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G protein-coupled receptors (GPCRs) are the largest family of plasma membrane-embedded signaling proteins. These receptors are involved in a wide array of physiological processes, marking them as attractive targets for drug development. Bitopic ligands, which are comprised of a pharmacophore that targets the receptor orthosteric site and a linked moiety that binds to a separate site, have considerable potential for addressing GPCR function. Here, we report the synthesis and evaluation of novel bitopic conjugates consisting of a small molecule pharmacophore that activates the adenosine A2A receptor (A2AR) linked to antibody fragments (nanobodies, Nbs). This approach leverages the high-affinity and specificity binding of Nbs to non orthosteric sites on engineered A2AR variants to provide bitopic Nb-ligand conjugates that stimulate strong and enduring signaling responses. We further demonstrate that such bitopic conjugates can induce activation by spanning two distinct receptor protomers. This property enables the selective targeting of receptor pairs over either individual receptor, as a form of “logic-gated” activity. We showcase the broad applicability of bitopic conjugates in this context by demonstrating their activity in targeting several pairs of co-expressed receptors, including GPCR monomers from different classes. Furthermore, we demonstrate that this dual-targeting strategy initiates signaling responses that diverge from those induced by monovalent ligands. The ability to target receptor pairs using Nb-ligand conjugates offers a powerful strategy with potential for cell type-selective signaling and implications for GPCR drug discovery efforts more broadly.

Antibodies conjugated to bioactive compounds allow targeted delivery of therapeutics to cell types of choice based on that antibody’s specificity. Here we develop a new type of conjugate that consists of a nanobody and a peptidic ligand for a G protein-coupled receptor (GPCR), fused via their C-termini. We address activation of parathyroid hormone receptor-1 (PTHR1) and improve the signaling activity and specificity of otherwise poorly active N-terminal peptide fragments of PTH by conjugating them to nanobodies (VHHs) that recognize PTHR1. These C-to-C conjugates show biological activity superior to that of the parent fragment peptide in vitro. In an exploratory experiment in mice, a VHH-PTH peptide conjugate showed biological activity, whereas the corresponding free peptide did not. The lead conjugate also possesses selectivity for PTHR1 superior to that of PTH(1-34). This design approach, dubbed “conjugation of ligands and antibodies for membrane proteins” (CLAMP), can yield ligands with high potency and specificity. Antibodies conjugated to bioactive compounds can allow targeted delivery of therapeutics. Here the authors present a strategy for fusing nanobodies to suboptimal GPCR peptide ligands to potently and selectively activate receptors.

Ligand-induced activation of G protein-coupled receptors (GPCRs) can initiate signaling through multiple distinct pathways with differing biological and physiological outcomes. There is intense interest in understanding how variation in GPCR ligand structure can be used to promote pathway selective signaling (“biased agonism”) with the goal of promoting desirable responses and avoiding deleterious side effects. Here we present an approach in which a conventional peptide ligand for the type 1 parathyroid hormone receptor (PTHR1) is converted from an agonist which induces signaling through all relevant pathways to a compound that is highly selective for a single pathway. This is achieved not through variation in the core structure of the agonist, but rather by linking it to a nanobody tethering agent that binds with high affinity to a separate site on the receptor not involved in signal transduction. The resulting conjugate represents the most biased agonist of PTHR1 reported to date. This approach holds promise for facile generation of pathway selective ligands for other GPCRs. Activated GPCRs signal through multiple pathways. Ligands that signal through a single pathway are highly valued. The authors demonstrate that tethering ligands to receptors via conjugation with binding nanobodies enables pathway-specific signaling.

The activity of a peptide agonist on a G protein–coupled receptor is readily modulated by altering its backbone structure without changing its side chains. Systematic modification of the backbone of bioactive polypeptides through β-amino acid residue incorporation could provide a strategy for generating molecules with improved drug properties, but such alterations can result in lower receptor affinity and potency. Using an agonist of parathyroid hormone receptor-1 (PTHR1), a G protein–coupled receptor in the B-family, we present an approach for α→β residue replacement that enables both high activity and improved pharmacokinetic properties in vivo .

Expansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags linked to proteins of interest and recognized by existing binders such as nanobodies facilitate protein studies by obviating the need to isolate new antibodies directed against them. Nanobodies have several advantages over conventional antibodies, as they can be expressed and used as tools for visualization and manipulation of proteins in vivo. Here, we characterize two short (<15aa) NanoTag epitopes, 127D01 and VHH05, and their corresponding high-affinity nanobodies. We demonstrate their use in Drosophila for in vivo protein detection and re-localization, direct and indirect immunofluorescence, immunoblotting, and immunoprecipitation. We further show that CRISPR-mediated gene targeting provides a straightforward approach to tagging endogenous proteins with the NanoTags. Single copies of the NanoTags, regardless of their location, suffice for detection. This versatile and validated toolbox of tags and nanobodies will serve as a resource for a wide array of applications, including functional studies in Drosophila and beyond.

The immune system’s ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition. SynTacs, when labeled with positron-emitting isotopes, can noninvasively image antigen-specific CD8 + T cells in vivo. Using radiolabeled synTacs loaded with the appropriate peptides, we imaged human papillomavirus-specific CD8 + T cells by positron emission tomography in mice bearing human papillomavirus-positive tumors, as well as influenza A virus–specific CD8 + T cells in the lungs of influenza A virus–infected mice. It is thus possible to visualize antigen-specific CD8 + T-cell populations in vivo, which may serve prognostic and diagnostic roles. Antigen-specific CD8 + T cells can be imaged by immunoPET with the help of synTacs, MHC-based tools that bind to relevant T-cell receptors.

Parathyroid hormone (PTH) analogs with improved actions in vivo could lead to optimized treatments for bone and mineral ion diseases. Rapid clearance from the circulation and short dwell times on the PTH receptor limit the efficacies of conventional PTH peptides currently in medical use. Here, we seek to enhance PTH peptide efficacy using two distinct peptide lipidation strategies. First, we append a lipid chain to the peptide’s C-terminus in a fashion to promote binding to serum albumin and hence prolong the peptide’s circulation half-life in vivo. Second, we append a lipid chain to a lysine side chain in a fashion designed to anchor the peptide to the cell membrane as the ligand is bound to the receptor and hence increase its dwell time on the receptor. We find that both strategies of lipidation can profoundly enhance the efficacy of PTH peptides in vitro and in mice. Our results could lead to the development of modified PTH analogs with optimized therapeutic utility. Current parathyroid hormone (PTH) peptides have limited efficacy due to rapid clearance and short receptor binding. Here, the authors show that lipidation strategies can extend circulation time and receptor engagement, enhancing PTH peptide efficacy in vitro and in vivo

Several chemical and enzymatic methods have been used to link antibodies to moieties that facilitate visualization of cognate targets. Emerging evidence suggests that the extent of labeling, dictated by the type of chemistry used, has a substantial impact on performance, especially in the context of antibodies used for the visualization of tumors in vivo. These effects are particularly pronounced in studies using small antibody fragments, such as single‐domain antibodies, or nanobodies. Here, we leverage a new variety of conjugation chemistry, based on a nanobody that forms a crosslink with a specialized high‐affinity epitope analogue, to label target‐specific nanobody constructs with functionalities of choice, including fluorophores, chelators, and click chemistry handles. Using heterodimeric nanobody conjugates, comprised of an antigen recognition module and a self‐labeling module, enables us to attach the desired functional group at a location distal to the site of antigen recognition. Constructs generated using this approach bound to antigens expressed on xenograft murine models of liver cancer and allowed for non‐invasive diagnostic imaging. The modularity of our approach using a self‐labeling nanobody offers a novel method for site‐specific functionalization of biomolecules and can be extended to other applications for which covalent labeling is required. Several methods have been used to label antibodies to facilitate visualization of cognate targets. Here, we leverage a new variety of conjugation chemistry, based on a crosslinking nanobody, to label target‐specific nanobody constructs with functionalities of choice. Constructs generated using this approach bound to antigens expressed on xenograft murine models of liver cancer and allowed for non‐invasive diagnostic imaging.

