Multiplex Detection of Fluorescent Chemokine Binding to CXC Chemokine Receptors by NanoBRET.

NanoLuc-mediated bioluminescence resonance energy transfer (NanoBRET) has gained popularity for its ability to homogenously measure ligand binding to G protein-coupled receptors (GPCRs), including the subfamily of chemokine receptors. These receptors, such as ACKR3, CXCR4, CXCR3, play a crucial role in the regulation of the immune system, are associated with inflammatory diseases and cancer, and are seen as promising drug targets. The aim of this study was to optimize NanoBRET-based ligand binding to NLuc-ACKR3 and NLuc-CXCR4 using different fluorescently labeled chemokine CXCL12 analogs and their use in a multiplex NanoBRET binding assay of two chemokine receptors at the same time. The four fluorescent CXCL12 analogs (CXCL12-AZD488, -AZD546, -AZD594, -AZD647) showed high-affinity saturable binding to both NLuc-ACKR3 and NLuc-CXCR4, with relatively low levels of non-specific binding. Additionally, the binding of all AZDye-labeled CXCL12s to Nluc receptors was inhibited by pharmacologically relevant unlabeled chemokines and small molecules. The NanoBRET binding assay for CXCL10-AZD488 binding to Nluc-CXCR3 was also successfully established and successfully employed for the simultaneous measurement of the binding of unlabeled small molecules to NLuc-CXCR3 and NLuc-CXCR4. In conclusion, multiplexing the NanoBRET-based competition binding assay is a promising tool for testing unlabeled (small) molecules against multiple GPCRs simultaneously.

Pharmacological Characterization and Radiolabeling of VUF15485, a High-Affinity Small-Molecule Agonist for the Atypical Chemokine Receptor ACKR3.

Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study, we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this ß-arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC50 = 8.3) to human ACKR3, as measured in [125I]CXCL12 competition binding experiments. Moreover, in a bioluminescence resonance energy transfer-based ß-arrestin2 recruitment assay VUF15485 acts as a potent ACKR3 agonist (pEC50 = 7.6) and shows a similar extent of receptor activation compared with CXCL12 when using a newly developed, fluorescence resonance energy transfer-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485, tested against a (atypical) chemokine receptor panel (agonist and antagonist mode), proves to be selective for ACKR3. VUF15485 labeled with tritium at one of its methoxy groups ([3H]VUF15485), binds ACKR3 saturably and with high affinity (K d = 8.2 nM). Additionally, [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (<2 minutes) for binding to ACKR3. The selectivity of [3H]VUF15485 for ACKR3, was confirmed by binding studies, whereupon CXCR3, CXCR4, and ACKR3 small-molecule ligands were competed for binding against the radiolabeled agonist. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [3H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose that VUF15485 binds in the major and the minor binding pocket of ACKR3. SIGNIFICANCE STATEMENT: The atypical chemokine receptor atypical chemokine receptor 3 (ACKR3) is considered an interesting drug target in relation to cancer and multiple sclerosis. The study reports on new chemical biology tools for ACKR3, i.e., a new agonist that can also be radiolabeled and a new ACKR3 conformational sensor, that both can be used to directly study the interaction of ACKR3 ligands with the G protein-coupled receptor.

NanoB2 to monitor interactions of ligands with membrane proteins by combining nanobodies and NanoBRET.

The therapeutic potential of ligands targeting disease-associated membrane proteins is predicted by ligand-receptor binding constants, which can be determined using NanoLuciferase (NanoLuc)-based bioluminescence resonance energy transfer (NanoBRET) methods. However, the broad applicability of these methods is hampered by the restricted availability of fluorescent probes. We describe the use of antibody fragments, like nanobodies, as universal building blocks for fluorescent probes for use in NanoBRET. Our nanobody-NanoBRET (NanoB2) workflow starts with the generation of NanoLuc-tagged receptors and fluorescent nanobodies, enabling homogeneous, real-time monitoring of nanobody-receptor binding. Moreover, NanoB2 facilitates the assessment of receptor binding of unlabeled ligands in competition binding experiments. The broad significance is illustrated by the successful application of NanoB2 to different drug targets (e.g., multiple G protein-coupled receptors [GPCRs] and a receptor tyrosine kinase [RTK]) at distinct therapeutically relevant binding sites (i.e., extracellular and intracellular).

Optical control of the ß2-adrenergic receptor with opto-prop-2: A cis-active azobenzene analog of propranolol.

