Illuminating Neuropeptide Y Y4 Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools.

The neuropeptide Y (NPY) Y4 receptor (Y4R), a member of the family of NPY receptors, is physiologically activated by the linear 36-amino acid peptide pancreatic polypeptide (PP). The Y4R is involved in the regulation of various biological processes, most importantly pancreatic secretion, gastrointestinal motility, and regulation of food intake. So far, Y4R binding affinities have been mostly studied in radiochemical binding assays. Except for a few fluorescently labeled PP derivatives, fluorescence-tagged Y4R ligands with high affinity have not been reported. Here, we introduce differently fluorescence-labeled (Sulfo-Cy5, Cy3B, Py-1, Py-5) Y4R ligands derived from recently reported cyclic hexapeptides showing picomolar Y4R binding affinity. With pKi values of 9.22-9.71 (radioligand competition binding assay), all fluorescent ligands (16-19) showed excellent Y4R affinity. Y4R saturation binding, binding kinetics, and competition binding with reference ligands were studied using different fluorescence-based methods: flow cytometry (Sulfo-Cy5, Cy3B, and Py-1 label), fluorescence anisotropy (Cy3B label), and NanoBRET (Cy3B label) binding assays. These experiments confirmed the high binding affinity to Y4R (equilibrium pKd: 9.02-9.9) and proved the applicability of the probes for fluorescence-based Y4R competition binding studies and imaging techniques such as single-receptor molecule tracking.

Activation of Multiple G Protein Pathways to Characterize the Five Dopamine Receptor Subtypes Using Bioluminescence Technology.

G protein-coupled receptors show preference for G protein subtypes but can recruit multiple G proteins with various downstream signaling cascades. This functional selection can guide drug design. Dopamine receptors are both stimulatory (D1-like) and inhibitory (D2-like) with diffuse expression across the central nervous system. Functional selectivity of G protein subunits may help with dopamine receptor targeting and their downstream effects. Three bioluminescence-based assays were used to characterize G protein coupling and function with the five dopamine receptors. Most proximal to ligand binding was the miniG protein assay with split luciferase technology used to measure recruitment. For endogenous and selective ligands, the G-CASE bioluminescence resonance energy transfer (BRET) assay measured G protein activation and receptor selectivity. Downstream, the BRET-based CAMYEN assay quantified cyclic adenosine monophosphate (cAMP) changes. Several dopamine receptor agonists and antagonists were characterized for their G protein recruitment and cAMP effects. G protein selectivity with dopamine revealed potential Gq coupling at all five receptors, as well as the ability to activate subtypes with the "opposite" effects to canonical signaling. D1-like receptor agonist (+)-SKF-81297 and D2-like receptor agonist pramipexole showed selectivity at all receptors toward Gs or Gi/o/z activation, respectively. The five dopamine receptors show a wide range of potentials for G protein coupling and activation, reflected in their downstream cAMP signaling. Targeting these interactions can be achieved through drug design. This opens the door to pharmacological treatment with more selectivity options for inducing the correct physiological events.

Stereochemistry-Driven Interactions of α,γ-Peptide Ligands with the Neuropeptide Y Y4-Receptor.

The G-protein-coupled Y4-receptor (Y4R) and its endogenous ligand, pancreatic polypeptide (PP), suppress appetite in response to food intake and, thus, are attractive drug targets for body-weight control. The C-terminus of human PP (hPP), T32-R33-P34-R35-Y36-NH2, penetrates deep into the binding pocket with its tyrosine-amide and di-arginine motif. Here, we present two C-terminally amidated α,γ-hexapeptides (1a/b) with sequence Ac-R31-γ-CBAA32-R33-L34-R35-Y36-NH2, where γ-CBAA is the (1R,2S,3R)-configured 2-(aminomethyl)-3-phenylcyclobutanecarboxyl moiety (1a) or its mirror image (1b). Both peptides bind the Y4R (Ki of 1a/b: 0.66/12 nM) and act as partial agonists (intrinsic activity of 1a/b: 50/39%). Their induced-fit binding poses in the Y4R pocket are unique and build ligand-receptor contacts distinct from those of the C-terminus of the endogenous ligand hPP. We conclude that energetically favorable interactions, although they do not match those of the native ligand hPP, still guarantee high binding affinity (with 1a rivaling hPP) but not the maximum receptor activation.

Monitoring the Reversibility of GPCR Signaling by Combining Photochromic Ligands with Label-free Impedance Analysis.