The parathyroid hormone receptor type 1 (PTH1R) is a G protein-coupled receptor that plays key roles in regulating calcium homeostasis and skeletal development via binding the ligands, PTH and PTH-related protein (PTHrP), respectively. Eiken syndrome is a rare disease of delayed bone mineralization caused by homozygous PTH1R mutations. Of the three mutations identified so far, R485X, truncates the PTH1R C-terminal tail, while E35K and Y134S alter residues in the receptor’s amino-terminal extracellular domain. Here, using a variety of cell-based assays, we show that R485X increases the receptor’s basal rate of cAMP signaling and decreases its capacity to recruit β-arrestin2 upon ligand stimulation. The E35K and Y134S mutations each weaken the binding of PTHrP leading to impaired β-arrestin2 recruitment and desensitization of cAMP signaling response to PTHrP but not PTH. Our findings support a critical role for interaction with β-arrestin in the mechanism by which the PTH1R regulates bone formation. Three mutations in parathyroid hormone receptor type 1 which are present in patients with Eiken syndrome are shown to influence cAMP signaling and β-arrestin recruitment via gain-of-function effects.

Key Points Parathyroid hormone (PTH)/parathyroid hormone-related protein (PTHrP) receptor (PTHR1) mediates the biological actions of two endogenous ligands, PTH and PTHrP and has key roles in regulating blood calcium levels and tissue development PTH and PTHrP interact with PTHR1 through similar, although not identical mechanisms, and preferentially stabilize distinct receptor conformations Certain structurally distinct PTH and PTHrP ligand analogues, which stabilize distinct receptor conformations, induce altered signalling responses that differ in signal type and duration Prolonged signalling by certain PTH ligand analogues correlates temporally with ligand–receptor complexes located in endosomes, which suggests mechanisms of signal generation and termination distinct from those described by traditional G-protein-coupled receptor models Consideration of ligand-based mechanisms that control signal duration provide insight into the processes of receptor dysfunction, as wells as guidance for addressing PTHR1-related diseases Identification and incorporation of specific structural features that promote or prevent long-lasting biological responses hold promise for the design of treatments for hypoparathyroidism and osteoporosis, respectively Parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR1) is a family B G-protein-coupled receptor and is involved in the regulation of skeletal development, bone turnover and mineral ion homeostasis. This Review discusses fundamental aspects of ligand-binding and signalling mechanisms at PTHR1, highlighting the relationship between ligand structural modification and variation in PTHR1 signalling responses. The action of these signalling mechanisms in disease states in which PTHR1 function has an important role are also discussed. Parathyroid hormone/parathyroid hormone-related protein receptor (PTH/PTHrP type 1 receptor; commonly known as PTHR1) is a family B G-protein-coupled receptor (GPCR) that regulates skeletal development, bone turnover and mineral ion homeostasis. PTHR1 transduces stimuli from PTH and PTHrP into the interior of target cells to promote diverse biochemical responses. Evaluation of the signalling properties of structurally modified PTHR1 ligands has helped to elucidate determinants of receptor function and mechanisms of downstream cellular and physiological responses. Analysis of PTHR1 responses induced by structurally modified ligands suggests that PTHR1 can continue to signal through a G-protein-mediated pathway within endosomes. Such findings challenge the longstanding paradigm in GPCR biology that the receptor is transiently activated at the cell membrane, followed by rapid deactivation and receptor internalization. Evaluation of structurally modified PTHR1 ligands has further led to the identification of ligand analogues that differ from PTH or PTHrP in the type, strength and duration of responses induced at the receptor, cellular and organism levels. These modified ligands, and the biochemical principles revealed through their use, might facilitate an improved understanding of PTHR1 function in vivo and enable the treatment of disorders resulting from defects in PTHR1 signalling. This Review discusses current understanding of PTHR1 modes of action and how these findings might be applied in future therapeutic agents.