Computational protocol: Identification of Histamine H3 Receptor Ligands Using a New Crystal Structure Fragment based Method

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Protocol publication

[…] A structure-based sequence alignment of the H1 and H3 receptors was downloaded from GPCRdb, and a model of the H3 receptor transmembrane domain was built in Modeller using the H1 crystal structure (PDB: 3RZE) template. The pharmacophore was designed using our previously described crystal structure-based pharmacophore method, which is based on the manual annotation of a library of structural fragments, pairs of a receptor residue and interacting ligand moiety, from GPCR crystal structure complexes. Herein, we uploaded our H3 receptor model to GPCRdb to identify conserved residues represented in this library, and to superpose the backbones of the corresponding fragments. The pharmacophore elements were placed using Phase at the highest density of the (multiple) fragment moieties. The vectors of the hydrogen bonding features were defined after optimization of ligand moiety – receptor residue interactions. Furthermore, the H3 pharmacophore was extended with an additional element not covered by the fragment library, but instead defined by matching reference ligands to the pharmacophore as well as docking them into a H3 structure model. The additional pharmacophore element represents a cationic ligand functionality that interacts with a Glu residue in position 5.46 × 461. This residue has been shown by mutagenesis studies be important for binding of imidazole- and pyridine-containing ligands, including histamine, . [...] Histamine H3 receptor reference ligands were downloaded from ChEMBL and the IUPHAR guide to pharmacology databases. We used only the ligands with submicromolar dose-response affinity or activity values (K i, pK i, EC50, pEC50, IC50 and pIC50), and the highest assay confidence scores: 8 or 9. The screening database, eMolecules plus, was prepared with LigPrep to desalt, add hydrogen atoms and generate tautomers, stereoisomers (max 32) and 3D conformations (max 10 ring conformations). Epik and the OPLS 2005 force field were applied to generate charge states at pH: 7.0 ± 1.0. LigFilter was used to remove structures with reactive functional groups and match the properties (Supplementary Table ) of the reference ligands. [...] The Phase database, containing both reference ligands and screening compounds, was prepared with 100 maximum conformers, up to 10 conformations per rotatable bond, thorough conformational sampling, conformational variation of amide bonds and a maximum relative energy difference of 6.0 kcal/mol. A minimum of four matching pharmacophore elements was required and a preference was set for partial matches involving more sites. Hits were sorted by fitness score and clustered with Canvas to select diverse representative structures. As a secondary assessment of compound structures, we used SiteMap on a H3 structure model. After the first assaying round small structure-activity relationship analyses were conducted and the compounds sorted into lead ligand series. The selections of analogues were based on substructures drawn in MarvinSketch and queried n the eMolecules database loaded into Instant JChem (Marvin 5.12.3, 2014 and Instant JChem 6.2.0, 2014, ChemAxon, www.chemaxon.com). […]

Pipeline specifications

Software tools MODELLER, SuperPose, eMolecules, LigPrep, Epik, Canvas, SiteMap, Marvin
Databases ChEMBL GPCRDB IUPHAR
Organisms Homo sapiens
Chemicals Histamine