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The histamine H1 receptor (H1R) has recently been implicated in mediating cell proliferation and cancer progression; therefore, high-affinity H1R-selective fluorescent ligands are desirable tools for further investigation of this behavior in vitro and in vivo. We previously reported a H1R fluorescent ligand, bearing a peptide-linker, based on antagonist VUF13816 and sought to further explore structure-activity relationships (SARs) around the linker, orthostere, and fluorescent moieties. Here, we report a series of high-affinity H1R fluorescent ligands varying in peptide linker composition, orthosteric targeting moiety, and fluorophore. Incorporation of a boron-dipyrromethene (BODIPY) 630/650-based fluorophore conferred high binding affinity to our H1R fluorescent ligands, remarkably overriding the linker SAR observed in corresponding unlabeled congeners. Compound 31a, both potent and subtype-selective, enabled H1R visualization using confocal microscopy at a concentration of 10 nM. Molecular docking of 31a with the human H1R predicts that the optimized peptide linker makes interactions with key residues in the receptor.

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.