Frizzleds act as dynamic pharmacological entities.

The Frizzled family of transmembrane receptors (FZD1-10) belongs to the class F of G protein-coupled receptors (GPCRs). FZDs bind to and are activated by Wingless/Int1 (WNT) proteins. The WNT/FZD signaling system regulates crucial aspects of developmental biology and stem-cell regulation. Dysregulation of WNT/FZD communication can lead to developmental defects and diseases such as cancer and fibrosis. Recent insight into the activation mechanisms of FZDs has underlined that protein dynamics and conserved microswitches are essential for FZD-mediated information flow and build the basis for targeting these receptors pharmacologically. In this review, we summarize recent advances in our understanding of FZD activation, and how novel concepts merge and collide with existing dogmas in the field.

Structural and functional insight into the interaction of Clostridioides difficile toxin B and FZD7.

The G protein-coupled receptors of the Frizzled (FZD) family, in particular FZD1,2,7, are receptors that are exploited by Clostridioides difficile toxin B (TcdB), the major virulence factor responsible for pathogenesis associated with Clostridioides difficile infection. We employ a live-cell assay examining the affinity between full-length FZDs and TcdB. Moreover, we present cryoelectron microscopy structures of TcdB alone and in complex with full-length FZD7, which reveal that large structural rearrangements of the combined repetitive polypeptide domain are required for interaction with FZDs and other TcdB receptors, constituting a first step for receptor recognition. Furthermore, we show that bezlotoxumab, an FDA-approved monoclonal antibody to treat Clostridioides difficile infection, favors the apo-TcdB structure and thus disrupts binding with FZD7. The dynamic transition between the two conformations of TcdB also governs the stability of the pore-forming region. Thus, our work provides structural and functional insight into how conformational dynamics of TcdB determine receptor binding.

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, FZD7 , 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 FZD7 , preserving the native signal peptide. These cells were used to study eGFP-Wnt-3a association with endogenous and overexpressed HiBiT-FZD7 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-FZD7 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 FZD7 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-FZD7 binding should be performed using receptors expressed under endogenous promotion.

NanoBRET and NanoBiT/BRET-Based Ligand Binding Assays Permit Quantitative Assessment of Small Molecule Ligand Binding to Smoothened.

Smoothened (SMO) is a G protein-coupled receptor (GPCR) that mediates Hedgehog (Hh) signaling. SMO activity is regulated following the binding of Hh to the transmembrane protein Patched. Overactive SMO signaling is oncogenic, and hence this receptor is a target for several marketed drugs. However, development of new SMO ligands has been hampered by the fact that current radioligand and fluorescence-based binding assays are not high-throughput scalable. Here, we demonstrate two Nanoluciferase (Nluc) bioluminescence resonance energy transfer-based ligand binding assays (NanoBRET and NanoBiT/BRET) which provide a sensitive and high-throughput-compatible tool in drug screening efforts. In the described assays, SMO is N-terminally tagged either with full-length nanoluciferase or the partial HiBiT sequence, and subsequently binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between the receptor and the fluorescent ligand. Additionally, the assay allows performing competition binding experiments using commercially available SMO ligands, such as the SMO agonist SAG1.3.

Employing Genetically Encoded, Biophysical Sensors to Understand WNT/Frizzled Interaction and Receptor Complex Activation.

The Frizzled (FZD) family of WNT receptors consists of ten paralogues in mammals. They belong to the superfamily of G protein-coupled receptors and regulate crucial processes during embryonic development. Dysregulated FZD signaling leads to disease, most prominently to diverse forms of cancer, which renders these receptors attractive for drug discovery. Recent advances in assay development and the design of genetically encoded biosensors monitoring ligand-receptor interaction, conformational dynamics, and protein-protein interaction have allowed for a better pharmacological understanding of WNT/FZD signal transduction and open novel avenues for mechanism-based drug discovery and screening. In this chapter, we summarize the recent progress in the molecular dissection of FZD activation based on advanced biosensors.

Quantitative Profiling of WNT-3A Binding to All Human Frizzled Paralogues in HEK293 Cells by NanoBiT/BRET Assessments.

The WNT signaling system governs critical processes during embryonic development and tissue homeostasis, and its dysfunction can lead to cancer. Details concerning selectivity and differences in relative binding affinities of 19 mammalian WNTs to the cysteine-rich domain (CRD) of their receptors-the ten mammalian Frizzleds (FZDs)-remain unclear. Here, we used eGFP-tagged mouse WNT-3A for a systematic analysis of WNT interaction with every human FZD paralogue in HEK293A cells. Employing HiBiT-tagged full-length FZDs, we studied eGFP-WNT-3A binding kinetics, saturation binding, and competition binding with commercially available WNTs in live HEK293A cells using a NanoBiT/BRET-based assay. Further, we generated receptor chimeras to dissect the contribution of the transmembrane core to WNT-CRD binding. Our data pinpoint distinct WNT-FZD selectivity and shed light on the complex WNT-FZD binding mechanism. The methodological development described herein reveals yet unappreciated details of the complexity of WNT signaling and WNT-FZD interactions, providing further details with respect to WNT-FZD selectivity.

