A MiniG Protein Recruitment Assay for the Histamine Receptor Family to Functionally Characterize Histamine Receptor Ligands

In addition to the determination of binding affinities, the functional characterization of biologically active compounds is a prerequisite in drug discovery in order to identify lead structures and to optimize potential scaffolds regarding their mode of action and potency. Therefore, our aim was to develop a high‐throughput screening (HTS) compatible assay procedure yielding functional parameters of histamine receptor ligands at a proximal level avoiding signal amplification. For this purpose, we applied the NanoLuc split‐luciferase technology [1] to the histamine receptor subtypes H 1 , H 2 , H 3 and H 4 and the corresponding G protein surrogates miniG q , miniG s and miniG i [2] . The small fragment of the NanoLuc (NlucC) was fused to the C‐terminus of the respective receptor to retain the integrity of the receptors regarding binding affinities and intracellular signalling, whereas the large fragment of the NanoLuc (NlucN) was N‐terminally fused to the miniG proteins (see fig. 1 ). Upon activation by a ligand the corresponding miniG protein is recruited to the receptor which then leads to the formation of a functional NanoLuc enzyme enabling the registration of a concentration dependent luminescence signal (see fig. 1 ). Compared to reported data, standard ligands for histamine receptors provide appropriate potencies, intrinsic activities and modes of action .By radioligand saturation binding experiments it could be shown that there was no alteration in the binding affinities of the radioligands due to the minor receptor modification by the C‐terminally fused NlucC. Additionally, this assay principle should be appropriate for HTS assessed by the investigation of the Z′ factor, which was in an excellent range for all histamine receptor subtypes (0.5 < Z′ < 1). From these results we conclude that the application of the presented methodology is an excellent approach to be implemented in routine and detailed functional characterisation of putative histamine receptor ligands synthesized in our group. Moreover, the assay offers an opportunity to draw conclusions on biased signalling of investigated ligands in combination with the results obtained in β‐arrestin recruitment assays [3] . Support or Funding Information This project is funded by the DFG research training group GRK1910. Schematic illustration of the assay principle. Two complementing NanoLuc fragments fused to the receptor and the mini G protein form a functional NanoLuc upon activation of the GPCR. Figure 1