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

Shedding Light on the D1-Like Receptors: A Fluorescence-Based Toolbox for Visualization of the D1 and D5 Receptors

Dopamine D1-like receptors are the most abundant type of dopamine receptors in the central nervous system and, even after decades of discovery, still highly interesting for the study of neurological diseases. We herein describe the synthesis of a new set of fluorescent ligands, structurally derived from D1R antagonist SCH-23390 and labeled with two different fluorescent dyes, as tool compounds for the visualization of D1-like receptors. Pharmacological characterization in radioligand binding studies identified UR-NR435 (25) as a high-affinity ligand for D1-like receptors (pKi (D1R) = 8.34, pKi (D5R) = 7.62) with excellent selectivity towards D2-like receptors. Compound 25 proved to be a neutral antagonist at the D1R and D5R in a Gs heterotrimer dissociation assay, an important feature to avoid receptor internalization and degradation when working with whole cells. The neutral antagonist 25 displayed rapid association and complete dissociation to the D1R in kinetic binding studies using confocal microscopy verifying its applicability for fluorescence microscopy. Moreover, molecular brightness studies determined a single-digit nanomolar binding affinity of the ligand, which was in good agreement with radioligand binding data. For this reason, this fluorescent ligand is a useful tool for a sophisticated characterization of native D1 receptors in a variety of experimental setups

Structural and Molecular Insight into Piperazine and Piperidine Derivatives as Histamine H3 and Sigma-1 Receptor Antagonists with Promising Antinociceptive Properties

In an attempt to extend recent studies showing that some clinically evaluated histamine H3 receptor (H3R) antagonists possess nanomolar affinity at sigma-1 receptors (σ1R), we selected 20 representative structures among our previously reported H3R ligands to investigate their affinity at σRs. Most of the tested compounds interact with both sigma receptors to different degrees. However, only six of them showed higher affinity toward σ1R than σ2R with the highest binding preference to σ1R for compounds 5, 11, and 12. Moreover, all these ligands share a common structural feature: the piperidine moiety as the fundamental part of the molecule. It is most likely a critical structural element for dual H3/σ1 receptor activity as can be seen by comparing the data for compounds 4 and 5 (hH3R Ki = 3.17 and 7.70 nM, σ1R Ki = 1531 and 3.64 nM, respectively), where piperidine is replaced by piperazine. We identified the putative protein–ligand interactions responsible for their high affinity using molecular modeling techniques and selected compounds 5 and 11 as lead structures for further evaluation. Interestingly, both ligands turned out to be high-affinity histamine H3 and σ1 receptor antagonists with negligible affinity at the other histamine receptor subtypes and promising antinociceptive activity in vivo. Considering that many literature data clearly indicate high preclinical efficacy of individual selective σ1 or H3R ligands in various pain models, our research might be a breakthrough in the search for novel, dual-acting compounds that can improve existing pain therapies. Determining whether such ligands are more effective than single-selective drugs will be the subject of our future studies

Synthesis, biological evaluation, and computational studies of tri- and tetracyclic nitrogen-bridgehead compounds as potent dual-acting AChE inhibitors and hH3 receptor antagonists

Combination of AChE inhibiting and histamine H3 receptor antagonizing properties in a single molecule might show synergistic effects to improve cognitive deficits in Alzheimer's disease, since both pharmacological actions are able to enhance cholinergic neurotransmission in the cortex. However, whereas AChE inhibitors prevent hydrolysis of acetylcholine also peripherally, histamine H3 antagonists will raise acetylcholine levels mostly in the brain due to predominant occurrence of the receptor in the central nervous system. In this work, we designed and synthesized two novel classes of tri- and tetracyclic nitrogen-bridgehead compounds acting as dual AChE inhibitors and histamine H3 antagonists by combining the nitrogen-bridgehead moiety of novel AChE inhibitors with a second N-basic fragment based on the piperidinylpropoxy pharmacophore with different spacer lengths. Intensive structure-activity relationships (SARs) with regard to both biological targets led to compound 41 which showed balanced affinities as hAChE inhibitor with IC50 = 33.9 nM, and hH3R antagonism with Ki = 76.2 nM with greater than 200-fold selectivity over the other histamine receptor subtypes. Molecular docking studies were performed to explain the potent AChE inhibition of the target compounds and molecular dynamics studies to explain high affinity at the hH3R