A virtual library of small molecules mimicking dipeptides.

Virtual combinatorial libraries are prevalent in drug discovery due to improvements in the prediction of synthetic reactions that can be performed. This has gone hand in hand with the development of virtual screening capabilities to effectively screen the large chemical spaces spanned by exhaustive enumeration of reaction products. In this study, we generated a small-molecule dipeptide mimic library to target proteins binding small peptides. The library was created based on the general idea of peptide synthesis, that is, amino acid mimics were reacted in silico to form the dipeptide mimics, yielding 2,036,819 unique compounds. After docking calculations, two compounds from the library were synthesized and tested against WD repeat-containing protein 5 (WDR5) and histamine receptors H1-H4 to evaluate whether these molecules are viable in assays. The compounds showed the highest potency at the histamine H3 receptor, with Ki values in the two-digit micromolar range.

Cryo-EM structure of cell-free synthesized human histamine 2 receptor/Gs complex in nanodisc environment.

Here we describe the cryo-electron microscopy structure of the human histamine 2 receptor (H2R) in an active conformation with bound histamine and in complex with Gs heterotrimeric protein at an overall resolution of 3.4 Å. The complex was generated by cotranslational insertion of the receptor into preformed nanodisc membranes using cell-free synthesis in E. coli lysates. Structural comparison with the inactive conformation of H2R and the inactive and Gq-coupled active state of H1R together with structure-guided functional experiments reveal molecular insights into the specificity of ligand binding and G protein coupling for this receptor family. We demonstrate lipid-modulated folding of cell-free synthesized H2R, its agonist-dependent internalization and its interaction with endogenously synthesized H1R and H2R in HEK293 cells by applying a recently developed nanotransfer technique.

The dark sides of the GPCR tree - research progress on understudied GPCRs.

A large portion of the human GPCRome is still in the dark and understudied, consisting even of entire subfamilies of GPCRs such as odorant receptors, class A and C orphans, adhesion GPCRs, Frizzleds and taste receptors. However, it is undeniable that these GPCRs bring an untapped therapeutic potential that should be explored further. Open questions on these GPCRs span diverse topics such as deorphanisation, the development of tool compounds and tools for studying these GPCRs, as well as understanding basic signalling mechanisms. This review gives an overview of the current state of knowledge for each of the diverse subfamilies of understudied receptors regarding their physiological relevance, molecular mechanisms, endogenous ligands and pharmacological tools. Furthermore, it identifies some of the largest knowledge gaps that should be addressed in the foreseeable future and lists some general strategies that might be helpful in this process.

Fluorescent Tools for the Imaging of Dopamine D2 -Like Receptors.

The family of dopamine D2 -like receptors represents an interesting target for a variety of neurological diseases, e. g. Parkinson's disease (PD), addiction, or schizophrenia. In this study we describe the synthesis of a new set of fluorescent ligands as tools for visualization of dopamine D2 -like receptors. Pharmacological characterization in radioligand binding studies identified UR-MN212 (20) as a high-affinity ligand for D2 -like receptors (pKi (D2long R)=8.24, pKi (D3 R)=8.58, pKi (D4 R)=7.78) with decent selectivity towards D1 -like receptors. Compound 20 is a neutral antagonist in a Go1 activation assay at the D2long R, D3 R, and D4 R, which is an important feature for studies using whole cells. The neutral antagonist 20, equipped with a 5-TAMRA dye, displayed rapid association to the D2long R in binding studies using confocal microscopy demonstrating its suitability for fluorescence microscopy. Furthermore, in molecular brightness studies, the ligand's binding affinity could be determined in a single-digit nanomolar range that was in good agreement with radioligand binding data. Therefore, the fluorescent compound can be used for quantitative characterization of native D2 -like receptors in a broad variety of experimental setups.

Shedding Light on the D1 -Like Receptors: A Fluorescence-Based Toolbox for Visualization of the D1 and D5 Receptors.

