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In drug discovery, assays with proximal readout are of great importance to study target-specific effects of potential drug candidates. In the field of G protein-coupled receptors (GPCRs), the determination of GPCR-G protein interactions and G protein activation by means of radiolabeled GTP analogs ([35S]GTP&gamma, S, [&gamma, 32P]GTP) has widely been used for this purpose. Since we were repeatedly faced with insufficient quality of radiolabeled nucleotides, there was a requirement to implement a novel proximal functional assay for the routine characterization of putative histamine receptor ligands. We applied the split-NanoLuc to the four histamine receptor subtypes (H1R, H2R, H3R, H4R) and recently engineered minimal G (mini-G) proteins. Using this method, the functional response upon receptor activation was monitored in real-time and the four mini-G sensors were evaluated by investigating selected standard (inverse) agonists and antagonists. All potencies and efficacies of the studied ligands were in concordance with literature data. Further, we demonstrated a significant positive correlation of the signal amplitude and the mini-G protein expression level in the case of the H2R, but not for the H1R or the H3R. The pEC50 values of histamine obtained under different mini-G expression levels were consistent. Moreover, we obtained excellent dynamic ranges (Z&rsquo, factor) and the signal spans were improved for all receptor subtypes in comparison to the previously performed [35S]GTP&gamma, S binding assay.

The gastrointestinal microbiota is emerging as a unique and inexhaustible source for metabolites with potential to modulate G-protein coupled receptors (GPCRs). The ghrelin receptor [growth hormone secretagogue receptor (GHSR)-1a] is a GPCR expressed throughout both the gut and the brain and plays a crucial role in maintaining energy balance,metabolism, and the centralmodulation of food intake,motivation, reward, andmood. To date, few studies have investigated the potential of the gastrointestinal microbiota and its metabolites to modulate GPCR signaling. Here we investigate the ability of short-chain fatty acids (SCFAs), lactate, and different bacterial strains, including Bifidobacteriumand Lactobacillus genera, tomodulateGHSR-1a signaling.We identify, forwhat is to our knowledge the first time, a potent effect ofmicrobiota-derivedmetabolites on GHSR-1a signaling with potential significant consequences for host metabolism and physiology. We show that SCFAs, lactate, and bacterial supernatants are able to attenuate ghrelin-mediated signaling through the GHSR-1a.We suggest a novel route of communication between the gut microbiota and the host via modulation of GHSR-1a receptor signaling. Together, this highlights the emerging therapeutic potential in the exploration of themicrobiota metabolome in the specific targeting of key GPCRs, with pleiotropic actions that span both the CNS and periphery., This study was supported by Science Foundation Ireland Research Centre Grant SFI/12/RC/2273 to the Alimentary Pharmabiotic Centre (APC) Microbiome Ireland, Student/Faculty Interaction (SFI) Grant 12/RC/2276 DBP and APC Microbiome Ireland industry partner Cremo SA

On the basis of the long-known prototypic pharmacophore 3-(1H-imidazol-4-yl) propylguanidine (SK&F 91486, 2), monomeric, homodimeric, and heterodimeric bisalkylguanidine-type histamine H-2 receptor (H2R) agonists with various alkyl spacers were synthesized. Aiming at increased H2R selectivity of the ligands, the imidazol-4-yl moiety was replaced by imidazol-1-yl, 2-aminothiazol-5-yl or 2-amino-4-methylthiazol-5-yl according to a bioisosteric approach. All compounds turned out to be partial or full agonists at the h/gp/rH(2)R. The most potent analogue, the thiazole-type heterodimeric ligand 63 (UR-Po461), was a partial agonist (E-max = 88%) and 250 times more potent than histamine (pEC(50): 8.56 vs 6.16, gpH(2)R, atrium). The homodimeric structures 56 (UR-Po395) and 58 (UR-Po448) exhibited the highest hH(2)R affinities (pK(i): 7.47, 7.33) in binding studies. Dimeric amino(methyl) thiazole derivatives, such as 58, generated an increased hH(2)R selectivity compared to the monomeric analogues, e.g., 139 (UR-Po444). Although monomeric ligands showed up lower affinities and potencies at the H2R, compounds with a short alkylic side chain like 129 (UR-Po194) proved to be highly affine hH(4)R ligands., zu den Nachnutzungsbedingungen: "ACS AuthorChoice - This is an open access article published under an ACS AuthorChoice License (https://pubs.acs.org/page/policy/authorchoice_termsofuse.html), which permits copying and redistribution of the article or any adaptations for non-commercial purposes