In this study, we synthesized and evaluated new photoswitchable ligands for the beta-adrenergic receptors ß1-AR and ß2-AR, applying an azologization strategy to the first-generation beta-blocker propranolol. The resulting compounds (Opto-prop-1, -2, -3) have good photochemical properties with high levels of light-induced trans-cis isomerization (>94%) and good thermal stability (t 1/2 > 10 days) of the resulting cis-isomer in an aqueous buffer. Upon illumination with 360-nm light to PSS cis , large differences in binding affinities were observed for photoswitchable compounds at ß1-AR as well as ß2-AR. Notably, Opto-prop-2 (VUF17062) showed one of the largest optical shifts in binding affinities at the ß2-AR (587-fold, cis-active), as recorded so far for photoswitches of G protein-coupled receptors. We finally show the broad utility of Opto-prop-2 as a light-dependent competitive antagonist of the ß2-AR as shown with a conformational ß2-AR sensor, by the recruitment of downstream effector proteins and functional modulation of isolated adult rat cardiomyocytes.

BRET-Based Biosensors to Measure Agonist Efficacies in Histamine H1 Receptor-Mediated G Protein Activation, Signaling and Interactions with GRKs and ß-Arrestins.

The histamine H1 receptor (H1R) is a G protein-coupled receptor (GPCR) and plays a key role in allergic reactions upon activation by histamine which is locally released from mast cells and basophils. Consequently, H1R is a well-established therapeutic target for antihistamines that relieve allergy symptoms. H1R signals via heterotrimeric Gq proteins and is phosphorylated by GPCR kinase (GRK) subtypes 2, 5, and 6, consequently facilitating the subsequent recruitment of ß-arrestin1 and/or 2. Stimulation of a GPCR with structurally different agonists can result in preferential engagement of one or more of these intracellular signaling molecules. To evaluate this so-called biased agonism for H1R, bioluminescence resonance energy transfer (BRET)-based biosensors were applied to measure H1R signaling through heterotrimeric Gq proteins, second messengers (inositol 1,4,5-triphosphate and Ca2+), and receptor-protein interactions (GRKs and ß-arrestins) in response to histamine, 2-phenylhistamines, and histaprodifens in a similar cellular background. Although differences in efficacy were observed for these agonists between some functional readouts as compared to reference agonist histamine, subsequent data analysis using an operational model of agonism revealed only signaling bias of the agonist Br-phHA-HA in recruiting ß-arrestin2 to H1R over Gq biosensor activation.

Exploring the Effect of Cyclization of Histamine H1 Receptor Antagonists on Ligand Binding Kinetics.

There is an increasing interest in guiding hit optimization by considering the target binding kinetics of ligands. However, compared to conventional structure-activity relationships, structure-kinetics relationships have not been as thoroughly explored, even for well-studied archetypical drug targets such as the histamine H1 receptor (H1R), a member of the family A G-protein coupled receptor. In this study, we show that the binding kinetics of H1R antagonists at the H1R is dependent on the cyclicity of both the aromatic head group and the amine moiety of H1R ligands, the chemotypes that are characteristic for the first-generation H1R antagonists. Fusing the two aromatic rings of H1R ligands into one tricyclic aromatic head group prolongs the H1R residence time for benchmark H1R ligands as well as for tailored synthetic analogues. The effect of constraining the aromatic rings and the basic amines is systematically explored, leading to a coherent series and detailed discussions of structure-kinetics relationships. This study shows that cyclicity has a pronounced effect on the binding kinetics.

Route to Prolonged Residence Time at the Histamine H1 Receptor: Growing from Desloratadine to Rupatadine.

Drug-target binding kinetics are an important predictor of in vivo drug efficacy, yet the relationship between ligand structures and their binding kinetics is often poorly understood. We show that both rupatadine (1) and desloratadine (2) have a long residence time at the histamine H1 receptor (H1R). Through development of a [3H]levocetirizine radiolabel, we find that the residence time of 1 exceeds that of 2 more than 10-fold. This was further explored with 22 synthesized rupatadine and desloratadine analogues. Methylene-linked cycloaliphatic or ß-branched substitutions of desloratadine increase the residence time at the H1R, conveying a longer duration of receptor antagonism. However, cycloaliphatic substituents directly attached to the piperidine amine (i.e., lacking the spacer) have decreased binding affinity and residence time compared to their methylene-linked structural analogues. Guided by docking studies, steric constraints within the binding pocket are hypothesized to explain the observed differences in affinity and binding kinetics between analogues.