Dopamine D1 -like receptors are the most abundant type of dopamine receptors in the central nervous system and, even after decades of discovery, still highly interesting for the study of neurological diseases. We herein describe the synthesis of a new set of fluorescent ligands, structurally derived from D1 R antagonist SCH-23390 and labeled with two different fluorescent dyes, as tool compounds for the visualization of D1 -like receptors. Pharmacological characterization in radioligand binding studies identified UR-NR435 (25) as a high-affinity ligand for D1 -like receptors (pKi (D1 R)=8.34, pKi (D5 R)=7.62) with excellent selectivity towards D2 -like receptors. Compound 25 proved to be a neutral antagonist at the D1 R and D5 R in a Gs heterotrimer dissociation assay, an important feature to avoid receptor internalization and degradation when working with whole cells. The neutral antagonist 25 displayed rapid association and complete dissociation to the D1 R in kinetic binding studies using confocal microscopy verifying its applicability for fluorescence microscopy. Moreover, molecular brightness studies determined a single-digit nanomolar binding affinity of the ligand, which was in good agreement with radioligand binding data. For this reason, this fluorescent ligand is a useful tool for a sophisticated characterization of native D1 receptors in a variety of experimental setups.

Development of Fluorescent AF64394 Analogues Enables Real-Time Binding Studies for the Orphan Class A GPCR GPR3.

The orphan G protein-coupled receptor (oGPCR) GPR3 represents a potential drug target for the treatment of Alzheimer's disease and metabolic disorders. However, the limited toolbox of pharmacological assays hampers the development of advanced ligands. Here, we developed a signaling pathway-independent readout of compound-GPR3 interaction. Starting from computational binding pose predictions of the most potent GPR3 ligand, we designed a series of fluorescent AF64394 analogues and assessed their suitability for BRET-based binding studies. The most potent ligand, 45 (UR-MB-355), bound to GPR3 and closely related receptors, GPR6 and GPR12, with similar submicromolar affinities. Furthermore, we found that 45 engages GPR3 in a distinct mode compared to AF64394, and coincubation studies with the GPR3 agonist diphenyleneiodonium chloride revealed allosteric modulation of 45 binding. These insights provide new cues for the pharmacological manipulation of GPR3 activity. This novel binding assay will foster the development of future drugs acting through these pharmacologically attractive oGPCRs.

Conformational GPCR BRET Sensors Based on Bioorthogonal Labeling of Noncanonical Amino Acids.

Here we describe the application of genetic code expansion and site-specific incorporation of noncanonical amino acids that serve as anchor points for fluorescent labeling to generate bioluminescence resonance energy transfer (BRET)-based conformational sensors. Using a receptor with an N-terminal NanoLuciferase (Nluc) and a fluorescently labeled noncanonical amino acid in the receptor's extracellular part allows to analyze receptor complex formation, dissociation, and conformational rearrangements over time and in living cells. These BRET sensors can be used to investigate ligand-induced intramolecular (cysteine-rich domain [CRD] dynamics), but also intermolecular (dimer dynamics) receptor rearrangements. With the design of BRET conformational sensors based on the minimally invasive bioorthogonal labeling procedure, we describe a method that can be used in a microtiter plate format and can be easily adopted to investigate ligand-induced dynamics in various membrane receptors.

WNT stimulation induces dynamic conformational changes in the Frizzled-Dishevelled interaction.

Frizzleds (FZDs) are G protein-coupled receptors (GPCRs) that bind to WNT family ligands. FZDs signal through multiple effector proteins, including Dishevelled (DVL), which acts as a hub for several downstream signaling pathways. To understand how WNT binding to FZD stimulates intracellular signaling and influences downstream pathway selectivity, we investigated the dynamic changes in the FZD5-DVL2 interaction elicited by WNT-3A and WNT-5A. Ligand-induced changes in bioluminescence resonance energy transfer (BRET) between FZD5 and DVL2 or the isolated FZD-binding DEP domain of DVL2 revealed a composite response consisting of both DVL2 recruitment and conformational dynamics in the FZD5-DVL2 complex. The combination of different BRET paradigms enabled us to identify ligand-dependent conformational dynamics in the FZD5-DVL2 complex and distinguish them from ligand-induced recruitment of DVL2 or DEP to FZD5. The observed agonist-induced conformational changes at the receptor-transducer interface suggest that extracellular agonist and intracellular transducers cooperate through transmembrane allosteric interaction with FZDs in a ternary complex reminiscent of that of classical GPCRs.

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.

Functional modulation of PTH1R activation and signaling by RAMP2.

