Saturation Binding Experiments Research Articles

Discovery of a Tritiated Radioligand with High Affinity and Selectivity for the Histamine H3 Receptor.

Radioligands used previously for histamine H3 receptor (H3R) are accompanied by a number of disadvantages. In this study, we report the synthesis of the new H3R radioligand [3H]UR-MN259 ([3H]11) with high (radio)chemical purity and stability. The radioligand exhibits sub-nanomolar affinity for the target receptor (pKi (H3R) = 9.56) and displays an outstanding selectivity profile within the histamine receptor family (>100,000-fold selective). [3H]UR-MN259 is ideally suitable for the characterization of H3R ligands in competition binding and shows one-site binding to the H3R in saturation binding experiments. The radiotracer shows fast association to the receptor (τassoc = 6.11 min), as well as full dissociation from the receptor (τdissoc = 14.48 min) in kinetic binding studies. The distinguished profile of [3H]UR-MN259 makes it a highly promising pharmacological tool to further investigate the role of the H3R in the CNS.

Insertion of Nanoluc into the Extracellular Loops as a Complementary Method To Establish BRET-Based Binding Assays for GPCRs.

Luminescence-based techniques play an increasingly important role in all areas of biochemical research, including investigations on G protein-coupled receptors (GPCRs). One quite recent and popular addition has been made by introducing bioluminescence resonance energy transfer (BRET)-based binding assays for GPCRs, which are based on the fusion of nanoluciferase (Nluc) to the N-terminus of the receptor and the occurring energy transfer via BRET to a bound fluorescent ligand. However, being based on BRET, the technique is strongly dependent on the distance/orientation between the luciferase and the fluorescent ligand. Here we describe an alternative strategy to establish BRET-based binding assays for GPCRs, where the N-terminal fusion of Nluc did not result in functioning test systems with our fluorescent ligands (e.g., for the neuropeptide Y Y1 receptor (Y1R) and the neurotensin receptor type 1 (NTS1R)). Instead, we introduced Nluc into their second extracellular loop and we obtained binding data for the fluorescent ligands and reported standard ligands (in saturation and competition binding experiments, respectively) comparable to data from the literature. The strategy was transferred to the angiotensin II receptor type 1 (AT1R) and the M1 muscarinic acetylcholine receptor (M1R), which led to affinity estimates comparable to data from radioligand binding experiments. Additionally, an analysis of the binding kinetics of all fluorescent ligands at their respective target was performed using the newly described receptor/Nluc-constructs.

Streptococcal autolysin promotes dysfunction of swine tracheal epithelium by interacting with vimentin.

Streptococcus suis serotype 2 (SS2) is a major zoonotic pathogen resulting in manifestations as pneumonia and septic shock. The upper respiratory tract is typically thought to be the main colonization and entry site of SS2 in pigs, but the mechanism through which it penetrates the respiratory barrier is still unclear. In this study, a mutant with low invasive potential to swine tracheal epithelial cells (STECs) was screened from the TnYLB-1 transposon insertion mutant library of SS2, and the interrupted gene was identified as autolysin (atl). Compared to wild-type (WT) SS2, Δatl mutant exhibited lower ability to penetrate the tracheal epithelial barrier in a mouse model. Purified Atl also enhanced SS2 translocation across STEC monolayers in Transwell inserts. Furthermore, Atl redistributed the tight junctions (TJs) in STECs through myosin light chain kinase (MLCK) signaling, which led to increased barrier permeability. Using mass spectrometry, co-immunoprecipitation (co-IP), pull-down, bacterial two-hybrid and saturation binding experiments, we showed that Atl binds directly to vimentin. CRISPR/Cas9-targeted deletion of vimentin in STECs (VIM KO STECs) abrogated the capacity of SS2 to translocate across the monolayers, SS2-induced phosphorylation of myosin II regulatory light chain (MLC) and MLCK transcription, indicating that vimentin is indispensable for MLCK activation. Consistently, vimentin null mice were protected from SS2 infection and exhibited reduced tracheal and lung injury. Thus, MLCK-mediated epithelial barrier opening caused by the Atl-vimentin interaction is found to be likely the key mechanism by which SS2 penetrates the tracheal epithelium.