Probe dependency in the determination of ligand binding kinetics at a prototypical G protein-coupled receptor.

Drug-target binding kinetics are suggested to be important parameters for the prediction of in vivo drug-efficacy. For G protein-coupled receptors (GPCRs), the binding kinetics of ligands are typically determined using association binding experiments in competition with radiolabelled probes, followed by analysis with the widely used competitive binding kinetics theory developed by Motulsky and Mahan. Despite this, the influence of the radioligand binding kinetics on the kinetic parameters derived for the ligands tested is often overlooked. To address this, binding rate constants for a series of histamine H1 receptor (H1R) antagonists were determined using radioligands with either slow (low koff) or fast (high koff) dissociation characteristics. A correlation was observed between the probe-specific datasets for the kinetic binding affinities, association rate constants and dissociation rate constants. However, the magnitude and accuracy of the binding rate constant-values was highly dependent on the used radioligand probe. Further analysis using recently developed fluorescent binding methods corroborates the finding that the Motulsky-Mahan methodology is limited by the employed assay conditions. The presented data suggest that kinetic parameters of GPCR ligands depend largely on the characteristics of the probe used and results should therefore be viewed within the experimental context and limitations of the applied methodology.

The long duration of action of the second generation antihistamine bilastine coincides with its long residence time at the histamine H1 receptor.

Drug-target binding kinetics has recently attracted considerable interest in view of the potential predictive power for in vivo drug efficacy. The recently introduced antihistamine bilastine has a long duration of in vivo drug action, which outlasts pharmacological active bilastine concentrations in blood. To provide a molecular basis for the long duration of action, we explored the kinetics of bilastine binding to the human histamine H1 receptor using [3H]mepyramine binding studies and compared its pharmacodynamics properties to the reference compounds fexofenadine and diphenhydramine, which have a long (60 ±â€¯20 min) and short (0.41 ±â€¯0.1 min) residence time, respectively. Bilastine shows a long drug-target residence time at the H1 receptor (73 ±â€¯5 min) and this results in a prolonged H1 receptor antagonism in vitro (Ca2+ mobilization in Fluo-4 loaded HeLa cells), following a washout of unbound antagonist. Hence, the long residence time of bilastine can explain the observed long duration of drug action in vivo.

Development of novel fluorescent histamine H1-receptor antagonists to study ligand-binding kinetics in living cells.

The histamine H1-receptor (H1R) is an important mediator of allergy and inflammation. H1R antagonists have particular clinical utility in allergic rhinitis and urticaria. Here we have developed six novel fluorescent probes for this receptor that are very effective for high resolution confocal imaging, alongside bioluminescence resonance energy transfer approaches to monitor H1R ligand binding kinetics in living cells. The latter technology exploits the opportunities provided by the recently described bright bioluminescent protein NanoLuc when it is fused to the N-terminus of a receptor. Two different pharmacophores (mepyramine or the fragment VUF13816) were used to generate fluorescent H1R antagonists conjugated via peptide linkers to the fluorophore BODIPY630/650. Kinetic properties of the probes showed wide variation, with the VUF13816 analogues having much longer H1R residence times relative to their mepyramine-based counterparts. The kinetics of these fluorescent ligands could also be monitored in membrane preparations providing new opportunities for future drug discovery applications.

The Target Residence Time of Antihistamines Determines Their Antagonism of the G Protein-Coupled Histamine H1 Receptor.

The pharmacodynamics of drug-candidates is often optimized by metrics that describe target binding (Kd or Ki value) or target modulation (IC50). However, these metrics are determined at equilibrium conditions, and consequently information regarding the onset and offset of target engagement and modulation is lost. Drug-target residence time is a measure for the lifetime of the drug-target complex, which has recently been receiving considerable interest, as target residence time is shown to have prognostic value for the in vivo efficacy of several drugs. In this study, we have investigated the relation between the increased residence time of antihistamines at the histamine H1 receptor (H1R) and the duration of effective target-inhibition by these antagonists. Hela cells, endogenously expressing low levels of the H1R, were incubated with a series of antihistamines and dissociation was initiated by washing away the unbound antihistamines. Using a calcium-sensitive fluorescent dye and a label free, dynamic mass redistribution based assay, functional recovery of the H1R responsiveness was measured by stimulating the cells with histamine over time, and the recovery was quantified as the receptor recovery time. Using these assays, we determined that the receptor recovery time for a set of antihistamines differed more than 40-fold and was highly correlated to their H1R residence times, as determined with competitive radioligand binding experiments to the H1R in a cell homogenate. Thus, the receptor recovery time is proposed as a cell-based and physiologically relevant metric for the lead optimization of G protein-coupled receptor antagonists, like the H1R antagonists. Both, label-free or real-time, classical signaling assays allow an efficient and physiologically relevant determination of kinetic properties of drug molecules.