Residue 6.43 defines receptor function in class F GPCRs.

The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD1-10) subtypes and Smoothened (SMO), remains one of the most enigmatic GPCR families. While SMO relies on cholesterol binding to the 7TM core of the receptor to activate downstream signaling, underlying details of receptor activation remain obscure for FZDs. Here, we aimed to investigate the activation mechanisms of class F receptors utilizing a computational biology approach and mutational analysis of receptor function in combination with ligand binding and downstream signaling assays in living cells. Our results indicate that FZDs differ substantially from SMO in receptor activation-associated conformational changes. SMO manifests a preference for a straight TM6 in both ligand binding and functional readouts. Similar to the majority of GPCRs, FZDs present with a kinked TM6 upon activation owing to the presence of residue P6.43. Functional comparison of FZD and FZD P6.43F mutants in different assay formats monitoring ligand binding, G protein activation, DVL2 recruitment and TOPflash activity, however, underlines further the functional diversity among FZDs and not only between FZDs and SMO.

Geissospermiculatine, a New Alkaloid from Geissospermum reticulatum Bark.

A new alkaloid, geissospermiculatine was characterized in Geissospermum reticulatum A. H. Gentry bark (Apocynaceae). Here, following a simplified isolation protocol, the structure of the alkaloid was elucidated through GC-MS, LC-MS/MS, 1D, and 2D NMR (COSY, ROESY, HSQC, HMBC, 1H-15N HMBC). Cytotoxic properties were evaluated in vitro on malignant THP-1 cells, and the results demonstrated that the cytotoxicity of the alkaloid (30 µg/mL) was comparable with staurosporine (10 µM). Additionally, the toxicity was tested on zebrafish (Danio rerio) embryos in vivo by monitoring their development (0-72 h); toxicity was not evident at 30 µg/mL.

Molecular Pharmacology of Class F Receptor Activation.

The class Frizzled (FZD) or class F of G protein-coupled receptors consists of 10 FZD paralogues and Smoothened (SMO). FZDs coordinate wingless/Int-1 signaling and SMO mediates Hedgehog signaling. Class F receptor signaling is intrinsically important for embryonic development and its dysregulation leads to diseases, including diverse forms of tumors. With regard to the importance of class F signaling in human disease, these receptors provide an attractive target for therapeutics, exemplified by the use of SMO antagonists for the treatment of basal cell carcinoma. Here, we review recent structural insights in combination with a more detailed functional understanding of class F receptor activation, G protein coupling, conformation-based functional selectivity, and mechanistic details of activating cancer mutations, which will lay the basis for further development of class F-targeting small molecules for human therapy. SIGNIFICANCE STATEMENT: Stimulated by recent insights into the activation mechanisms of class F receptors from structural and functional analysis of Frizzled and Smoothened, we aim to summarize what we know about the molecular details of ligand binding, agonist-driven conformational changes, and class F receptor activation. A better understanding of receptor activation mechanisms will allow us to engage in structure- and mechanism-driven drug discovery with the potential to develop more isoform-selective and potentially pathway-selective drugs for human therapy.

A NanoBRET-Based Binding Assay for Smoothened Allows Real-time Analysis of Ligand Binding and Distinction of Two Binding Sites for BODIPY-cyclopamine.

Smoothened (SMO) is a GPCR that mediates hedgehog signaling. Hedgehog binds the transmembrane protein Patched, which in turn regulates SMO activation. Overactive SMO signaling is oncogenic and is therefore a clinically established drug target. Here we establish a nanoluciferase bioluminescence resonance energy transfer (NanoBRET)-based ligand binding assay for SMO providing a sensitive and high throughput-compatible addition to the toolbox of GPCR pharmacologists. In the NanoBRET-based binding assay, SMO is N terminally tagged with nanoluciferase (Nluc) and binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between receptor and ligand. The assay allowed kinetic analysis of ligand-receptor binding in living HEK293 cells, competition binding experiments using commercially available SMO ligands (SANT-1, cyclopamine-KAAD, SAG1.3 and purmorphamine), and pharmacological dissection of two BODIPY-cyclopamine binding sites. This high throughput-compatible assay is superior to commonly used SMO ligand binding assays in the separation of specific from non-specific ligand binding and, provides a suitable complement to chemical biology strategies for the discovery of novel SMO-targeting drugs. SIGNIFICANCE STATEMENT: We established a NanoBRET-based binding assay for SMO with superior sensitivity compared to fluorescence-based assays. This assay allows distinction of two separate binding sites for BODIPY-cyclopamine on the SMO transmembrane core in live cells in real time. The assay is a valuable complement for drug discovery efforts and will support a better understanding of Class F GPCR pharmacology.

