Stephen W. Doughty

Molecular dynamics simulations of the adenosine A2a receptor: structural stability, sampling, and convergence.

Molecular dynamics (MD) simulations of membrane-embedded G-protein coupled receptors (GPCRs) have rapidly gained popularity among the molecular simulation community in recent years, a trend which has an obvious link to the tremendous pharmaceutical importance of this group of receptors and the increasing availability of crystal structures. In view of the widespread use of this technique, it is of fundamental importance to ensure the reliability and robustness of the methodologies so they yield valid results and enable sufficiently accurate predictions to be made. In this work, 200 ns simulations of the A2a adenosine receptor (A2a AR) have been produced and evaluated in the light of these requirements. The conformational dynamics of the target protein, as obtained from replicate simulations in both the presence and absence of an inverse agonist ligand (ZM241385), have been investigated and compared using principal component analysis (PCA). Results show that, on this time scale, convergence of the replicates is not readily evident and dependent on the types of the protein motions considered. Thus rates of inter- as opposed to intrahelical relaxation and sampling can be different. When studied individually, we find that helices III and IV have noticeably greater stability than helices I, II, V, VI, and VII in the apo form. The addition of the inverse agonist ligand greatly improves the stability of all helices.

Homology modeling of the human 5-HT(1A), 5-HT (2A), D1, and D2 receptors: model refinement with molecular dynamics simulations and docking evaluation

Abstract5-HT1A serotonin and D1 dopamine receptor agonists have been postulated to be able to improve negative and cognitive impairment symptoms of schizophrenia, while partial agonists and antagonists of the D2 and 5-HT2A receptors have been reported to be effective in reducing positive symptoms. There is therefore a need for well-defined homology models for the design of more selective antipsychotic agents, since no three-dimensional (3D) crystal structures of these receptors are currently available. In this study, homology models were built based on the high-resolution crystal structure of the β2-adrenergic receptor (2RH1) and further refined via molecular dynamics simulations in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer system with the GROMOS96 53A6 united atom force field. Docking evaluations with representative agonists and antagonists using AutoDock 4.2 revealed binding modes in agreement with experimentally determined site-directed mutagenesis data and significant correlations between the computed and experimental pKi values. The models are also able to distinguish between antipsychotic agents with different selectivities and binding affinities for the four receptors, as well as to differentiate active compounds from decoys. Hence, these human 5-HT1A, 5-HT2A, D1 and D2 receptor homology models are capable of predicting the activities of novel ligands, and can be used as 3D templates for antipsychotic drug design and discovery.

Chromatographic retention behaviour of n-alkylbenzenes and pentylbenzene structural isomers on porous graphitic carbon and octadecyl-bonded silica studied using molecular modelling and QSRR

The retention behaviour of a series of 15 n-alkylbenzenes and pentylbenzene structural isomers and benzene were investigated using porous graphitic carbon (PGC) and octadecyl-bonded silica (ODS) stationary phases. Shorter chain n-alkylbenzenes and benzene (n=0-6), and all the pentylbenzene isomers were more strongly retained on ODS, although the selectivity was greater with PGC. For the pentylbenzene analytes the degree of branching in the alkyl chain at the position adjacent to the aromatic ring affects retention on PGC, with higher retention in less branched molecules. Molecular modelling studies have provided new insights into the geometry of aromatic π-π stacking interactions in retention on PGC. For alkylbenzenes with high branching at the position adjacent to the ring, the preferred geometry of association with the surface is with the branched chain directed away from the surface, a geometry not seen in the other alkylbenzenes. The most energetically favoured orientation for interaction between analytes and the PGC surface was found to be cofacial for toluene and ethylbenzene, whereas for other analytes this interaction was in a face-edge orientation. The alternative geometry of association observed with both toluene and ethylbenzene may explain the enhanced retention of these two analytes on PGC compared with their longer chain analogues. Quantitative structure-retention relationships revealed the importance of compactness in analyte structure during retention on PGC, with decreased compactness (associated with longer chain length and reduced chain branching) improving retention.

A quantitative assessment of inhaled drug particle-pulmonary surfactant interaction by atomic force microscopy

To date limited consideration has been given to the physical interaction between inhaled drug particles and pulmonary surfactant (PS). This study combines atomic force microscopy (AFM) with a Langmuir-Blodgett (LB) approach to quantify the force of adhesion between micronised budesonide particles and simulated PS monolayers. A LB approach was used to prepare Survanta monolayers at pre-determined surface pressures and AFM was employed to facilitate their visualisation. Adhesion measurements between drug particles and PS monolayers were executed via AFM. Contact angle measurements were performed to probe material wetting characteristics, the data confirmed that budesonide is hydrophobic and Survanta films at increasing surface pressure exhibit a rising hydrophobic character. AFM revealed that PS properties were governed by applied surface pressure and that the degree of interaction of budesonide was greater at higher surface pressure, where packing of the lipid film was increased; consistent with the point of exhalation. This correlates well with the accepted inhaler technique. The increasing hydrophobicity of the PS film, on increased pressure, was believed to be the primary reason for increased interaction with the hydrophobic budesonide. Surface chemistries of the drug particles and PS interface are considered to be important for inhaled drug delivery.

The application of molecular modelling to the interpretation of inverse gas chromatography data.

The use of molecular modelling in the interpretation of inverse gas chromatography data is discussed. Crystal faces can be visualised and likely cleavage planes calculated using the surface attachment energies. Assuming that the preferred cleavage plane is the crystal face with the smallest attachment energy then the predominant crystal faces of a crystalline particle can be predicted. Surface adsorption can be modelled using Van der Waals and electrostatic interactions to evaluate the interaction energies between individual atoms of the probe molecule and atoms of the test molecule orientated as in the surface. Using examples of pharmaceutical materials, modelling has been shown to be successful in the understanding of changes in the surface energetics.