Kinetics for Drug Discovery: an industry-driven effort to target drug residence time.

A considerable number of approved drugs show non-equilibrium binding characteristics, emphasizing the potential role of drug residence times for in vivo efficacy. Therefore, a detailed understanding of the kinetics of association and dissociation of a target-ligand complex might provide crucial insight into the molecular mechanism-of-action of a compound. This deeper understanding will help to improve decision making in drug discovery, thus leading to a better selection of interesting compounds to be profiled further. In this review, we highlight the contributions of the Kinetics for Drug Discovery (K4DD) Consortium, which targets major open questions related to binding kinetics in an industry-driven public-private partnership.

Identification of Ligand Binding Hot Spots of the Histamine H1 Receptor following Structure-Based Fragment Optimization.

Developments in G protein-coupled receptor (GPCR) structural biology provide insights into GPCR-ligand binding. Compound 1 (4-(2-benzylphenoxy)piperidine) with high ligand efficiency for the histamine H1 receptor (H1R) was used to design derivatives to investigate the roles of (i) the amine-binding region, (ii) the upper and lower aromatic region, and (iii) binding site solvation. SAR analysis showed that the amine-binding region serves as the primary binding hot spot, preferably binding small tertiary amines. In silico prediction of water network energetics and mutagenesis studies indicated that the displacement of a water molecule from the amine-binding region is most likely responsible for the increased affinity of the N-methylated analog of 1. Deconstruction of 1 showed that the lower aromatic region serves as a secondary binding hot spot. This study demonstrates that an X-ray structure in combination with tool compounds, assessment of water energetics, and mutagenesis studies enables SAR exploration to map GPCR-ligand binding hot spots.

BRET-based ß-arrestin2 recruitment to the histamine H1 receptor for investigating antihistamine binding kinetics.

Ligand residence time is thought to be a critical parameter for optimizing the in vivo efficacy of drug candidates. For the histamine H1 receptor (H1R) and other G protein-coupled receptors, the kinetics of ligand binding are typically measured by low throughput radioligand binding experiments using homogenized cell membranes expressing the target receptor. In this study, a real-time proximity assay between H1R and ß-arrestin2 in living cells was established to investigate the dynamics of antihistamine binding to the H1R. No receptor reserve was found for the histamine-induced recruitment of ß-arrestin2 to the H1R and the transiently recruited ß-arrestin2 therefore reflected occupancy of the receptor by histamine. Antihistamines displayed similar kinetic signatures on antagonizing histamine-induced ß-arrestin2 recruitment as compared to displacing radioligand binding from the H1R. This homogeneous functional method unambiguously determined the fifty-fold difference in the dissociation rate constant between mepyramine and the long residence time antihistamines levocetirizine and desloratadine.

Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo.

Alternative splicing generates a vast diversity of protein isoforms from a limited number of protein-coding genes, with many of the isoforms possessing unique, and even contrasting, functions. Fluorescence-based splicing reporters have the potential to facilitate studies of alternative splicing at the single-cell level and can provide valuable information on phenotypic transitions in almost real time. Fibroblast growth factor receptor 2 (FGFR2) pre-mRNA is alternatively spliced to form the epithelial-specific and mesenchymal-specific IIIb and IIIc isoforms, respectively, which are useful markers of epithelial-mesenchymal transitions (EMT). We have used our knowledge of FGFR2 splicing regulation to develop a fluorescence-based reporter system to visualize exon IIIc regulation in vitro and in vivo. Here we show the application of this reporter system to the study of EMT in vitro in cell culture and in vivo in transgenic mice harboring these splicing constructs. In explant studies, the reporters revealed that FGFR2 isoform switching is not required for keratinocyte migration during cutaneous wound closure. Our results demonstrate the value of the splicing reporters as tools to study phenotypic transitions and cell fates at single cell resolution. Moreover, our data suggest that keratinocytes migrate efficiently in the absence of a complete EMT.