The histamine H(4) receptor (H(4)R) is a G protein-coupled receptor (GPCR) that plays an important role in inflammation. Similar to the homologous histamine H(3) receptor (H(3)R), two acidic residues in the H(4)R binding pocket, D(3.32) and E(5.46), act as essential hydrogen bond acceptors of positively ionizable hydrogen bond donors in H(4)R ligands. Given the symmetric distribution of these complementary pharmacophore features in H(4)R and its ligands, different alternative ligand binding mode hypotheses have been proposed. The current study focuses on the elucidation of the molecular determinants of H(4)R-ligand binding modes by combining (3D) quantitative structure-activity relationship (QSAR), protein homology modeling, molecular dynamics simulations, and site-directed mutagenesis studies. We have designed and synthesized a series of clobenpropit (N-(4-chlorobenzyl)-S-[3-(4(5)-imidazolyl)propyl]isothiourea) derivatives to investigate H(4)R-ligand interactions and ligand binding orientations. Interestingly, our studies indicate that clobenpropit (2) itself can bind to H(4)R in two distinct binding modes, while the addition of a cyclohexyl group to the clobenpropit isothiourea moiety allows VUF5228 (5) to adopt only one specific binding mode in the H(4)R binding pocket. Our ligand-steered, experimentally supported protein modeling method gives new insights into ligand recognition by H(4)R and can be used as a general approach to elucidate the structure of protein-ligand complexes.

Cells express distinct G protein-coupled receptor (GPCR)subtypes on their surface, allowing them to react to a corresponding variety of extracellular stimuli. Cross-regulation between different ligand-GPCR pairs is essential to generate appropriate physiological responses. GPCRs can physically affect each other's functioning by forming heteromeric complexes, whereas cross-regulation between activated GPCRs also occurs through integration of shared intracellular signaling networks. Human herpesviruses utilize virally encoded GPCRs to hijack cellular signaling networks for their own benefit. Previously, we demonstrated that the Epstein-Barr virus-encoded GPCR BILF1 forms heterodimeric complexes with human chemokine receptors. Using a combination of bimolecular complementation and bioluminescence resonance energy transfer approaches, we now show the formation of hetero-oligomeric complexes between this viral GPCR and human CXCR4. BILF1 impaired CXCL12 binding to CXCR4 and, consequently, also CXCL12-induced signaling. In contrast, the G protein uncoupled mutant BILF1-K 3.50 A affected CXCL12-induced CXCR4 signaling to a much lesser extent, indicating that BILF1-mediated CXCR4 inhibition is a consequence of its constitutive activity. Co-expression of G␣ i1 with BILF1 and CXCR4 restored CXCL12-induced signaling. Likewise, BILF1 formed heteromers with the human histamine H 4 receptor (H 4 R). BILF1 inhibited histamine-induced G␣ i -mediated signaling by H 4 R, however, without affecting histamine binding to this receptor. These data indicate that functional cross-regulation of G␣ i -coupled GPCRs by BILF1 is at the level of G proteins, even though these GPCRs are assembled in hetero-oligomeric complexes.

Virtual fragment screening (VFS) is a promising new method that uses computer models to identify small, fragment-like biologically active molecules as useful starting points for fragment-based drug discovery (FBDD). Training sets of true active and inactive fragment-like molecules to construct and validate target customized VFS methods are however lacking. We have for the first time explored the possibilities and challenges of VFS using molecular fingerprints derived from a unique set of fragment affinity data for the histamine H(3) receptor (H(3)R), a pharmaceutically relevant G protein-coupled receptor (GPCR). Optimized FLAP (Fingerprints of Ligands and Proteins) models containing essential molecular interaction fields that discriminate known H(3)R binders from inactive molecules were successfully used for the identification of new H(3)R ligands. Prospective virtual screening of 156,090 molecules yielded a high hit rate of 62% (18 of the 29 tested) experimentally confirmed novel fragment-like H(3)R ligands that offer new potential starting points for the design of H(3)R targeting drugs. The first construction and application of customized FLAP models for the discovery of fragment-like biologically active molecules demonstrates that VFS is an efficient way to explore protein-fragment interaction space in silico.

