Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe.

Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs

The Molecular Basis for Biased Signalling by Human Cannabinoid CB2 Receptor

As part of our endocannabinoid system, human cannabinoid CB2 receptor is involved in regulation of many physiological processes. Due to its involvement in inflammation, CB2 is an interesting pharmacological target for treating conditions such as chronic pain, osteoporosis and Alzheimer’s disease. Although naturally existing cannabinoids have been used for millennia, and there are drugs targeting the endocannabinoid system, the mechanism of their action at molecular level is still not well understood. CB2 belongs to a large family of versatile membrane proteins, G protein‑coupled receptors (GPCRs). In response to extracellular stimuli, GPCRs undergo conformational changes, interact with intracellular signalling partners, such as G protein and arrestins, resulting in an appropriate intracellular response. GPCRs are known to be able to selectively activate distinct signalling pathways, in response to different biased ligands. Although it was shown that activation of only certain signalling pathways can be pharmacologically beneficial, it remains unknown how the signal is propagated within a GPCR, leading to biased signalling. Within my thesis, I studied the potential for biased signalling by cannabinoid CB1 and CB2 receptors, as well as the molecular basis for biased signalling by CB2, using biosensors based on bioluminescence resonance energy transfer (BRET). I screened a library of 35 structurally diverse ligands for the activation of CB1 and CB2. Furthermore, I tested signalling profiles of over 360 CB2 mutants, collecting over 7000 concentration‑response curves. As a result, I identified CB2 residues involved in selective activation of distinct signalling pathways and the maintenance of its constitutive activity. These residues important for signalling formed extensive allosteric networks connecting the ligand and the effector binding sites. Through a collaboration with a computational group performing molecular dynamics simulations on the same system, I was able to add a structural and dynamic information to the functional data. In addition, I compared my data with analogous data obtained for another GPCR, vasopressin V2 receptor, identifying conserved receptor regions of crucial importance for signal propagation, as well as unique ways of obtaining active conformations utilised by the two receptors. Taken together, my data allow determination of the molecular basis of biased signalling and constitutive activity in CB2 receptor, setting the foundation for the rational design of biased ligands with fewer side effects. Due to their high structural similarity, my results are likely to be transferrable to other GPCRs, improving our understanding of the mechanisms underlying their signalling

High-throughput mutagenesis using a two-fragment PCR approach

Site-directed scanning mutagenesis is a powerful protein engineering technique which allows studies of protein functionality at single amino acid resolution and design of stabilized proteins for structural and biophysical work. However, creating libraries of hundreds of mutants remains a challenging, expensive and time-consuming process. The efficiency of the mutagenesis step is the key for fast and economical generation of such libraries. PCR artefacts such as misannealing and tandem primer repeats are often observed in mutagenesis cloning and reduce the efficiency of mutagenesis. Here we present a high-throughput mutagenesis pipeline based on established methods that significantly reduces PCR artefacts. We combined a two-fragment PCR approach, in which mutagenesis primers are used in two separate PCR reactions, with an in vitro assembly of resulting fragments. We show that this approach, despite being more laborious, is a very efficient pipeline for the creation of large libraries of mutants.ISSN:2045-232

ThermoBRET: A Ligand‐Engagement Nanoscale Thermostability Assay Applied to GPCRs

Measurements of membrane protein thermostability reflect ligand binding. Current thermostability assays often require protein purification or rely on pre‐existing radiolabelled or fluorescent ligands, limiting their application to established targets. Alternative methods, such as fluorescence‐detection size exclusion chromatography thermal shift, detect protein aggregation but are not amenable for high‐throughput screening. Here, we present a ThermoBRET method to quantify the relative thermostability of G protein coupled receptors (GPCRs), using cannabinoid receptors (CB1 and CB2) and the b2‐adrenoceptor (b2AR) as model systems. ThermoBRET reports receptor unfolding, does not need labelled ligands and can be used with non‐purified proteins. It uses Bioluminescence Resonance Energy Transfer (BRET) between Nanoluciferase (Nluc) and a thiol‐reactive fluorescent dye that binds cysteines exposed by unfolding. We demonstrate that the melting point (Tm) of Nluc‐fused GPCRs can be determined in non‐purified detergent solubilised membrane preparations or solubilised whole cells, revealing differences in thermostability for different solubilising conditions and in the presence of stabilising ligands. We extended the range of the assay by developing the thermostable tsNLuc by incorporating mutations from the fragments of split‐Nluc (Tm of 87 ⁰C versus 59 ⁰C). ThermoBRET allows the determination of GPCR thermostability, which is useful for protein purification optimisation and for drug discovery screening

Drug Derived Fluorescent Probes for the Specific Visualization of Cannabinoid Type 2 Receptor - A Toolbox Approach

Cannabinoid type 2 receptor (CB2R) is a fundamental part of the endocannabinoid signaling system (eCB system), and is known to play an important role in tissue injury, inflammation, cancer and pain. In stark contrast to its significance, the underlying signaling mechanisms and tissue expression profiles are poorly understood. Due to its low expression in healthy tissue and lack of reliable chemical tools, CB2R visualization in live cells remains uncharted. Here we report the development of a drug derived toolbox of highly potent, CB2R-selective fluorescent probes based on reverse design. Extensive validation in several applications such as CB2R detection in flow cytometry and time-resolved imaging, and the development of a novel fluorescent-based TR-FRET assay to generate kinetic and equilibrium binding data demonstrate the high versatility of our toolbox. These probes are the first to preserve affinity and efficacy in both human and mouse CB2R, a crucial aspect for preclinical translatability, and to enable imaging of CB2R internalization in living cells using confocal microscopy

Highly Specific, Fluorescent Cannabinoid Type 2 Receptor Probes Enable Applications in Microscopy, Flow Cytometry and FRET-based Binding Assays

Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous conditions including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite its significance, researchers lack reliable tools to address questions concerning the complex mechanism of CB2R signaling and its downstream consequences, especially in cell-type and tissue-dependent contexts. Herein, we report highly specific CB2R fluorescent probes and their use in a variety of applications: flow cytometry with overexpressing as well as endogenously expressing cells, real-time confocal microscopy of living cells, and a novel FRET-based, CB2R binding assay amenable to high throughput screening.<br /

Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells

Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB2R signaling, especially in cell-type and tissue-dependent context. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB2R fluorescent probes, used successfully across applications, species and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB2R specificity was demonstrated by competition experiments in living cells expressing CB2R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer’s disease and to the detection of CB2R in human breast cancer cells