Receptor-activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that associate with different G protein-coupled receptors (GPCRs), including the parathyroid hormone 1 receptor (PTH1R), a class B GPCR and an important modulator of mineral ion homeostasis and bone metabolism. However, it is unknown whether and how RAMP proteins may affect PTH1R function. Using different optical biosensors to measure the activation of PTH1R and its downstream signaling, we describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique preactivated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signaling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signaling sensitivity. Additionally, RAMP2 increases both PTH- and PTHrP-triggered ß-arrestin2 recruitment to PTH1R. Employing homology modeling, we describe the putative structural molecular basis underlying our functional findings. These data uncover a critical role of RAMPs in the activation and signaling of a GPCR that may provide a new venue for highly specific modulation of GPCR function and advanced drug design.

Isoforms of GPR35 have distinct extracellular N-termini that allosterically modify receptor-transducer coupling and mediate intracellular pathway bias.

Within the intestine, the human G protein-coupled receptor (GPCR) GPR35 is involved in oncogenic signaling, bacterial infections, and inflammatory bowel disease. GPR35 is known to be expressed as two distinct isoforms that differ only in the length of their extracellular N-termini by 31 amino acids, but detailed insights into their functional differences are lacking. Through gene expression analysis in immune and gastrointestinal cells, we show that these isoforms emerge from distinct promoter usage and alternative splicing. Additionally, we employed optical assays in living cells to thoroughly profile both GPR35 isoforms for constitutive and ligand-induced activation and signaling of 10 different heterotrimeric G proteins, ligand-induced arrestin recruitment, and receptor internalization. Our results reveal that the extended N-terminus of the long isoform limits G protein activation yet elevates receptor-ß-arrestin interaction. To better understand the structural basis for this bias, we examined structural models of GPR35 and conducted experiments with mutants of both isoforms. We found that a proposed disulfide bridge between the N-terminus and extracellular loop 3, present in both isoforms, is crucial for constitutive G13 activation, while an additional cysteine contributed by the extended N-terminus of the long GPR35 isoform limits the extent of agonist-induced receptor-ß-arrestin2 interaction. The pharmacological profiles and mechanistic insights of our study provide clues for the future design of isoform-specific GPR35 ligands that selectively modulate GPR35-transducer interactions and allow for mechanism-based therapies against, for example, inflammatory bowel disease or bacterial infections of the gastrointestinal system.

A guide to adhesion GPCR research.

Adhesion G protein-coupled receptors (aGPCRs) are a class of structurally and functionally highly intriguing cell surface receptors with essential functions in health and disease. Thus, they display a vastly unexploited pharmacological potential. Our current understanding of the physiological functions and signaling mechanisms of aGPCRs form the basis for elucidating further molecular aspects. Combining these with novel tools and methodologies from different fields tailored for studying these unusual receptors yields a powerful potential for pushing aGPCR research from singular approaches toward building up an in-depth knowledge that will facilitate its translation to applied science. In this review, we summarize the state-of-the-art knowledge on aGPCRs in respect to structure-function relations, physiology, and clinical aspects, as well as the latest advances in the field. We highlight the upcoming most pressing topics in aGPCR research and identify strategies to tackle them. Furthermore, we discuss approaches how to promote, stimulate, and translate research on aGPCRs 'from bench to bedside' in the future.

Frizzled BRET sensors based on bioorthogonal labeling of unnatural amino acids reveal WNT-induced dynamics of the cysteine-rich domain.

Frizzleds (FZD1­10) are G protein­coupled receptors containing an extracellular cysteine-rich domain (CRD) binding Wingless/Int-1 lipoglycoproteins (WNTs). Despite the role of WNT/FZD signaling in health and disease, our understanding of how WNT binding is translated into receptor activation and transmembrane signaling remains limited. Current hypotheses dispute the roles for conformational dynamics. To clarify how WNT binding to FZD translates into receptor dynamics, we devised conformational FZD-CRD biosensors based on bioluminescence resonance energy transfer (BRET). Using FZD with N-terminal nanoluciferase (Nluc) and fluorescently labeled unnatural amino acids in the linker domain and extracellular loop 3, we show that WNT-3A and WNT-5A induce similar CRD conformational rearrangements despite promoting distinct signaling pathways and that CRD dynamics are not required for WNT/ß-catenin signaling. Thus, these FZD-CRD biosensors provide insights into binding, activation, and signaling processes in FZDs. The sensor design is broadly applicable to explore ligand-induced dynamics also in other membrane receptors.

