Role of the second and third extracellular loops of the histamine H4 receptor in receptor activation

The histamine H4 receptor subtype (H4R) belongs to the class 1 of G protein-coupled receptors and is involved in inflammatory and immunological processes. The aim of this study was to elucidate the importance of extracellular regions for the large species differences between human (h) and canine (c) H4R. Therefore, chimeric receptors were generated by replacing corresponding domains of the hH4R with canine N-terminus (hcNTH4R) and three canine extracellular loops, respectively (hcE1H4R, hcE2H4R and hcE3H4R). Wild-type and chimeric H4 receptors were expressed in Sf9 insect cells and subsequently characterized in [3H]histamine binding experiments and in steady-state GTPase activity assays, where standard H4R ligands histamine, 5-methylhistamine, thioperamide, 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine (JNJ7777120) and clozapine were examined. The exchange of N-terminus or first extracellular loop did not influence hH4R pharmacology. The effect of altered second extracellular loop (E2-loop) and third extracellular loop (E3-loop) was rather ligand-specific than agonist/inverse agonist-specific. At hcE3H4R, the potency of histamine and 5-methylhistamine significantly decreased. The efficacy of the inverse agonist thioperamide was strongly reduced at hcE2H4R and hcE3H4R. Surprisingly, JNJ7777120 as weak inverse agonist at hH4R exhibited partial agonistic activity at hcE2H4R and hcE3H4R. Molecular dynamic simulations suggest that the E2- and E3-loops are independently of each other involved in partial/inverse agonism of JNJ7777120 and that E2- as well as E3-loop do not directly interact with JNJ7777120 in the binding pocket. In conclusion, our study indicates an involvement of E2- and E3-loop in H4R activation process after binding of some but not all examined ligands

Pharmacological profile of astemizole-derived compounds at the histamine H1 and H4 receptor - H1/H4 receptor selectivity

Astemizole, a H1R antagonist shows high affinity to the histamine H1 receptor but only a moderate affinity to the histamine H4 receptor. This study aims to modify the astemizole to keep high affinity to the histamine H1 receptor and to increase affinity to the histamine H4 receptor. Therefore, 13 astemizole-derived compounds and astemizole-JNJ7777120-derived hybrid compounds were synthesized and pharmacologically characterized at the histamine H1 and H4 receptors. The new compounds show affinity to the histamine H1 receptor in the pK i range from 5.3 to 8.8, whereas the affinity of these compounds to the histamine H4 receptor was surprisingly rather low (pK i from 4.4 to 5.6). Three representative compounds were docked into the histamine H1 receptor and molecular dynamic studies were performed to explain the binding mode and the experimental results on a molecular level. Furthermore, taking into account the binding mode of compounds with high affinity to the histamine H4 receptor, a H1/H4-pharmacophore hypothesis was developed