The development of [18F]ACI‐12589, a high affinity and selective alpha‐synuclein radiotracer, as a biomarker for Parkinson’s disease and other synucleinopathies

AbstractBackgroundParkinson’s disease (PD) and other alpha‐synucleinopathies are characterized by the progressive accumulation of alpha‐synuclein (a‐syn) inclusions in the brain, which start in the pre‐motor stage of the disease and correlates with its severity. An imaging agent for a‐syn could allow differential diagnosis and support interventional trails. AC Immune has developed and is currently testing in a first in human (FiH) clinical trial the recently discovered compound, ACI‐12589, as a PET tracer with high affinity and selectivity for a‐syn.MethodScreening of our conformation‐specific, small‐molecular weight MorphomerTM library was followed by an iterative medicinal chemistry optimization program. The primary screening strategy included the development and use of a proprietary binding assay, that importantly involved aggregates derived from PD brain samples. Autoradiography experiments performed with human tissue samples from different alpha‐synucleinopathies provided target engagement and signal specificity data. Autoradiography techniques were also employed to assess selectivity, using tissue samples from Alzheimer’s disease or Frontotemporal lobar degeneration‐TDP donors. Selected compounds were 18F radiolabeled and evaluated for their pharmacokinetics in non‐human primates (NHPs) before proceeding to clinical studies.ResultRecent optimization efforts have led to the identification of several new, highly promising chemical scaffolds. From these, the compound ACI‐12589, has emerged with highly favorable characteristics including low nanomolar affinity and excellent target occupancy in saturation binding experiments using PD brain homogenates. Using high‐resolution autoradiography, ACI‐12589 illustrates target engagement across multiple alpha‐synucleinopathies including PD, Multiple System Atrophy, and Dementia with Lewy Bodies. In classical autoradiography experiments, ACI‐12589 demonstrates significantly higher specific signal in diseased versus non‐diseased tissue samples. Furthermore, ACI‐12589 has at least 30‐fold selectivity versus Abeta and shows no binding to Tau nor TDP‐43 aggregates. Studying [18F]ACI‐12589 in NHPs reveals rapid brain uptake and quick wash out. ACI‐12589 was considered safe in the IND‐enabling toxicology study allowing advancement into the FiH clinical study.ConclusionBased on experience with other tracers, [18F]ACI‐12589 meets the target product profile deemed necessary of a highly favorable candidate to succeed as a radiotracer for imaging a‐syn inclusions. As such, [18F]ACI‐12589 has advanced into the First in Human clinical trial in patients with Parkinson’s disease to be followed by evaluation in other alpha‐synucleinopathies.

1-Indanone and 1,3-indandione Derivatives as Ligands for Misfolded α-Synuclein Aggregates.

The development of imaging agents for in vivo detection of alpha-synuclein (α-syn) pathologies faces several challenges. A major gap in the field is the lack of diverse molecular scaffolds with high affinity and selectivity to α-syn fibrils for in vitro screening assays. Better in vitro scaffolds can instruct the discovery of better in vivo agents. We report the rational design, synthesis, and in vitro evaluation of a series of novel 1-indanone and 1,3-indandione derivatives from a Structure-Activity Relationship (SAR) study centered on some existing α-syn fibril binding ligands. Our results from fibril saturation binding experiments show that two of the lead candidates compounds 8 and 32 bind α-syn fibrils with binding constants (Kd ) of 9.0 and 18.8 nM, respectively, and selectivity of greater than 10× for α-syn fibrils compared with amyloid-β (Aβ) and tau fibrils. Our results demonstrate that the lead ligands avidly label all forms of α-syn on PD brain tissue sections, but only the dense core of senile plaques in AD brain tissue, respectively. These results are corroborated by ligand-antibody colocalization data from Syn211, which shows immunoreactivity toward all forms of α-syn aggregates, and Syn303, which displays preferential reactivity toward mature Lewy pathology. Our results reveal that 1-indanone derivatives have desirable properties for the biological evaluation of α-synucleinopathies.

