Development of a conformational histamine H(3) receptor biosensor for the synchronous screening of agonists and inverse agonists

The histamine H(3) receptor (H(3)R) represents a highly attractive drug target for the treatment of various central nervous system disorders, but the discovery of novel H(3)R targeting compounds relies on the assessment of highly amplified intracellular signaling events that do not only reflect H(3)R modulation and carry the risk of high false-positive and -negative screening rates. To address these limitations, we designed an intramolecular H(3)R biosensor based on the principle of bioluminescence resonance energy transfer (BRET) that reports the receptor's real-time conformational dynamics and provides an advanced tool to screen for both H(3)R agonists and inverse agonists in a live cell screening-compatible assay format. This conformational G-protein-coupled receptor (GPCR) sensor allowed us to characterize the pharmacological properties of known and new H(3) receptor ligands with unprecedented accuracy. Interestingly, we found that one newly developed H(3) receptor ligand possesses even stronger inverse agonistic activity than reference H(3)R inverse agonists including the current gold standard pitolisant. Taken together, we describe here the design and validation of the first screening-compatible H(3)R conformational biosensor that will aid in the discovery of novel H(3)R ligands and can be employed to gain deeper insights into the (in-)activation mechanism of this highly attractive drug target

Methods to study the molecular pharmacology of the histamine H4 receptor

The H4R is the latest addition of the histamine receptor family. This GPCR was found to be involved in a multitude of allergic and inflammatory diseases. Antagonizing H4Rs results in profound anti-inflammatory effects in various animal disease models, and a first phase 2a clinical trial in patients suffering from atopic dermatitis has already been conducted. In order to accelerate and expand current drug discovery at H4R, in-depth understanding of its molecular mechanisms and signaling pathways are key. Complexity of H4R signaling pathways was high- lighted by the discovery of so-called biased ligands, which can initiate differential efficacies in various GPCR responses. This chapter will provide an overview of commonly applied methods used to elucidate the molecular pharmacological aspects of the H4R from receptor-ligand-binding interactions to down- stream gene transcription

HCMV-encoded G-protein-coupled receptors as constitutively active modulators of cellular signaling networks

Several herpesviruses encode G-protein-coupled receptor (vGPCR) proteins that are homologous to human chemokine receptors. In contrast to chemokine receptors, many vGPCRs signal in a ligand-independent (constitutive) manner. Such constitutive signaling is of major significance because various pathologies are associated with activating GPCR mutations. Constitutive activity of the human herpesvirus 8-encoded GPCR (ORF74), for example, is essential for its oncogenic potential to cause angioproliferative Kaposi's sarcoma-like lesions. The human cytomegalovirus (HCMV) encodes four GPCRs, of which US28 and UL33 display constitutive activity in transfected, but also HCMV-infected, cells. In addition, US28 is activated by a broad spectrum of chemokines. Furthermore, both US28 and UL33 show promiscuous G-protein coupling, whereas chemokine receptors activate primarily

Adhesion CPCRs in immunology

Adhesion GPCRs (aGPCRs) form a subfamily of the large GPCR super family. Most aGPCRs are characterised by a non-covalent bipartite structure that consists of a large extracellular domain and a membrane-spanning 7 transmembrane domain. Typically, aGPCRs can combine cell adhesion by the large extracellular domain with intracellular signalling by the 7 transmembrane domain. Immune responses rely on cellular communication and subsequent defence reactions. Indeed, aGPCR ADGRB1 and members of the ADGRE class have been linked to processes like phagocytosis, leucocyte activation and migration. Nevertheless, research is hampered by absence of endogenous ligands, unknown activity of generated antibodies and non-identified signalling pathways. Yet, based on their membrane localisation and important function, aGPCRs could be novel drug targets to modulate leucocyte function

BRET-based beta-arrestin2 recruitmanet to the histamine H1 receptor for investigating antihistamine binding kinetics.

Ligand residence time is thought to be a critical parameter for optimizing the in vivo efficacy of drug candidates. For the histamine

Ligand-binding kinetics on histamine receptors

Equilibrium-binding affinities of ligands for a drug target do not always accurately reflect the success of drug candidates in the clinic. Affinity-based predictions concerning competitive antagonism on the target will only be accurate if equilibrium binding of both ligands is allowed. Unless equilibrium for ligand bind- ing is obtained really quickly, it is unlikely that equilibrium is established in vivo. Instead, concentrations of (endogenous) ligands rapidly fluctuate over time. Hence, the velocity in which binding equilibrium is reached and the duration of target occupancy by the ligand (also known as residence time) are thought to be more important predictors of drug in vivo efficacy. This chapter provides the theoretical background on ligand-binding kinetics and several experimental approaches to determine the target residence time of antihistamines on histamine receptors

Biochemical and pharmacological characterization of the histamine H4 receptor

The histamine H4 receptor (