Three-dimensional models of histamine H3 receptor antagonist complexes and their pharmacophore

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Abstract

Molecular modeling was used to analyze the binding mode and activities of histamine H3 receptor antagonists. A model of the H3 receptor was constructed through homology modeling methods based on the crystal structure of bovine rhodopsin. Known H3 antagonists were interactively docked into the putative antagonist binding pocket and the resultant model was subjected to molecular mechanics energy minimization and molecular dynamics simulations which included a continuum model of the lipid bilayer and intra- and extracellular aqueous environments surrounding the transmembrane helices. The transmemebrane helices stayed well embedded in the dielectric slab representing the lipid bilayer and the intra- and extracellular loops remain situated in the aqueous solvent region of the model during molecular dynamics simulations of up to 200 ps in duration. A pharmacophore model was calculated by mapping the features common to three active compounds three-dimensionally in space. The 3D pharmacophore model complements our atomistic receptor/ligand modeling. The H3 antagonist pharmacophore consists of two protonation sites (i.e. basic centers) connected by a central aromatic ring or hydrophobic region. These two basic sites can simultaneously interact with Asp 114 (3.32) in helix III and a Glu 206 (5.46) in helix V which are believed to be the key residues that histamine interacts with to stabilize the receptor in the active state. The interaction with Glu 206 is consistent with the enhanced activity resulting from the additional basic site. In addition to these two salt bridging interactions, the central region of these antagonists contains a lipophilic group, usually an aromatic ring, that is found to interact with several nearby hydrophobic side chains. The picture of antagonist binding provided by these models is consistent with earlier pharmacophore models for H3 antagonists with some exceptions.

Introduction

Histamine is a potent biogenic amine that elicits a variety of physiological effects, which are played out by one of four known histamine receptors (H1, H2, H3, and H4) [1], [2], [3], [4], [5]. These physiological effects include allergic reactions (H1), gastric acid secretion (H2), mediation of neurotransmitter release (H3) and immunological response (H4). Both the H1 and H2 receptors are well known and effective antagonists have been developed to control a range of maladies, including allergies and gastric ulcers, and constitute some of the most commercially successful drugs on the market today [6], [7]. The H3 and H4 receptors have been discovered fairly recently and their biological significance as well as antagonists that modulate them are an active area of investigation by industry and academia [8], [9].

The H3 receptor is found in the central and peripheral nervous system on the presynaptic cleft of neurons where it functions as a negative regulator of histamine as well as mediates the release of other neurotransmitters [10], [11]. Possible roles for an antagonist of this target include treatments for narcolepsy, arousal, ADHD, cognition, and memory disorders [12], [13]. The H3 receptor belongs to the G-coupled protein receptor (GPCR) super family containing a transmembrane region consisting of a seven alpha-helical bundle. This transmembrane region is often the site of ligand and drug interaction [14], [15]. This is particularly true for the aminoergic receptors which include histamine, dopamine, serotonin and andrenergic receptors [14], [15]. For each of these receptors the natural ligand is derived from an amino acid and contains a primary amine that is believed to interact with a highly conserved aspartic acid side-chain in the third helix inside the helical bundle [14], [15]. The same is true for many of their antagonists [14], [15].

Several groups have reported the discovery of potent and selective non-imidazole H3 receptor antagonists [16], [17], [18], [19], [20]. In general these compounds differ from H1, H2 and even H4 receptor antagonists in their chemical structures and features, and the general H3 pharmacophore has been described [17]. Specifically the non-imidazole based antagonists contain at least one basic amine as do most antagonists of aminoergic receptors, but in many instances a second basic site is present which significantly enhances activity. And these two basic centers are linked by a lipophilic group [17]. Several examples of H3 antagonists illustrating the features of this pharmacophore [16], [17], [18] are shown in Fig. 1. In each of these examples there is a primary basic group, either a piperidine or pyrolidine, which is connected by an alkyl linkage. In each chemical series there is an example of an antagonist with a second basic group. In each of the three cases presented in Fig. 1 (1 and 2, 3 and 4, 5 and 6) the addition of the secondary basic group increases the binding affinity by 100-, 10- and 8-fold, respectively. Each of these chemical series also contains at least one lipophilic aromatic ring.

