites, none of which is conserved in all proteins.

Prediction of activation energies for hydrogen abstraction by cytochrome p450.

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.

Structures of the high-valent metal-ion haem-oxygen intermediates in peroxidases, oxygenases and catalases.

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, Mössbauer 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.