Optimization of Peptide Linker-Based Fluorescent Ligands for the Histamine H(1) Receptor

[Image: see text] The histamine H(1) receptor (H(1)R) has recently been implicated in mediating cell proliferation and cancer progression; therefore, high-affinity H(1)R-selective fluorescent ligands are desirable tools for further investigation of this behavior in vitro and in vivo. We previously reported a H(1)R 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 H(1)R 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 H(1)R fluorescent ligands, remarkably overriding the linker SAR observed in corresponding unlabeled congeners. Compound 31a, both potent and subtype-selective, enabled H(1)R visualization using confocal microscopy at a concentration of 10 nM. Molecular docking of 31a with the human H(1)R predicts that the optimized peptide linker makes interactions with key residues in the receptor.