Overexpression of Orphan Receptor GPR61 Increases cAMP Levels upon Forskolin Stimulation in HEK293 Cells: in vitro and in silico Validation of 5-(Nonyloxy)Tryptamine as a Low-Affinity Inverse Agonist.

GPR61 is an orphan receptor that belongs to Class A of G-protein-coupled receptors. It has been reported that GPR61 has a constitutive activity and couples to Gαs. In the present study, we characterized GPR61 function and ligand binding by experimental and molecular docking studies. We demonstrated that heterologous expression of GPR61 in HEK293 cells enhanced the cAMP synthesis response to forskolin, whereas the basal cAMP synthesis was unaffected. 5-(Nonyloxy)tryptamine inhibited forskolin-stimulated cAMP production in GPR61-expressing HEK293 cells. These studies highlight that the intrinsic activity of this receptor is only measurable following its synergy with Gαs.

A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection.

Class F receptors are considered valuable therapeutic targets due to their role in human disease, but structural changes accompanying receptor activation remain unexplored. Employing population and cancer genomics data, structural analyses, molecular dynamics simulations, resonance energy transfer-based approaches and mutagenesis, we identify a conserved basic amino acid in TM6 in Class F receptors that acts as a molecular switch to mediate receptor activation. Across all tested Class F receptors (FZD4,5,6,7, SMO), mutation of the molecular switch confers an increased potency of agonists by stabilizing an active conformation as assessed by engineered mini G proteins as conformational sensors. Disruption of the switch abrogates the functional interaction between FZDs and the phosphoprotein Dishevelled, supporting conformational selection as a prerequisite for functional selectivity. Our studies reveal the molecular basis of a common activation mechanism conserved in all Class F receptors, which facilitates assay development and future discovery of Class F receptor-targeting drugs.

Antioxidant, Cytotoxic, and Antiproliferative Activities and Total Polyphenol Contents of the Extracts of Geissospermum reticulatum Bark.

Geissospermum species are medically important plants due to their health-promoting effects. The objective of this study was to determine the antioxidant ability and antiproliferative and cytotoxic effects of infusions, tinctures, and ethanolic extracts of Geissospermum reticulatum barks in relation to the contents of total phenolics and flavonoids. Seven samples of barks were collected in various regions of Peruvian Amazonia. We found that the amount of total phenolics in the studied products varied from 212.40 ± 0.69 to 1253.92 ± 11.20 mg GAE/kg. In our study there is a correlation (R2 = 0.7947) between the results of antioxidants assays: FRAP and ORAC for tinctures, infusions, and ethanolic extracts of G. reticulatum barks. We have also observed antiproliferative activities of the ethanolic extracts on normal T-cells. These extracts have caused death on malignant cell lines (THP-1 and HL-60) and this data correlates well with their antioxidant capacity measured by ORAC method. Interestingly, the highest concentration of the ethanolic extract was not toxic in the zebrafish embryo developmental assay. Our results indicate that G. reticulatum is rich in antioxidants and have cytotoxic and antiproliferative properties. The data suggests potential immunosuppressive role of the extracts. This is the first study presenting the results of chemical and biological analysis of multiple preparations from G. reticulatum.

Novel vitamin D analogues; cytotoxic and anti-proliferative activity against a diffuse large B-cell lymphoma cell line and B-cells from healthy donors.

Calcitriol (1,25-dihydroxyvitamin D3, 1,25D3) and vitamin D side-chain modified analogs (VDAs) have gained considerable attention as potential drugs in the treatment of acute myeloid leukemia (AML), yet studies of the impact of 1,25D3 and VDAs upon other haematological malignancies are more limited. To address this gap in knowledge, we have examined the action of 1,25D3 and VDAs on a human cell line (DOHH2, K422) typifying diffuse large B-cell lymphoma (DLBCL) and also peripheral blood B-cells isolated from healthy donors. 1,25D3 and certain VDAs displayed moderate cytotoxic and pro-apoptotic actions upon DLBCL cells. 1,25D3 and VDAs (100nM) caused the death of approximately 40% DOHH2 cells after 24h stimulation, similar to their impact on HL-60 cells (acute myeloid leukaemia cell line). In addition, 1,25D3 and VDAs displayed concentration and time-dependent anti-proliferative actions upon stimulated B-cells from healthy donors. The VDAs inhibited proliferation by approximately 30%. Hence VDAs may offer therapeutic potential for the treatment of DLBCL or conditions benefitted by B-cell depletion.