NanoBiT ‐ and NanoBiT/BRET ‐based assays allow the analysis of binding kinetics of Wnt‐3a to endogenous Frizzled 7 in a colorectal cancer model

Background and Purpose Wnt binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on Wnt–FZD pharmacology using overexpressed HEK293 cells. However, assessing ligand binding at endogenous receptor expression levels is important due to differential binding behaviour in a native environment. Here, we study FZD paralogue, FZD$_{7}$, and analyse its interactions with Wnt-3a in live CRISPR-Cas9-edited SW480 cells typifying colorectal cancer. Experimental Approach SW480 cells were CRISPR-Cas9-edited to insert a HiBiT tag on the N-terminus of FZD$_{7}$, preserving the native signal peptide. These cells were used to study eGFP-Wnt-3a association with endogenous and overexpressed HiBiT-FZD$_{7}$ using NanoBiT/bioluminescence resonance energy transfer (BRET) and NanoBiT to measure ligand binding and receptor internalization. Key Results With this new assay the binding of eGFP-Wnt-3a to endogenous HiBiT-FZD$_{7}$ was compared with overexpressed receptors. Receptor overexpression results in increased membrane dynamics, leading to an apparent decrease in binding on-rate and consequently in higher, up to 10 times, calculated Kd. Thus, measurements of binding affinities to FZD$_{7}$ obtained in overexpressed cells are suboptimal compared with the measurements from endogenously expressing cells. Conclusions and Implications Binding affinity measurements in the overexpressing cells fail to replicate ligand binding affinities assessed in a (patho)physiologically relevant context where receptor expression is lower. Therefore, future studies on Wnt–FZD$_{7}$ binding should be performed using receptors expressed under endogenous promotion

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

Class Frizzled GPCRs in GtoPdb v.2023.1

Receptors of the Class Frizzled (FZD, nomenclature as agreed by the NC-IUPHAR subcommittee on the Class Frizzled GPCRs [180]), are GPCRs originally identified in Drosophila [20], which are highly conserved across species. While SMO shows structural resemblance to the 10 FZDs, it is functionally separated as it is involved in the Hedgehog signaling pathway [180]. SMO exerts its effects by activating heterotrimeric G proteins or stabilization of GLI by sequestering catalytic PKA subunits [186, 6, 58]. While SMO itself is bound by sterols and oxysterols [27, 94], FZDs are activated by WNTs, which are cysteine-rich lipoglycoproteins with fundamental functions in ontogeny and tissue homeostasis. FZD signalling was initially divided into two pathways, being either dependent on the accumulation of the transcription regulator β-catenin or being β-catenin-independent (often referred to as canonical vs. non-canonical WNT/FZD signalling, respectively). WNT stimulation of FZDs can, in cooperation with the low density lipoprotein receptors LRP5 (O75197) and LRP6 (O75581), lead to the inhibition of a constitutively active destruction complex, which results in the accumulation of β-catenin and subsequently its translocation to the nucleus. β-catenin, in turn, modifies gene transcription by interacting with TCF/LEF transcription factors. WNT/β-catenin-dependent signalling can also be activated by FZD subtype-specific WNT surrogates [138]. β-catenin-independent FZD signalling is far more complex with regard to the diversity of the activated pathways. WNT/FZD signalling can lead to the activation of heterotrimeric G proteins [34, 183, 155], the elevation of intracellular calcium [189], activation of cGMP-specific PDE6 [2] and elevation of cAMP as well as RAC-1, JNK, Rho and Rho kinase signalling [57]. Novel resonance energy transfer-based tools have allowed the study of the GPCR-like nature of FZDs in greater detail. Upon ligand stimulation, FZDs undergo conformational changes and signal via heterotrimeric G proteins [244, 245, 107, 179, 104]. Furthermore, the phosphoprotein Dishevelled constitutes a key player in WNT/FZD signalling towards planar-cell-polarity-like pathways. Importantly, FZDs exist in at least two distinct conformational states that regulate pathway selection [245]. As with other GPCRs, members of the Frizzled family are functionally dependent on the arrestin scaffolding protein for internalization [23], as well as for β-catenin-dependent [14] and -independent [91, 15] signalling. The pattern of cell signalling is complicated by the presence of additional ligands, which can enhance or inhibit FZD signalling (secreted Frizzled-related proteins (sFRP), Wnt-inhibitory factor (WIF), sclerostin or Dickkopf (DKK)), as well as modulatory (co)-receptors with Ryk, ROR1, ROR2 and Kremen, which may also function as independent signalling proteins

Development of a Neurotensin-Derived 68Ga-Labeled PET Ligand with High In Vivo Stability for Imaging of NTS1 Receptor-Expressing Tumors

Overexpression of the neurotensin receptor type 1 (NTS1R), a peptide receptor located at the plasma membrane, has been reported for a variety of malignant tumors. Thus, targeting the NTS1R with 18F- or 68Ga-labeled ligands is considered a straightforward approach towards in vivo imaging of NTS1R-expressing tumors via positron emission tomography (PET). The development of suitable peptidic NTS1R PET ligands derived from neurotensin is challenging due to proteolytic degradation. In this study, we prepared a series of NTS1R PET ligands based on the C-terminal fragment of neurotensin (NT(8–13), Arg8-Arg9-Pro10-Tyr11-Ile12-Leu13) by attachment of the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) via an Nω-carbamoylated arginine side chain. Insertion of Ga3+ in the DOTA chelator gave potential PET ligands that were evaluated concerning NTS1R affinity (range of Ki values: 1.2–21 nM) and plasma stability. Four candidates were labeled with 68Ga3+ and used for biodistribution studies in HT-29 tumor-bearing mice. [68Ga]UR-LS130 ([68Ga]56), containing an N-terminal methyl group and a β,β-dimethylated tyrosine instead of Tyr11, showed the highest in vivo stability and afforded a tumor-to-muscle ratio of 16 at 45 min p.i. Likewise, dynamic PET scans enabled a clear tumor visualization. The accumulation of [68Ga]56 in the tumor was NTS1R-mediated, as proven by blocking studies