OAB-044: Anti-CD38 nanobodies as theranostic agents for multiple myeloma

<h3>Background</h3> Theranostic agents include molecules with a combined diagnostic and therapeutic capability and nanobodies (Nbs) are increasingly used in this field. Nbs are single-domain antigen-binding fragments that are derived from Camelidae heavy-chain antibodies and have several advantages such as their small size leading to better tissue penetration, their favourable pharmacological properties and their ability to recognise small, buried epitopes. <h3>Methods</h3> The current project aims to use nanobodies directed against CD38 as diagnostic and therapeutic tools in the management of multiple myeloma (MM) disease. The differentiation marker CD38 is highly expressed on myeloma cells isolated from patients with a newly diagnosed or a relapsing disease. Our Nb, retained for its affinity, stability, its favorable biodistribution and its capacity to recognize myeloma cells in a xenograft model of MM, was coupled to Indium-111 and Lutetium-177 and intravenously injected in tumor bearing mice to assess its diagnostic and therapeutic value. Tumor development was assessed by bioluminescence. <h3>Results</h3> The binding capacities of 111In-DTPA-Nb2F8 was verified by saturation binding experiments (using serial dilutions of the conjugated Nb) on CD38+ RPMI-8226 cells and internalisation experiments were performed in order to assess their therapeutic potential. A minor internalisation (about 20% of the initial bound activity) was observed in the first hours and remained stable during 24H. Micro-SPECT/CT images of mice bearing CD38+ RPMI-8226 tumors and injected with 111In-DPTA-Nb2F8 showed specific tumor targeting 1 hour and at least until 48 hours after injection with a low background signal already 1 hour post-injection (p.i.), except kidneys. The in and ex vivo biodistribution data revealed uptake values in tumor of 3.1% IA/g at 1h post injection, while the uptake of a labelled control Nb was low 0.54% IA/g confirming the specific targeting of our anti-CD38 Nb. Similar biodistributions were found with 177Lu-DTPA-Nb2F8. In order to assess the therapeutic potential of this nanobody, mice with subcutaneously injected RPMI-8226 cells were randomly categorised into 3 groups (n=10). Mice in each group received 3 consecutive intravenous administrations of a high (17.5MBq) or a low radioactive dose (8.6MBq) 177Lu-DTPA-2F8 or an equal volume of vehicle solution. A dose-dependent regression was observed for both therapeutic regimens, which translated in a prolonged median survival from 43 days for vehicle-treated mice, compared to 62 days (p=0.027) for the low and 65 days for the high (p=0.0007) radioactive dose regimen respectively. <h3>Conclusions</h3> In conclusion, this is the first report on CD38-binding Nbs used for the identification and treatment of MM cells by conjugating them to diagnostic and therapeutic isotopes.

The skeletal muscle relaxer cyclobenzaprine is a potent non‐competitive histamine H1 receptor antagonist

Cyclobenzaprine (Flexeril®) is a tricyclic dimethylpropanamine skeletal muscle relaxant that is used clinically to decrease muscle spasm and hypercontractility, without altering homeostatic muscle function. Although the absolute mechanism of action of cyclobenzaprine is unknown, it acts centrally to reduce tonic somatic motor function, likely via modulation of serotonergic and/or noradrenergic systems. While cyclobenzaprine is effective as a muscle relaxant, greater than 30% of patients experience drowsiness and sedative effects, yet, the mechanisms that cause these side effects are also undescribed. Based on this common adverse effect profile and the structural similarity of cyclobenzaprine to tricyclic antidepressants, we hypothesized that cyclobenzaprine facilitates sedative effects via off-target antagonism of central histamine H1 receptors (H1R). Here, for the first time, we present data that demonstrate that cyclobenzaprine exhibits low nanomolar affinity for the cloned human H1R, as well as that expressed in both rat and mouse brain. Using saturation binding experiments, our results demonstrate that cyclobenzaprine binds to the H1R in a non-competitive manner. Finally, using functional Ca+2 influx assays and novel TRUPATH BRET sensors, we show that cyclobenzaprine also blocks histamine-mediated H1R functional activity in a non-competitive manner. Given that cyclobenzaprine readily crosses the blood-brain barrier and its muscle relaxant effects occur centrally, our data suggest that off-target central antagonism of H1R by cyclobenzaprine facilitate the significant sedative effect of this agent seen in patients.