G protein-coupled cell surface receptors (GPCR) trigger complex intracellular signaling cascades upon agonist binding. Classic pharmacological assays provide information about binding affinities, activation or blockade at different stages of the signaling cascade, but real time dynamics and reversibility of these processes remain often disguised. We show that combining photochromic NPY receptor ligands, which can be toggled in their receptor activation ability by irradiation with light of different wavelengths, with whole cell label-free impedance assays allows observing the cell response to receptor activation and its reversibility over time. The concept demonstrated on NPY receptors may be well applicable to many other GPCRs providing a deeper insight into the time course of intracellular signaling processes.

Cardiac Effects of Novel Histamine H2 Receptor Agonists.

In an integrative approach, we studied cardiac effects of recently published novel H2 receptor agonists in the heart of mice that overexpress the human H2 receptor (H2-TG mice) and littermate wild type (WT) control mice and in isolated electrically driven muscle preparations from patients undergoing cardiac surgery. Under our experimental conditions, the H2 receptor agonists UR-Po563, UR-MB-158, and UR-MB-159 increased force of contraction in left atrium from H2-TG mice with pEC50 values of 8.27, 9.38, and 8.28, respectively, but not in WT mice. Likewise, UR-Po563, UR-MB-158, and UR-MB-159 increased the beating rate in right atrium from H2-TG mice with pEC50 values of 9.01, 9.24, and 7.91, respectively, but not from WT mice. These effects could be antagonized by famotidine, a H2 receptor antagonist. UR-Po563 (1 µM) increased force of contraction in Langendorff-perfused hearts from H2-TG but not WT mice. Similarly, UR-Po563, UR-MB-158, or UR-MB-159 increased the left ventricular ejection fraction in echocardiography of H2-TG mice. Finally, UR-Po563 increased force of contraction in isolated human right atrial muscle strips. The contractile effects of UR-Po563 in H2-TG mice were accompanied by an increase in the phosphorylation state of phospholamban. In summary, we report here three recently developed agonists functionally stimulating human cardiac H2 receptors in vitro and in vivo. We speculate that these compounds might be of some merit to treat neurologic disorders if their cardiac effects are blocked by concomitantly applied receptor antagonists that cannot pass through the blood-brain barrier or might be useful to treat congestive heart failure in patients. SIGNIFICANCE STATEMENT: Recently, a new generation of histamine H2 receptor (H2R) agonists has been developed as possible treatment option for Alzheimer's disease. Here, possible cardiac (side) effects of these novel H2R agonists have been evaluated.

Specific Engineered G Protein Coupling to Histamine Receptors Revealed from Cellular Assay Experiments and Accelerated Molecular Dynamics Simulations.

G protein-coupled receptors (GPCRs) are targets of extracellular stimuli and hence occupy a key position in drug discovery. By specific and not yet fully elucidated coupling profiles with α subunits of distinct G protein families, they regulate cellular responses. The histamine H2 and H4 receptors (H2R and H4R) are prominent members of Gs- and Gi-coupled GPCRs. Nevertheless, promiscuous G protein and selective Gi signaling have been reported for the H2R and H4R, respectively, the molecular mechanism of which remained unclear. Using a combination of cellular experimental assays and Gaussian accelerated molecular dynamics (GaMD) simulations, we investigated the coupling profiles of the H2R and H4R to engineered mini-G proteins (mG). We obtained coupling profiles of the mGs, mGsi, or mGsq proteins to the H2R and H4R from the mini-G protein recruitment assays using HEK293T cells. Compared to H2R-mGs expressing cells, histamine responses were weaker (pEC50, Emax) for H2R-mGsi and -mGsq. By contrast, the H4R selectively bound to mGsi. Similarly, in all-atom GaMD simulations, we observed a preferential binding of H2R to mGs and H4R to mGsi revealed by the structural flexibility and free energy landscapes of the complexes. Although the mG α5 helices were consistently located within the HR binding cavity, alternative binding orientations were detected in the complexes. Due to the specific residue interactions, all mG α5 helices of the H2R complexes adopted the Gs-like orientation toward the receptor transmembrane (TM) 6 domain, whereas in H4R complexes, only mGsi was in the Gi-like orientation toward TM2, which was in agreement with Gs- and Gi-coupled GPCRs structures resolved by X-ray/cryo-EM. These cellular and molecular insights support (patho)physiological profiles of the histamine receptors, especially the hitherto little studied H2R function in the brain, as well as of the pharmacological potential of H4R selective drugs.

