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The structural analysis of the ligandbinding pocket is important for understanding subtype specificity of receptors and, therefore, is essential for rational design of novel ligands. Identification of amino-acids residues, which are involved in binding to a ligand, is the main challenge in the design of the new drugs because the exclusive binding properties of ligands to amino-acids residues determine the drugs specificity, selectivity, affinity and eventually effectiveness. Both, first and second generation drugs designed as antagonists of histamine 1 receptors are not perfect yet as they show considerable side effects such as sedation, dry mouth and arrhythmia because of penetration across the blood-brain barrier and low receptor selectivity [1]. The optimization of drugs is aimed at reducing side effects associated with the compounds of first and second generation and improving selectivity while maintaining their effective antagonistic properties. The analysis of amino-acid residues in the ligand-binding pocket involved in the binding of already existent H1R antagonists may help to improve the desired features. Through conducting a literature search describing the structure of human histamine H1 receptor and its ligands we identified 4 amino-acid residues essential for ligand binding. Overall, the ligand-binding pocket exhibits hydrophobic nature and contains mainly well-conserved regions. The non-conserved residues Trp1584.56 and Asn1985.46 in the pocket make minor hydrophobic interactions. Existing antagonists bind to a pocket mainly by building interactions with amino acids located
on the helices III, V and VI [2]. Four main amino-acids residues interacting with antagonists are Asp107, Lys191, Lys 179 and Trp428. Interaction with Asp1073.32 has been reported to be essential for ligand binding in several mutational studies [3-5]. Asp1073.32 is highly conserved side chain in aminergic receptor located on the third helix [5]. Asp1073.32 was shown to be crucial in both agonist and antagonist binding. Mutations of Asp1073.32 to alanine result in a loss of binding properties [5]. Lys179 and Lys191 are two residues which contribute to a novel
Figure 1. Location of Lys191, Asn198 and Asp 107, amino acids residues essential for histamine binding. feature of ligand-binding complex which is an anion-binding site at its entrance. Their interaction was shown to enhance the specificity of second generation drugs and lower affinity to other aminergic receptors. The mutational study have determined that Lys191 on the 5th helix and Lys179 located on the extracellular
Figure 2. Binding interactions of doxepin showing importance of Asp107 and Trp 428. loop are important for the selectivity of compounds [6]. Finally, Trp428 located on the 6th helix was shown to be important for activation-related conformational changes. A direct interaction with this residue provides stabilization of the receptor and consequently its permanent deactivation.
Identified amino acids residues help to describe the main pharmacophore features in the ligand-binding pocket. The most crucial interaction exhibited by all existing H1R antagonists is the formation of an anchor salt bridge between the Asp1073.32 and the amine moiety on the ligand. This interaction ensures stability and the affinity of a ligand [2]. Lys191 and Lys179 were proven to be crucial for selectivity and affinity of second generation drugs. An anion-binding site provides binding for a phosphate ion which plays as a positive modulator of ligand binding and affects the binding of some ligands and stability of H1R. Interaction with extracellular domain of H1R through binding of carboxyl group to Lys191 and Lys179 forming salt bridges increases specificity of compounds [2]. This entails that the less deep binding position is profound for some ligands. Lastly, interaction with Trp428 is important in mechanism of H1R inactivation [6]. Extensive hydrophobic interactions with Trp428 rings stabilizes hydrophobic packing around helix VI and its switch is very efficient in bringing H1R to its inactive conformation [2]. Overall, both transmemembrane residues and residues located on the extracellular loops should be included in the pharmacophore models when designing compunds.
References 1. Yanai, K., et al. (1995). Histamine H1 receptor occupancy in human brains after single oral doses of histamine H1 antagonists measured by positron emission tomography. BJP, 1649-1655 2. Shimamura T, Shiroishi M, Weyand S, Tsujimoto H, Winter G, Katritch V, Abagyan R, Cherezov V, Liu W, Han GW, Kobayashi T,
Figure 3. Binding interactions of second generation drugs showing importance of Lys179 and Lys191 residues.
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Stevens, RC, Iwata, S (2011) Structure of the human histamine H1 receptor complex with doxepin. Nature 475: 65-72. Ohta K, et al. (1994). Site-directed mutagenesis of the histamine H1 receptor: roles of aspartic acid107, asparagine198 and threonine194. Biochem Biophys Res Commun. 203:10961101. Nonaka H, et al.(1998) Unique binding pocket for KW-4679 in the histamine H1 receptor. Eur J Pharmacol. 345:111117. Kiss, R.; Kovri, Z.; Keser, G. M. (2204). Homology modelling and binding site mapping of the human histamine H1 receptor. Eur. J. Med. Chem., 39, 959-967 Totrov M, Abagyan R. Flexible protein-ligand docking by global energy optimization in internal coordinates. Proteins. 1997;(Supp l 1):215220.