Fluorescence polarization as an approach to study the molecular interaction between lupin seed γ-conglutin and insulin

γ-Conglutin is an unusual protein found lupin in seeds. Numerous scientific data indicate that this protein is capable of reducing glucose levels in the blood. Unfortunately, the molecular mechanisms underlying this unique feature remain poorly understood, mostly due to experimental obstacles. Interestingly, γ-conglutin shows also insulin-binding ability, however, there is limited data about the kinetics of this interaction. Moreover, the available information comes from studies in which γ-conglutin was immobilized or partially unfolded. Herein, the fluorescence polarization assay application allowed for a successful determination of the insulin-γ-conglutin complex's binding parameters. Saturation and competitive binding experiments were done in a solution where γ-conglutin and insulin retained their native structure. The dissociation (Kd) and inhibition (Ki) constant of the complex was 9.16 × 10−6 M and 7.34 × 10−6 M, respectively. It is expected that knowing the quantitative interaction parameters can help in explaining the γ-conglutin hypoglycemic activity.

Intra-individual dynamic comparison of 18F-PSMA-11 and 68Ga-PSMA-11 in LNCaP xenograft bearing mice

Recently, a 18F-labeled derivative of the widely used 68Ga-PSMA-11 was developed for PET imaging of prostate cancer. Although 18F-PSMA-11 has already been evaluated in a Phase I and Phase II clinical trial, preclinical evaluation of this radiotracer is important for further understanding its dynamic behavior. Saturation binding experiments were conducted by incubation of LNCaP cells with 18F-PSMA-11 or 68Ga-PSMA-11 for 1 h, followed by determination of the specific and aspecific binding. Mice bearing LNCaP or PC-3 xenografts each received ± 3.7 MBq 18F-PSMA-11 and 68Ga-PSMA-11 followed by dynamic acquisition of 2.5 h as well as ± 15 MBq 18F-FDG followed by static acquisition at 1 h post injection (p.i.). Uptake was evaluated by comparison of uptake parameters (SUVmean, SUVmax, TBRmean and TBRmax). Mice underwent ex vivo biodistribution where 18F-PSMA-11 activity was measures in excretory organs (kidneys, bladder and liver) as well as bone fragments (femur, humerus, sternum and skull) to evaluate bone uptake. The dissociation constant (Kd) of 18F-PSMA-11 and 68Ga-PSMA-11 was 2.95 ± 0.87 nM and 0.49 ± 0.20 nM, respectively. Uptake parameters were significantly higher in LNCaP compared to PC-3 xenografts for both 18F-PSMA-11 and 68Ga-PSMA-11, while no difference was found for 18F-FDG uptake (except for SUVmax). Tumor uptake of 18F-PSMA-11 showed a similar trend over time as 68Ga-PSMA-11, although all uptake parameter curves of the latter were considerably lower. When comparing early (60 min p.i.) to delayed (150 min p.i.) imaging for both radiotracers individually, TBRmean and TBRmax were significantly higher at the later timepoint, as well as the SUVmax of 68Ga-PSMA-11. The highest %ID/g was determined in the kidneys (94.0 ± 13.6%ID/g 1 h p.i.) and the bladder (6.48 ± 2.18%ID/g 1 h p.i.). No significant increase in bone uptake was seen between 1 and 2 h p.i. Both radiotracers showed high affinity for the PSMA receptor. Over time, all uptake parameters were higher for 18F-PSMA-11 compared to 68Ga-PSMA-11. Delayed imaging with the latter may improve tumor visualization, while no additional benefits could be found for late 18F-PSMA-11 imaging. Ex vivo biodistribution demonstrated fast renal clearance of 18F-PSMA-11 as well as no significant increase in bone uptake.

A2A adenosine receptors are involved in the reparative response of tendon cells to pulsed electromagnetic fields.

