Bioluminescence-based biosensors to study histamine receptor activity

Histamine receptors are important drug targets due to their role in various (patho) physiological processes and account for 14% of the approved GPCR drugs. In this thesis, biased signaling at histamine receptors was studied using a variety of biosensors. In the field of drug discovery, biased agonism has drawn considerable interest in the last decade, since selective modulation distinct cellular pathways might lead to new drugs with fewer side effects. Hence, biased agonism holds the promise of developing entire new classes of “smarter or safer” drugs. In chapter 2 and 3, we have developed/implemented bioluminescent-based approaches to study β-arrestin2 recruitment to histamine H1 and H4 receptors (H1R and H4R), and evaluated the pharmacological property of the previously identified biased agonist JNJ7777120 at H4R using different approaches. Our results show that JNJ7777120 can act either neutral antagonist or partial agonist with respect to β-arrestin2 recruitment depending on the methods used. Therefore, the selection of appropriate assays to identify potential biased ligands become important and should be taken into consideration in the future. Several approaches and not a single approach should be employed for identification of biased agonists , which can thereby help us comprehensively understand the mechanisms underlying differentiated pharmacologyof a specific biased agonist. Next to classical G protein and β-arrestin2 signaling pathways, various GIPs have in the literature been reported to interact with various GPCRs in constitutive or ligand-induced fashion. GIPs have been reported to involve in GPCR targeting, trafficking and signaling. In chapter 4, 43 potential GIPs including two tetraspanin members TSPAN4 and CD63 were identified to interact with H4R following a MYTH screen of a Jurkat T cell cDNA library under basal condition. Histamine was demonstrated to negatively modulate the interaction between H4R and either tetraspanin member TSPAN4 or CD63 in MYTH liquid growth assay. The interaction of TSPAN4 with H4R and regulation by histamine were subsequently validated using BRET, BiFC and Co-IP technologies in an over-expression system. Furthermore, we found that TSPAN4 does not affect histamine affinity and H4R mediated G protein signaling. The use of CRISPR/Cas9 genomic editing to inhibit TSPAN4 expression in the future can help to better understand the role of TSPAN4 in H4R signaling cascade. Also the validation of other identified GIPs in the future will provide more information on how GIPs may modulate H4R signaling/functioning. In contrast to signaling-dependent assays, conformational biosensors are independent downstream signaling methods that allow to detect the receptor’s conformational changes directly upon ligand binding and without any signal amplification. Therefore, they represent a direct way to measure the efficacy and potency of ligands. A number of RET-based conformational GPCR biosensors that can determine the pharmacological profiles of GPCR ligands have been generated in the past two decades. In chapter 5 and 6, we have successfully generated a H3R biosensor which consists of a Nluc donor at its C terminus and a self-labeling protein Halotag acceptor in the ICL3. This H3R biosensor allows simultaneous screening of H3R agonists and inverse agonists in living cells and H3R biosensor membrane preparation can work as an alternative way for radio-ligand binding assay to evaluate their affinity via its potency as a highly correlation was obtained between potency and affinity values in membranes. The application of this sensor design technology to other GPCRs will aid in the future development of novel receptor ligands. In summary, our studies on biased signaling of histamine receptors provide new insights and theoretical basis for GPCR bias mechanism which thereby in the future can contribute to the drug discovery process.