SMARTCyp is an in silico method that predicts the sites of cytochrome P450-mediated metabolism of druglike molecules. The method is foremost a reactivity model, and as such, it shows a preference for predicting sites that are metabolized by the cytochrome P450 3A4 isoform. SMARTCyp predicts the site of metabolism directly from the 2D structure of a molecule, without requiring calculation of electronic properties or generation of 3D structures. This is a major advantage, because it makes SMARTCyp very fast. Other advantages are that experimental data are not a prerequisite to create the model, and it can easily be integrated with other methods to create models for other cytochrome P450 isoforms. Benchmarking tests on a database of 394 3A4 substrates show that SMARTCyp successfully identifies at least one metabolic site in the top two ranked positions 76% of the time. SMARTCyp is available for download at http://www.farma.ku.dk/p450.

No abstract

A method is developed for the combination of quantum chemical geometry optimizations and crystallographic structure refinement. The method is implemented by integrating the quantum chemical software Turbomole with the crystallographic software Crystallography and NMR System (CNS), using three small procedures transferring information between the two programs. The program (COMQUM-X)is used to study the binding of the inhibitor N-methylmesoporphyrin to ferrochelatase, and we show that the method behaves properly and leads to an improvement of the structure of the inhibitor. It allows us to directly quantify in energy terms how much the protein distort the structure of the bound inhibitor compared to the optimum vacuum structure (4-6 kJ/mol). The approach improves the standard combined quantum chemical and molecular mechanics (QC/MM) approach by guaranteeing that the final structure is in accordance with experimental data (the reflections) and avoiding the risk of propagating errors in the crystal coordinates. The program can also be seen as an improvement of standard crystallographic refinement, providing an accurate empirical potential function for any group of interest. The results can be directly interpreted in standard crystallographic terms (e.g., R factors or electron density maps). The method can be used to interpret crystal structures (e.g., the protonation status of metal-bound water molecules) and even to locally improve them.

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.

The orphan glutamate-like receptor GluR␦2 is predominantly expressed in Purkinje cells of the central nervous system. The classification of GluR␦2 to the ionotropic glutamate receptor family is based on sequence similarities, because GluR␦2 does not form functional homomeric glutamate-gated ion channels in transfected cells. Studies in GluR␦2 ؊/؊ knockout mice as well as in mice with naturally occurring mutations in the GluR␦2 gene have demonstrated an essential role of GluR␦2 in cerebellar long-term depression, motor learning, motor coordination, and synaptogenesis. However, the lack of a known agonist has hampered investigations on the function of GluR␦2. In this study, the ligand-binding core of GluR␦2 (GluR␦2-S1S2) was found to bind neutral amino acids such as D-serine and glycine, as demonstrated by isothermal titration calorimetry. Direct evidence for binding of D-serine and structural rearrangements in the binding cleft of GluR␦2-S1S2 is provided by x-ray structures of GluR␦2-S1S2 in its apo form and in complex with D-serine. Functionally, D-serine and glycine were shown to inactivate spontaneous ion-channel conductance in GluR␦2 containing the lurcher mutation (EC 50 values, 182 and 507 M, respectively). These data demonstrate that the GluR␦2 ligand-binding core is capable of binding ligands and that cleft closure of the ligandbinding core can induce conformational changes that alter ion permeation.crystal structure ͉ electrophysiology ͉ isothermal titration calorimetry ͉ ligand-binding core

2-Oxoglutarate and iron dependent oxygenases are therapeutic targets for human diseases. Using a representative 2OG oxygenase panel, we compare the inhibitory activities of 5-carboxy-8-hydroxyquinoline (IOX1) and 4-carboxy-8-hydroxyquinoline (4C8HQ) with that of two other commonly used 2OG oxygenase inhibitors, -oxalylglycine (NOG) and 2,4-pyridinedicarboxylic acid (2,4-PDCA). The results reveal that IOX1 has a broad spectrum of activity, as demonstrated by the inhibition of transcription factor hydroxylases, representatives of all 2OG dependent histone demethylase subfamilies, nucleic acid demethylases and γ-butyrobetaine hydroxylase. Cellular assays show that, unlike NOG and 2,4-PDCA, IOX1 is active against both cytosolic and nuclear 2OG oxygenases without ester derivatisation. Unexpectedly, crystallographic studies on these oxygenases demonstrate that IOX1, but not 4C8HQ, can cause translocation of the active site metal, revealing a rare example of protein ligand-induced metal movement.

The serotonin transporter (SERT)2 is an integral membrane protein that facilitates transport of serotonin (5-hydroxytryptamine, 5HT) across cellular membranes (1). In addition to peripheral endocrine functions, 5HT is a neurotransmitter in the brain; it is involved in control of several important physiological functions such as mood, appetite, and sexual behavior. Expressed mainly in the membrane of serotonergic neurons, SERT utilizes energetically favorable cotransport of Na ϩ to remove released 5HT from the extracellular space. Human SERT (hSERT) belongs to the solute carrier 6 (SLC6) transporter family along with highly homologous transporters for the neurotransmitters ␥-aminobutyric acid, glycine, dopamine, and norepinephrine (2-4). These transporters are important drug targets for treatment of a wide range of neurological diseases. In particular, hSERT is the molecular target for widely used drugs for treatment of depression and anxiety. Also, psychostimulants such as amphetamine and 3,4-methylenedioxy-N-methylamphetamine ("ecstasy") have hSERT as the molecular target (5-7). The selective serotonin re-uptake inhibitors (SSRIs) are a class of antidepressant and anti-anxiety drugs that function as highly selective competitive inhibitors of hSERT (8). Although SSRIs are highly important for treatment of affective disorders (6), the molecular basis for their function, including location and structure of drug binding pockets, is largely unknown and a matter of debate (9, 10). Such information is important for understanding essential aspects of drug action, ranging from selectivity profile to therapeutic efficacy. Moreover, such information is indispensable for the development of new and improved drugs targeting hSERT. The primary impediment for elucidation of the structural mechanisms of hSERT inhibition is the lack of a three-dimensional structure of the protein. Still, several residues in SERT have been identified mainly by mutagenesis studies that modulate antidepressants potency (11)(12)(13)(14)(15)(16)(17). The use of comparative molecular modeling to create structural models of ligand-hSERT interactions has previously been hampered by the low phylogenetic and functional similarity between hSERT and available template proteins (18 -21). However, high resolution crystal structures of a bacterial homolog to mammalian SLC6 transporters, LeuT (22,23), have proven excellent templates for constructing experimentally validated models of substrate and drug binding pockets in human SLC6 transporters, including the human transporters for dopamine and ␥-aminobutyric acid (24 -32).In this study, we provide an experimentally validated threedimensional model of the binding site in hSERT for the SSRI (S)-citalopram (Lexapro) using mutational analysis of hSERT paired with structure-activity data for (S)-citalopram analogs. LeuT structures are used to create homology models of hSERT, followed by docking of (S)-citalopram. Validation of binding models was performed based on the mutational dataset from 64 hSERT point mutants ...

Supplementary data are available at Bioinformatics online.