Tendinopathy is a degenerative disease in which inflammatory mediators have been found to be sometimes present. The interaction between inflammation and matrix remodeling in human tendon cells (TCs) is supported by the secretion of cytokines such as IL-1β, IL-6 and IL-33. In this context, it has been demonstrated that pulsed electromagnetic fields (PEMFs) were able to reduce inflammation and promote tendon marker synthesis. The aim of this study was to evaluate the anabolic and anti-inflammatory PEMF-mediated response on TCs in an in vitro model of inflammation. Moreover, since PEMFs enhance the anti-inflammatory efficacy of adenosine through the adenosine receptors (ARs), the study also focused on the role of A2AARs. Human TCs were exposed to PEMFs for 48 hours. After stimulation, A2AAR saturation binding experiments were performed. Along with 48 hours PEMF stimulation, TCs were treated with IL-1β and A2AAR agonist CGS-21680. IL-1Ra, IL-6, IL-8, IL-10, IL-33, VEGF, TGF-β1, PGE2 release and SCX, COL1A1, COL3A1, ADORA2A expression were quantified. PEMFs exerted A2AAR modulation on TCs and promoted COL3A1 upregulation and IL-33 secretion. In presence of IL-1β, TCs showed an upregulation of ADORA2A, SCX and COL3A1 expression and an increase of IL-6, IL-8, PGE2 and VEGF secretion. After PEMF and IL-1β exposure, IL-33 was upregulated, whereas IL-6, PGE2 and ADORA2A were downregulated. These findings demonstrated that A2AARs have a role in the promotion of the TC anabolic/reparative response to PEMFs and to IL-1β.

Toll-like receptor 4-mediated inflammation triggered by extracellular IFI16 is enhanced by lipopolysaccharide binding.

Damage-associated molecular patterns (DAMPs) are endogenous molecules activating the immune system upon release from injured cells. Here we show that the IFI16 protein, once freely released in the extracellular milieu of chronically inflamed tissues, can function as a DAMP either alone or upon binding to lipopolysaccharide (LPS). Specifically, using pull-down and saturation binding experiments, we show that IFI16 binds with high affinity to the lipid A moiety of LPS. Remarkably, IFI16 DAMP activity is potentiated upon binding to subtoxic concentrations of strong TLR4-activating LPS variants, as judged by TLR4-MD2/TIRAP/MyD88-dependent IL-6, IL-8 and TNF-α transcriptional activation and release in stimulated monocytes and renal cells. Consistently, using co-immunoprecipitation (co-IP) and surface plasmon resonance (SPR) approaches, we show that IFI16 is a specific TLR4-ligand and that IFI16/LPS complexes display a faster stimulation turnover on TLR4 than LPS alone. Altogether, our findings point to a novel pathomechanism of inflammation involving the formation of multiple complexes between extracellular IFI16 and subtoxic doses of LPS variants, which then signal through TLR4.

The Saturation Game: Teaching Protein–Ligand Binding with a Playing Card Analogy

The dissociation constant of a protein–ligand complex is commonly measured by a saturation binding experiment, in which a protein is progressively saturated by repeated additions of a ligand. Stude...

NanoBRET binding assay for histamine H2 receptor ligands using live recombinant HEK293T cells

Fluorescence/luminescence-based techniques play an increasingly important role in the development of test systems for the characterization of future drug candidates, especially in terms of receptor binding in the field of G protein-coupled receptors (GPCRs). In this article, we present the establishment of a homogeneous live cell-based BRET binding assay for the histamine H2 receptor with different fluorescently labeled squaramide-type compounds synthesized in the course of this study. Py-1-labeled ligand 8 (UR-KAT478) was found to be most suitable in BRET saturation binding experiments with respect to receptor affinity (pKd = 7.35) and signal intensity. Real-time kinetic experiments showed a full association of 8 within approximately 30 min and a slow dissociation of the ligand from the receptor. Investigation of reference compounds in BRET-based competition binding with 8 yielded pKi values in agreement with radioligand binding data. This study shows that the BRET binding assay is a versatile test system for the characterization of putative new ligands at the histamine H2 receptor and represents a valuable fluorescence-based alternative to canonical binding assays.

Fluorescent H2 Receptor Squaramide-Type Antagonists: Synthesis, Characterization, and Applications.

Fluorescence labeled ligands have been gaining importance as molecular tools, enabling receptor-ligand-binding studies by various fluorescence-based techniques. Aiming at red-emitting fluorescent ligands for the hH2R, a series of squaramides labeled with pyridinium or cyanine fluorophores (19-27) was synthesized and characterized. The highest hH2R affinities in radioligand competition binding assays were obtained in the case of pyridinium labeled antagonists 19-21 (pK i: 7.71-7.76) and cyanine labeled antagonists 23 and 25 (pK i: 7.67, 7.11). These fluorescent ligands proved to be useful tools for binding studies (saturation and competition binding as well as kinetic experiments), using confocal microscopy, flow cytometry, and high content imaging. Saturation binding experiments revealed pK d values comparable to the pK i values. The fluorescent probes 21, 23, and 25 could be used to localize H2 receptors in HEK cells and to determine the binding affinities of unlabeled compounds.