Quantitative assessment of constitutive G protein-coupled receptor activity with BRET-based G protein biosensors.

Heterotrimeric G proteins constitute the primary transducers of G protein­coupled receptor (GPCR) signaling. In addition to mediating ligand-induced GPCR activation, G proteins transduce basal signaling in various physiological and pathophysiological settings evoked by constitutively active, native GPCRs or disease-related receptor mutants. Optical biosensors have been developed and optimized to monitor GPCR ligand­induced activation of G proteins, but these biosensors cannot be used to detect constitutively active GPCRs. Here, we designed and validated eight bioluminescence resonance energy transfer (BRET)­based G protein sensors that can measure the activity of all four major families of G proteins. We also established a protocol to identify constitutive GPCR or G protein signaling in live cells. These G protein­based, tricistronic activity sensors (G-CASE) rely on the encoding of all three G protein subunits by a single plasmid, enabling their expression at the desired relative amounts and resulting in reduced signal variability in mammalian cells. We also present an experimental protocol to use the G-CASE sensor toolbox to quantify constitutive signaling of native and mutated GPCRs through these heterotrimeric transducers. This approach will help to characterize constitutively active GPCRs and their role in health and disease.

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.

A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H3 Receptor.

The histamine H3 receptor (H3R) is considered an attractive drug target for various neurological diseases. We here report the synthesis of UR-NR266, a novel fluorescent H3R ligand. Broad pharmacological characterization revealed UR-NR266 as a sub-nanomolar compound at the H3R with an exceptional selectivity profile within the histamine receptor family. The presented neutral antagonist showed fast association to its target and complete dissociation in kinetic binding studies. Detailed characterization of standard H3R ligands in NanoBRET competition binding using UR-NR266 highlights its value as a versatile pharmacological tool to analyze future H3R ligands. The low nonspecific binding observed in all experiments could also be verified in TIRF and confocal microscopy. This fluorescent probe allows the highly specific analysis of native H3R in various assays ranging from optical high throughput technologies to biophysical analyses and single-molecule studies in its natural environment. An off-target screening at 14 receptors revealed UR-NR266 as a selective compound.

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.

Bioluminescence in G Protein-Coupled Receptors Drug Screening Using Nanoluciferase and Halo-Tag Technology.

Here we describe the stepwise application of bioluminescence resonance energy transfer (BRET)-based conformational receptor biosensors to study GPCR activation in intact cells. This technology can be easily adopted to various plate reader devices and microtiter plate formats. Due to the high sensitivity of these BRET-based receptor biosensors and their ability to quantify simultaneously receptor activation/de-activation kinetics as well as compound efficacy and potency, these optical tools provide the most direct and unbiased approach to monitor GPCR activity in a high-throughput-compatible assay format, representing a novel promising tool for the discovery of potential GPCR therapeutics.

Deconvolution of WNT-induced Frizzled conformational dynamics with fluorescent biosensors.

The G protein-coupled receptors Frizzled1-10 (FZD1-10) act as molecular checkpoints mediating intracellular signaling induced by 19 mammalian, secreted Wingless/Int-1 lipoglycoproteins (WNTs). Despite the vital roles of these signaling components in health and disease, our knowledge about WNT/FZD selectivity, and the mechanisms of receptor activation and intracellular signal propagation by individual ligand/receptor pairs is limited due to the current lack of suitable biophysical techniques. Here, we developed fluorescence-based biosensors that detect WNT-induced FZD conformational changes in living cells in order to assess WNT action via FZDs at the most proximal level, i.e. the receptor conformation. By testing a panel of recombinant ligands on conformational biosensors representing all four homology clusters of FZDs, we discover yet unappreciated selectivities of WNTs to their receptors and, surprisingly, identify distinct ligand-induced receptor conformations. Furthermore, we demonstrate that FZDs can undergo conformational changes upon WNT binding without being dependent on the WNT co-receptors LRP5/6. This sensor toolbox provides an advanced platform for a thorough investigation of the 190 possible WNT/FZD pairings and for future screening campaigns targeting synthetic FZD ligands. Furthermore, our findings shed new light on the complexity of the WNT/FZD signaling system and have substantial implications for our understanding of fundamental biological processes including embryonal development and tumorigenesis.