Dibenzodiazepinone-type muscarinic receptor antagonists conjugated to basic peptides: Impact of the linker moiety and unnatural amino acids on M2R selectivity.

The family of human muscarinic acetylcholine receptors (MRs) is characterized by a high sequence homology among the five subtypes (M1R-M5R), being the reason for a lack of subtype selective MR ligands. In continuation of our work on dualsteric dibenzodiazepinone-type M2R antagonists, a series of M2R ligands containing a dibenzodiazepinone pharmacophore linked to small basic peptides was synthesized (64 compounds). The linker moiety was varied with respect to length, number of basic nitrogens (0-2) and flexibility. Besides proteinogenic basic amino acids (Lys, Arg), shorter homologues of Lys and Arg, containing three and two methylene groups, respectively, as well as D-configured amino acids were incorporated. The type of linker had a marked impact on M2R affinity and also effected M2R selectivity. In contrast, the structure of the basic peptide rather determined M2R selectivity than M2R affinity. For example, the most M2R selective compound (UR-CG188, 89) with picomolar M2R affinity (pKi 9.60), exhibited a higher M2R selectivity (ratio of Ki M1R/M2R/M3R/M4R/M5R: 110:1:5200:55:2300) compared to the vast majority of reported M2R preferring MR ligands. For selected ligands, M2R antagonism was confirmed in a M2R miniG protein recruitment assay.

Structural modifications in the distal, regulatory region of histamine H3 receptor antagonists leading to the identification of a potent anti-obesity agent.

A series of 4-pyridylpiperazine derivatives with varying regulatory region substituents proved to be potent histamine H3 receptor (H3R) ligands in the nanomolar concentration range. The most influential modification that affected the affinity toward the H3R appeared by introducing electron-withdrawing moieties into the distal aromatic ring. In order to finally discuss the influence of the characteristic 4-pyridylpiperazine moiety on H3R affinity, two Ciproxifan analogues 2 and 3 with a slight modification in their basic part were obtained. The replacement of piperazine in 3 with piperidine in compound 2, led to slightly reduced affinity towards the H3R (Ki = 3.17 and 7.70 nM, respectively). In fact, 3 showed the highest antagonistic properties among all compounds in this series, hence affirming our previous assumptions, that the 4-pyridylpiperazine moiety is the key element for suitable interaction with the human histamine H3 receptor. While its structural replacement to piperidine is also tolerated for H3R binding, the heteroaromatic 4-pyridyl moiety seems to be essential for proper ligand-receptor interaction. The putative protein-ligand interactions responsible for their high affinity were demonstrated using molecular modeling techniques. Furthermore, selectivity, intrinsic activity at the H3R, as well as drug-like properties of ligands were evaluated using in vitro methods. Moreover, pharmacological in vivo test results of compound 9 (structural analogue of Abbott's A-331440) clearly indicate that it may affect the amount of calories consumed, thus act as an anorectic compound.

A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine Receptor Subtypes.

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]GTPγS, [γ-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' factor) and the signal spans were improved for all receptor subtypes in comparison to the previously performed [35S]GTPγS binding assay.

Discovery of a G Protein-Biased Radioligand for the Histamine H2 Receptor with Reversible Binding Properties.

Currently employed histamine H2 receptor (H2R) radioligands possess several drawbacks, for example, high non-specificity, insurmountable binding, or short half-life. We report the synthesis and the chemical and pharmacological characterization of the highly stable carbamoylguanidine-type radioligand [3H]UR-KAT479 ([3H]23), a subtype selective histamine H2 receptor G protein-biased agonist. [3H]23 was characterized by saturation, kinetic, and competition binding assays at the human, guinea pig, and mouse H2 receptors (co-)expressed in HEK293(T) cells. [3H]23 reversibly bound to the respective H2Rs with moderate to high affinity (human/guinea pig/mouse Kd: 24/28/94 nM). In order to investigate the applicability of carbamoylguanidine-type ligands in animal studies elucidating the role of the H2R in the brain, we performed a preliminary partitioning experiment in the whole human/mouse blood, which indicated a low binding of [3H]23 to red blood cells. These properties turn [3H]23 into a powerful tool for the determination of binding affinities and demonstrate the promising pharmacokinetic profile of carbamoylguanidine-type ligands.