Adenosinergic System Involvement in Ischemic Stroke Patients' Lymphocytes.

Adenosine modulates many physiological processes through the interaction with adenosine receptors (ARs) named as A1, A2A, A2B, and A3ARs. During ischemic stroke, adenosine mediates neuroprotective and anti-inflammatory effects through ARs activation. One of the dominant pathways generating extracellular adenosine involves the dephosphorylation of ATP by ecto-nucleotidases CD39 and CD73, which efficiently hydrolyze extracellular ATP to adenosine. The aim of the study is to assess the presence of ARs in lymphocytes from ischemic stroke patients compared to healthy subjects and to analyze changes in CD39 and CD73 expression in CD4+ and CD8+ lymphocytes. Saturation binding experiments revealed that A2AARs affinity and density were significantly increased in ischemic stroke patients whilst no differences were found in A1, A2B, and A3ARs. These results were also confirmed in reverse transcription (RT)-polymerase chain reaction (PCR) assays where A2AAR mRNA levels of ischemic stroke patients were higher than in control subjects. In flow cytometry experiments, the percentage of CD73+ cells was significantly decreased in lymphocytes and in T-lymphocyte subclasses CD4+ and CD8+ obtained from ischemic stroke patients in comparison with healthy individuals. These data corroborate the importance of the adenosinergic system in ischemic stroke and could open the way to more targeted therapeutic approaches and biomarker development for ischemic stroke.

Red-Emitting Dibenzodiazepinone Derivatives as Fluorescent Dualsteric Probes for the Muscarinic Acetylcholine M2 Receptor

Fluorescently labeled dibenzodiazepinone-type muscarinic acetylcholine receptor (MR) antagonists, including dimeric ligands, were prepared using red-emitting cyanine dyes. Probes containing a fluorophore with negative charge showed high M2R affinities (pKi (radioligand competition binding): 9.10-9.59). Binding studies at M1 and M3-M5 receptors indicated a M2R preference. Flow cytometric and high-content imaging saturation and competition binding (M1R, M2R, and M4R) confirmed occupation of the orthosteric site. Confocal microscopy revealed that fluorescence was located mainly at the cell membrane (CHO-hM2R cells). Results from dissociation and saturation binding experiments (M2R) in the presence of allosteric M2R modulators (dissociation: W84, LY2119620, and alcuronium; saturation binding: W84) were consistent with a competitive mode of action between the fluorescent probes and the allosteric ligands. Taken together, these lines of evidence indicate that these ligands are useful fluorescent molecular tools to label the M2R in imaging and binding studies and suggest that they have a dualsteric mode of action.

Tritiated JNJ‐63514230 is a novel GluN2B‐selective radioligand with no affinity to sigma receptors

IntroductionCommonly used GluN2B radioligands exhibit limited selectivity towards GluN2B receptors as they interact with sigma and other off‐target receptors. Here we describe development and characterization of [3H] JNJ‐63514230, a novel radioligand selective for GluN2B‐containing NMDA receptors.MethodsThe selectivity JNJ 63514230 was determined using functional assays and CEREP panel. Saturation binding and competitive radioligand binding experiments were used to characterize [3H]‐JNJ 63514230 properties. We have also tested utility of [3H]‐JNJ 63514230 as a tracer for ex‐vivo autoradiography in rat brain.ResultsWe confirmed that widely used tracers [3H] Ifenprodil and [3H] Ro 25‐6981 have substantial affinity to sigma receptors as measured in radioligand competition experiments (CEREP). In contrast, new tracer [3H] JNJ‐63514230 exhibits no affinity to sigma 1 and sigma 2 receptors in CEREP assays. Saturation binding in rat brain membranes yielded a Kd value of 2.53 nM and Bmax of 2531 fmol/mg. Association constant Kon, dissociation constant Koff, and calculated equilibrium binding constant (Kon/Koff) were 0.0067±0.001 min−1 nM‐1, 0.021±0.0002 min−1, and 3.28±0.61 nM, respectively (average of 3 individual experiments each ± SD). Enhanced selectivity for GluN2B‐containing receptors translates to reduced non‐specific binding and better signal to noise ratio in both in vitro radioligand binding assays and ex vivo autoradiography.ConclusionsWe showed that new radioligand [3H] JNJ 63514230 is superior to previous previously described GluN2B tracers as it permits accurate measurement of affinity to GluN2B‐containing receptors (Ki) without using additional blockers to mask native sigma receptors.Support or Funding InformationAuthors are full time employees of Janssen Research &amp; Development, LLC. The research was funded by the company.

