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 K_i = 3.17 and 7.70 nM, σ₁R K_i = 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.

The histamine H₃ receptor (H₃R) represents a highly attractive drug target for the treatment of various central nervous disorders but the discovery of novel H₃R targeting compounds relies on the assessment of highly amplified intracellular signaling events that do not only reflect H₃R modulation and carry the risk of high false-positive and -negative screening rates. To address these limitations, we designed an intramolecular H₃R 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, H₃R 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 H₃ receptor ligands with unprecedented accuracy. Intriguingly, we found that one newly developed H₃ receptor ligand possesses even stronger inverse agonistic activity than reference H₃R inverse agonists including the current gold standard pitolisant. Taken together, we describe here the design and validation of the first screening-compatible H₃R conformational biosensor that will aid in the discovery of novel H₃R ligands and can be employed to gain deeper insights into the (in-)activation mechanism of this highly attractive drug target.

On the basis of the long-known prototypic pharmacophore 3-(1H-imidazol-4-yl)propylguanidine (SK&F 91486, 2), monomeric, homodimeric, and heterodimeric bisalkylguanidine-type histamine H₂ receptor (H₂R) agonists with various alkyl spacers were synthesized. Aiming at increased H₂R selectivity of the ligands, the imidazol-4-yl moiety was replaced by imidazol-1-yl, 2-aminothiazol-5-yl or 2-amino-4-methylthiazol-5-yl according to a bioisosteric approach. All compounds turned out to be partial or full agonists at the h/gp/rH₂R. The most potent analogue, the thiazole-type heterodimeric ligand 63 (UR-Po461), was a partial agonist (E_max = 88%) and 250 times more potent than histamine (pEC₅₀: 8.56 vs 6.16, gpH₂R, atrium). The homodimeric structures 56 (UR-Po395) and 58 (UR-Po448) exhibited the highest hH₂R affinities (pK_i: 7.47, 7.33) in binding studies. Dimeric amino(methyl)thiazole derivatives, such as 58, generated an increased hH₂R selectivity compared to the monomeric analogues, e.g., 139 (UR-Po444). Although monomeric ligands showed up lower affinities and potencies at the H₂R, compounds with a short alkylic side chain like 129 (UR-Po194) proved to be highly affine hH₄R ligands.