BackgroundGq is a heterotrimeric G protein that plays an important role in numerous physiological processes. To delineate the molecular mechanisms and kinetics of signalling through this protein, its activation should be measurable in single living cells. Recently, fluorescence resonance energy transfer (FRET) sensors have been developed for this purpose.ResultsIn this paper, we describe the development of an improved FRET-based Gq activity sensor that consists of a yellow fluorescent protein (YFP)-tagged Gγ2 subunit and a Gαq subunit with an inserted monomeric Turquoise (mTurquoise), the best cyan fluorescent protein variant currently available. This sensor enabled us to determine, for the first time, the kon (2/s) of Gq activation. In addition, we found that the guanine nucleotide exchange factor p63RhoGEF has a profound effect on the number of Gq proteins that become active upon stimulation of endogenous histamine H1 receptors. The sensor was also used to measure ligand-independent activation of the histamine H1 receptor (H1R) upon addition of a hypotonic stimulus.ConclusionsOur observations reveal that the application of a truncated mTurquoise as donor and a YFP-tagged Gγ2 as acceptor in FRET-based Gq activity sensors substantially improves their dynamic range. This optimization enables the real-time single cell quantification of Gq signalling dynamics, the influence of accessory proteins and allows future drug screening applications by virtue of its sensitivity.

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We have explored the possibilities and challenges of structure-based virtual screening (SBVS) against the human histamine H 4 receptor (H 4 R), a key player in inflammatory responses. Several SBVS strategies, employing different H 4 R ligand conformations, were validated and optimized with respect to their ability to discriminate small fragment-like H 4 R ligands from true inactive fragments, and compared to ligand-based virtual screening (LBVS) approaches. SBVS studies with a molecular interaction fingerprint (IFP) scoring method enabled the identification of H 4 R ligands that were not identified in LBVS runs, demonstrating the scaffold hopping potential of combining molecular docking and IFP scoring. Retrospective VS evaluations against H 4 R homology models based on the histamine H 1 receptor (H 1 R) crystal structure did not give higher enrichments of H 4 R ligands than H 4 R models based on the beta-2 adrenergic receptor (β 2 R). Complementary prospective SBVS studies against β 2 R-based and H 1 R-based H 4 R homology models led to the discovery of different new fragment-like H 4 R ligand chemotypes. Of the 37 tested compounds, 9 fragments (representing 5 different scaffolds) had affinities between 0.14 and 6.3 μM at the H 4 R.

After the recent description of ␤-arrestin2 recruitment to the human histamine H 4 receptor (hH 4 R) in response to the well known H 4 R antagonist 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methyl-piperazine (JNJ 7777120), we evaluated in this study the efficacy of 31 known hH 4 R ligands to induce G␣ i protein signaling and ␤-arrestin2 recruitment by the hH 4 R. The selected hH 4 R ligands belong to nine different structural classes that partly cover (pre)clinical trial candidates. We have identified hH 4 R ligands with a significant bias for the G␣ i protein or ␤-arrestin2 pathway on the basis of efficacy differences. In addition, hH 4 R antagonists that did not show positive efficacy in either functional readouts were found. A common trend in pathway preference for the nine different ligand classes could not be observed. In particular, the isothiourea class shows very diverse results, varying from G␣ i protein-biased or ␤-arrestin2-biased to nonbiased antagonists upon minor structural changes. The identified biased hH 4 R ligands are important pharmacological tools to unravel the significance of biased hH 4 R signaling in H 4 R pharmacology.

Rearrangement of transmembrane domains (TMs) 3 and 5 after agonist binding is necessary for stabilization of the active state of class A G protein-coupled receptors (GPCRs). Using sitedirected mutagenesis and functional assays, we provide the first evidence that the TAS(I/V) sequence motif at positions 3.37 to 3.40, highly conserved in aminergic receptors, plays a key role in the activation of the histamine H 1 receptor. By combining these data with structural information from X-ray crystallography and computational modeling, we suggest that Thr 3.37 interacts with TM5, stabilizing the inactive state of the receptor, whereas the hydrophobic side chain at position 3.40, highly conserved in the whole class A GPCR family, facilitates the reorientation of TM5. We propose that the structural change of TM5 during the process of GPCR activation involves a local Pro 5.50 -induced unwinding of the helix, acting as a hinge, and the highly conserved hydrophobic Ile 3.40 side chain, acting as a pivot.