The Concise Guide to PHARMACOLOGY 2023/24: G protein-coupled receptors.

peer reviewedThe Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.16177. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate

Luminescence-based methods for the investigation of ligand binding and GRK2 recruitment to GPCRs

The development of novel ligands for G protein-coupled receptors (GPCRs), representing important drug targets, requires the determination of ligand-receptor affinities. This has primarily been done by radioligand competition binding experiments. However, the use of radioligands is disadvantageous with respect to safety concerns, legal handling regulations and waste disposal. Thus, alternative and radioactivity-free methods for studying ligand-receptor binding are needed. Over the last decades, techniques using fluorescent ligands have emerged as a promising option. A recently reported luminescence-based binding assay makes use of the phenomenon of BRET (bioluminescence resonance energy transfer). The N-terminus of the receptor of interest was tagged with a small luciferase, which generates bioluminescence upon the addition of its substrate (e.g. furimazine). When a fluorescent ligand binds to the receptor, BRET can occur due to the close proximity of the luciferase and the fluorescent ligand. The BRET signal correlates with the amount of receptor-bound ligand and, in contrast to radioligand binding assays, ligand binding can be measured in real time with high temporal resolution. The aim of this thesis was the development of BRET-based binding assays for different GPCRs as alternative methods to radioligand binding assays. First, a BRET-based binding assay was developed for the histamine H2 receptor (H2R). For this purpose, the H2R, N-terminally fused to the NanoLuc® (NLuc), was stably expressed in HEK293T cells. Out of three differently labeled fluorescent H2R ligands, which were investigated in BRET saturation binding experiments, the Py-1-labeled ligand 2.1 turned out to be the most favorable in terms of ligand affinity and signal-to-background (S/B) ratio. The receptor binding kinetics of 2.1 were studied in real time and competition binding experiments with 2.1 and several reported H2R ligands afforded H2R binding data in accordance with literature values. Furthermore, a BRET-based binding assay was developed for the M2 muscarinic acetylcholine receptor (M2R). Two TAMRA-labeled M2R ligands (3.1 and 3.2) were characterized by saturation binding, association and dissociation kinetic (in real time) and, for 3.1, competition binding experiments with standard MR ligands. As the fluorophore TAMRA is also suited for fluorescence anisotropy (FA) studies, the BRET-based M2R binding assay was compared with an FA-based real-time binding assay, also using 3.1 and 3.2 as probes. Both methods, representing different methodologies, yielded similar results (ligand binding affinities from saturation binding and competition binding studies). Kinetic data from both assays revealed a complex binding behavior for 3.1 and 3.2 (e.g. biphasic dissociation kinetics), being most pronounced for 3.2. In the case of the neuropeptide Y Y1 receptor (Y1R) or the neurotensin receptor 1 (NTS1R), both exhibiting long N-termini, application of the reported concept (N-terminal fusion of the GPCR to NLuc) failed to establish BRET-based binding assays for these receptors. Obviously, their longer N-termini led to an increased distance or an unfavorable position of the luciferase relative to the fluorescent ligand, precluding efficient BRET. Therefore, an alternative strategy was developed by inserting the luciferase into the second extracellular loop of the Y1R and the NTS1R. This novel approach resulted in feasible BRET binding assays. The same strategy, which is considered a widely applicable method, was applied to two other GPCRs with comparably short N-termini, the angiotensin II receptor type 1 (AT1R) and the M1 muscarinic acetylcholine receptor (M1R). When compared with the N-terminally NLuc-tagged AT1R and M1R, insertion of NLuc into ECL2 of the receptors led to ligand affinities in better accordance with literature data, and/or higher S/B ratios. Finally, a split luciferase-based assay was developed to assess the recruitment of the G protein coupled receptor kinase 2 (GRK2) to three different receptors. Besides the possibility of generating concentration-response curves, the novel assay allowed analyzing GRK2 recruitment in a time-dependent manner. It revealed different recruitment kinetics for the NTS1R, which showed a more sustained interaction with GRK2, when compared with the M1R and the M5R, both showing a short-lasting interaction with GRK2. This suggested a potential classification of GPCRs based on their differential interaction with GRKs

Truth tables for modal logics T and S4, by using three-valued non-deterministic level semantics

Novel three-valued non-deterministic level semantics for modal logics T and S4 are presented. A criterion for partial level valuations is given, making it possible to create truth tables. Additionally, semantics and truth tables for 0 (defined as PC plus rule of necessitation) and 0T with only two values are based on Ivlev’s work. We need Kearns’ notion of level valuations: a generalization of Dugundji’s theorem shows that there is no non-deterministic semantics for modal logics up to S5⁠, containing the rule of necessitation

Finding Semantic Bugs Fast

Finding semantic bugs in code is difficult and requires precious expert time. Lacking comprehensive formal specifications, deductive verification is not an option. We propose an incremental specification procedure: With the help of automatic verification tools, a domain expert is guided through program runs and source code locations. The expert validates a run at certain locations and creates lightweight annotations. Formal methods training is not required. We demonstrate by example that this approach is capable to quickly detect different kinds of semantic bugs. We position our approach in the middle ground between fully-fledged deductive verification and bug finding without semantic guidance