Three-dimensional atomistic models of antagonist-receptor complexes have been used to investigate the details of ligand and drug interactions with GPCRs and have been successful in providing important insights regarding their binding [21], [22], [23], [24]. These models are usually based on a homology model built from the X-ray structure of bovine rhodopsin [25], but several first principle approaches to building the receptor model are being used as well [26]. Docking of the ligand is performed by automated or manual docking using either site directed mutagenesis data and/or sequence homology knowledge as to where the molecule binds. Usually the ligand is energy minimized in the binding region with some or complete relaxation of the receptor model but it is rare to perform lengthy dynamics since this requires a representation of the lipid bilayer and the surrounding aqueous environment, which is very costly to include in the dynamics calculations. Although there are a number of studies which include explicit bilayer and solvent using a periodic boundary condition they are in general the exception in modeling GPCR ligand interactions [14], [26]. Recently, continuum dielectric models, based on the Born approximation, have been extended to handle the unique situation of membrane bound proteins [27], [28]. These implicit membrane models were successfully applied to the study of the dynamics of a small helical peptide at a aqueous/lipid interface and the static orientation of bovine and bacterial rhodopsin crystal structures in their native bilayer environment [27], [28]. However, no full relaxation or molecular dynamics of a GPCR structure using this novel implicit membrane model have been reported to date.

Pharmacophore modeling can provide a qualitative picture of ligand receptor binding by identifying the important features for binding and their spatial arrangements [29]. This approach has been particularly successful for investigating GPCR/ligand binding modes and is complementary to 3D receptor/ligand modelling [30]. Consequently, two papers describing pharmacophore models of H3 receptor antagonists have been published [31], [32]. In both studies only imidazole based antagonists were considered. To date no quantitative pharmacophore models based on non-imidazole antagonist compounds have been reported.

We present a computational study of the binding of several H3 antagonists to the H3 receptor using a homology model for the receptor and employing this novel continuum dielectric model for the surrounding bilayer environment. This work represents the first use of this continuum method in which complete relaxation and molecular dynamics were run with the ligands bound to a GPCR. We examine the utility of the implicit membrane model to study GPCR drug interactions. We use sequence analysis and the results of existing site directed mutagenesis studies on the closely related histamine H1, H2 and H4 receptors, which were used to define the internal site of agonism and antagonism of known ligands and drugs, to define the putative antagonist binding site in our model. We then used this information to dock and simulate known H3 antagonist compounds bound to the receptor model. In addition we developed a pharmacophore model by three-dimensional alignment of the common features of several active compounds. We use the resulting models to investigate ligand binding in the H3 receptor and the nature of the two basic and the lipophilic features of the H3 phamacophore model and compare and contrast their similarities and differences.

Section snippets

Sequence alignment and homology modeling

The transmembrane portion of the H3 receptor was built by homology modeling techniques based on the 2.8 Å resolution crystal structure of Bovine Rhodopsin [25] (pdb entry 1F88) which is the most accurate rhodopsin structure available. The primary sequence of the H3 receptor was aligned with bovine rhodopsin based upon highly conserved amino acid residues in the seven helices. All homology models were constructed with the MODELER program [33], [34]. Seven of the eight extra- and intra-cellular

Agonist/antagonist binding site

The putative agonist and antagonist binding sites of the H1, H2 and H4 receptors have been investigated by site directed mutagenesis [45], [46], [47], [48]. A study on the H1 receptor found [45] that the Asp 107 (3.32) in helix III and Thr 194 (5.42) and Asn 198 (5.46) in helix V are important for histamine binding, while only Asp 107 was important for antagonist binding. The antagonist used is this study was mepyramine, a classic H1 antagonist with two aromatic rings and a basic amine arrayed

Conclusions

We have used three-dimensional atomistic modeling to explore the putative antagonist binding site in the histamine H3 receptor. Starting with a homology model for the H3 receptor we used molecular mechanics and dynamics in conjunction with a novel continuum dielectric model that accounts for the aqueous solvent and lipid bilayer regions. This was the first time this novel continuum dielectric model was applied to the study of GPCR antagonist binding. This implicit membrane model has several

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