Structure-based identification of aporphines with selective 5-HT2A receptor binding activity

Selective blockade of the serotonin 5‐HT2A receptor is a useful therapeutic approach for a number of disorders, including schizophrenia, insomnia and ischaemic heart disease. A series of aporphines were docked into a homology model of the rat 5‐HT2A receptor using AutoDock. Selected compounds with high in silico binding affinities were screened in vitro using radioligand‐binding assays against rat serotonin (5‐HT1A and 5‐HT2A) and dopamine (D1 and D2) receptors. (R)‐Roemerine and (±)‐nuciferine were found to have high affinity for the 5‐HT2A receptor (Ki = 62 and 139 nm, respectively), with (R)‐roemerine showing 20‐ to 400‐fold selectivity for the 5‐HT2A receptor over the 5‐HT1A, D1 and D2 receptors. Investigation into the ligand–receptor interactions suggested that the selectivity of (R)‐roemerine is due to it having stronger H‐bonding and dipole–dipole interactions with several of the key residues in the 5‐HT2A receptor‐binding site.

Inhibition of cobalamin-dependent methionine synthase by substituted benzo-fused heterocycles

The cobalamin–dependent cytosolic enzyme, methionine synthase (EC.2.1.1.13), catalyzes the remethylation of homocysteine to methionine using 5‐methyltetrahydrofolate as the methyl donor. The products of this remethylation – methionine and tetrahydrofolate – participate in the active methionine and folate pathways. Impaired methionine synthase activity has been implicated in the pathogenesis of anaemias, cancer and neurological disorders. Although the need for potent and specific inhibitors of methionine synthase has been recognized, there is a lack of such agents. In this study, we designed, synthesized and evaluated the inhibitory activity of a series of substituted benzimidazoles and small benzothiadiazoles. Kinetic analysis revealed that the benzimidazoles act as competitive inhibitors of the rat liver methionine synthase, whilst the most active benzothiadiazole (IC50 = 80 µm) exhibited characteristics of uncompetitive inhibition. A model of the methyltetrahydrofolate‐binding site of the rat liver methionine synthase was constructed; docking experiments were designed to elucidate, in greater detail, the binding mode and reveal structural requirements for the design of inhibitors of methionine synthase. Our results indicate that the potency of the tested compounds is related to a planar region of the inhibitor that can be positioned in the centre of the active site, the presence of a nitro functional group and two or three probable hydrogen‐bonding interactions.

Toward activated homology models of the human M1 muscarinic acetylcholine receptor

Structure-based virtual screening offers a good opportunity for the discovery of selective M1 muscarinic acetylcholine receptor (mAChR) agonists for the treatment of Alzheimers disease. However, no 3-D structure of an M1 mAChR is yet available and the homology models that have been previously reported are only able to identify antagonists in virtual screening experiments. In this study, we generated a homology model of the human M1 mAChR, based on the crystal structure of an M3 mAChR as the template. This initial model was modified, using the agonist-bound crystal structure of a β2-adrenergic receptor as a guide, to give two possible activated structures. The T192 side chain was adjusted in both structures and one of the structures also had the whole of transmembrane (TM) 5 rotated and tilted toward the inner channel of the transmembrane region. The binding sites of all three structures were then refined by induced-fit docking (IFD) with acetylcholine. Virtual screening experiments showed that all three refined models could efficiently differentiate agonists from decoy molecules, with the TM5-modified models also giving good agonist/antagonist selectivity. The whole range of agonists and antagonists was observed to bind within the orthosteric site of the structure obtained by IFD refinement alone, implying that it has inactive state character. In contrast, the two TM5-modified structures were unable to accommodate the antagonists, supporting the proposition that they possess activated state character.

Flavonoids with M1 Muscarinic Acetylcholine Receptor Binding Activity

Muscarinic acetylcholine receptor-active compounds have potential for the treatment of Alzheimer’s disease. In this study, a series of natural and synthetic flavones and flavonols was assayed in vitro for their ability to inhibit radioligand binding at human cloned M1 muscarinic receptors. Several compounds were found to possess competitive binding affinity (Ki = 40–110 µM), comparable to that of acetylcholine (Ki = 59 µM). Despite the fact that these compounds lack a positively-charged ammonium group under physiological conditions, molecular modelling studies suggested that they bind to the orthosteric site of the receptor, mainly through non-polar interactions.

The influence of substituted phenols on the sol:gel transition of hydroxypropyl methylcellulose (HPMC) aqueous solutions

The influence of the physicochemical parameters of substituted aromatic molecules on the phase transition from sol to gel of hydroxypropyl methylcellulose (HPMC) has been investigated using a homologous series of substituted phenols. Using a turbimetric methodology, concentration dependent suppression of phase transition temperature of HPMC was observed for phenol and its derivatives, including methyl-, nitro- and chloro-substituted molecules. Although no strong direct relationship between single molecular physicochemical properties of the phenolic compounds (such as pKa, LogP and other molecular descriptors) and ΔCPT was found for the compounds tested, a successful prediction of behaviour could be obtained by using a combination of parameters. This suggested that the interaction mechanism between HPMC and the substituted aromatic moiety is a complex summation of the different molecular physicochemical properties. Identification of these potentially deleterious chemical moieties may be of value in a pharmaceutical context when considering preformulation of drug structures containing them. An incompatibility between drug and polymer may be indicative of deleterious effects resulting from formulation with hydrophilic matrix dosage forms containing cellulose ethers such as HPMC.