Principle Investigator National Institutes of Health Bethesda, Maryland, United States
Introduction: Cell-to-cell communication occurs in large part through the release of signaling proteins (or peptides), also known as ligands, that act on protein receptors expressed on the surface of recipient cells. G protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors and are involved in virtually every aspect of intercellular communication and cellular sensing. The function of GPCRs is precisely regulated; however, dysregulation of signaling is a common cause of disease, as evidenced by the high proportion (>30%) of FDA approved drugs that target GPCRs. Despite this success, there remain many GPCR-associated diseases for which effective interventions are not available. Further, many naturally occurring GPCR ligands show low specificity for their target of interest, complicating their application as biological tools or as therapeutic candidates. We propose that the generation of new reagents to target GPCRs and dissect their function will enable further understanding of disease processes and highlight paths towards therapeutic development.
Materials and
Methods: We have developed a toolbox of modular, protein-derived reagents that modulate or reprogram GPCR function. These tools are comprised of proteins derived from the immune system (antibodies and fragments such as nanobodies) that are equipped with specialized features using synthetic and enzymatic chemistry. Such constructs have been applied both as novel ligands for GPCRs and as engineered variants of GPCRs. New GPCR ligands are produced through the recombinant expression of nanobodies, site-specific labeling using enzymatic (sortase A) chemistry, and subsequent elaboration with synthetic molecules prepared using organic chemistry. These semi-synthetic conjugates show unique and useful feature in targeting GPCRs, as discussed below. New engineered GPCR variants were generated through engrafting DNA sequences encoding nanobodies into constructs encoding GPCRs. Chimeric nanobody-GPCR constructs possess the recognition capacity of antibodies combined with the ligand-responsiveness of GPCRs, allowing unique patterns of ligand responsiveness and signaling profiles.
Results, Conclusions, and Discussions: Semi-synthetic nanobody-ligand conjugates that target GPCRs exhibited several useful properties including improved receptor subtype specificity, high potency, and prolonged duration of biological action. These trends were observed in applying conjugates to target several GPCRs including parathyroid hormone receptor, glucagon-like peptide-1 receptor, neurokinin receptor-1, and others. These improvements were most pronounced when using GPCR ligands that possess low affinity. This finding suggests that ligands provided by nature with suboptimal specificity or activity might be augmented through linkage with a target specific antibody to provide conjugates with profiles more amenable to tool or therapeutic development. Further evaluation demonstrated that these conjugates induced a GPCR-signaling response that involved only one out of several possible pathways, exhibiting a property known as “biased agonism”, which is of substantial interest for the development of therapeutics with reduced side effects.
Chimeric, engineered GPCRs have also proven useful for dissecting and designing receptor function. A variant of the A2A adenosine receptor (a GPCR) was fused with a nanobody that recognizes a peptide epitope tag. This engineered receptor was shown to exhibit very potent responses to engineered analogues of adenosine linked to the nanobody-bound peptide epitope. This approach offers a path towards the modular design of GPCRs that show preferential responses to engineered ligands, which should be applicable to any receptor for which ligands can be linked to carrier peptides or proteins. This chimeric nanobody-A2A-receptor was also probed using an analogue of the adenosine-peptide conjugate in which covalent crosslinking occurred between peptide and receptor. These tools demonstrated that covalent linkage of an adenosine analogue to its receptor enabled prolonged biological responses, thus offering a new tool to probe receptor pharmacology and physiological consequences of enduring GPCR activation.
These findings highlight the utility of modular building blocks (nanobodies, peptides, GPCR ligands) for dissecting and modulating receptor function. We expect the reach of this approach to expand as more receptor-specific nanobodies are characterized and new chemistries to create conjugates become available.