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 H1, H2, H3 and H4 and the corresponding G protein surrogates miniGq, miniGs and miniGi [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 &lt; Z′ &lt; 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 InformationThis 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

Identification of inhibitors of the RGS homology domain of GRK2 by docking-based virtual screening

AimsG protein-coupled receptor (GPCR) kinases (GRKs) are mainly involved in the desensitization of GPCRs. Among them, GRK2 has been described to be upregulated in many pathological conditions and its crucial role in cardiac hypertrophy, hypertension, and heart failure promoted the search for pharmacological inhibitors of its activity. There have been several reports of potent and selective inhibitors of GRK2, most of them directed to the kinase domain of the protein. However, the homologous to the regulator of G protein signaling (RH) domain of GRK2 has also been shown to regulate GPCRs signaling. Herein, we searched for potential inhibitors of receptor desensitization mediated by RH domain of GRK2. Materials and methodsWe performed a docking-based virtual screening utilizing the crystal structure of GRK2 to search for potential inhibitors of the interaction between GRK2 and Gαq protein. To evaluate the biological activity of compounds we measured, calcium response of histamine H1 receptor (H1R) using Fura-2AM dye and H1R internalization by saturation binding experiments in A549 cells. GRK2(45–178)GFP translocation was determined in HeLa cells through confocal fluorescence imaging. Key findingsWe identified inhibitors of GRK2 able to reduce the RH mediated desensitization of the histamine H1 receptor and GRK2 translocation to plasma membrane. Also candidates presented adequate lipophilia and cytotoxicity profile. SignificanceWe obtained compounds with the ability of reducing RH mediated actions of GRK2 that can be useful as a starting point in the development of novel drug candidates aimed to treat pathologies were GRK2 plays a key role.

Apelin-36-[L28A] and Apelin-36-[L28C(30kDa-PEG)] peptides that improve diet induced obesity are G protein biased ligands at the apelin receptor

BackgroundApelin signalling pathways have important cardiovascular and metabolic functions. Recently, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)], were reported to function independent of the apelin receptor in vivo to produce beneficial metabolic effects without modulating blood pressure. We aimed to show that these peptides bound to the apelin receptor and to further characterise their pharmacology in vitro at the human apelin receptor. Methods[Pyr1]apelin-13 saturation binding experiments and competition binding experiments were performed in rat and human heart homogenates using [125I]apelin-13 (0.1 nM), and/or increasing concentrations of apelin-36, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] (50pM-100μM). Apelin-36 and its analogues apelin-36-[F36A], apelin-36-[L28A], apelin-36-[L28C(30kDa-PEG)], apelin-36-[A28 A13] and [40kDa-PEG]-apelin-36 were tested in forskolin-induced cAMP inhibition and β–arrestin assays in CHO-K1 cells heterologously expressing the human apelin receptor. Bias signaling was quantified using the operational model for bias. ResultsIn both species, [Pyr1]apelin-13 had comparable subnanomolar affinity and the apelin receptor density was similar. Apelin-36, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] competed for binding of [125I]apelin-13 with nanomolar affinities. Apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] inhibited forskolin-induced cAMP release, with nanomolar potencies but they were less potent compared to apelin-36 at recruiting β-arrestin. Bias analysis suggested that these peptides were G protein biased. Additionally, [40kDa-PEG]-apelin-36 and apelin-36-[F36A] retained nanomolar potencies in both cAMP and β-arrestin assays whilst apelin-36-[A13 A28] exhibited a similar profile to apelin-36-[L28C(30kDa-PEG)] in the β–arrestin assay but was more potent in the cAMP assay. ConclusionsApelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] are G protein biased ligands of the apelin receptor, suggesting that the apelin receptor is an important therapeutic target in metabolic diseases.