Nanobodies as Probes and Modulators of Cardiovascular G Protein–Coupled Receptors

Abstract

Understanding the activation of G protein-coupled receptors (GPCRs) is of paramount importance to the field of cardiovascular medicine due to the critical physiological roles of these receptors and their prominence as drug targets. Although many cardiovascular GPCRs have been extensively studied as model receptors for decades, new complexities in their regulation continue to emerge. As a result, there is an ongoing need to develop novel approaches to monitor and to modulate GPCR activation. In less than a decade, nanobodies, or recombinant single-domain antibody fragments from camelids, have become indispensable tools for interrogating GPCRs both in purified systems and in living cells. Nanobodies have gained traction rapidly due to their biochemical tractability and their ability to recognize defined states of native proteins. Here, we review how nanobodies have been adopted to elucidate the structure, pharmacology, and signaling of cardiovascular GPCRs, resolving long-standing mysteries and revealing unexpected mechanisms. We also discuss how advancing technologies to discover nanobodies with tailored specificities may expand the impact of these tools for both basic science and therapeutic applications.

Nanobodies as Diagnostic and Therapeutic Tools for Cardiovascular Diseases (CVDs)

“…Another very important target for cardiovascular medicine is the G-protein coupled receptors (GPCRs) and various nanobody-based strategies have been employed in order to study their structure, pharmacokinetics, and expression modulation [ 58 ]. By using a library of synthetic camelid antibody fragments, a series of nanobodies that can interact with angiotensin II type 1 receptor (AT1R) were discovered.…”

“…Further studies showed that, for instance, clone NbAT110 helped determine the structural characteristics of the AT1R receptor and revealed how peptides bind and activate it, which until now, because of the size and flexibility of the peptides that bind to this receptor, was not very clear. Furthermore, some new information regarding receptor-peptide binding was revealed, such as a partial β-barrel at the N-terminal end of the receptor, a β-hairpin, and the N-terminus of the binding peptide [ 58 ].…”

Allosteric Regulation of G-Protein-Coupled Receptors: From Diversity of Molecular Mechanisms to Multiple Allosteric Sites and Their Ligands

“…Along with highly specialized components of GPCR signaling, endogenous allosteric regulators of GPCR include some classes of antibodies produced against extracellular regions of receptors, small proteins, oligopeptides, steroids, fatty acids, amino acids, and even simple ions [91,[94][95][96][97][98][99][100]. Some allosteric regulators specifically affect a particular receptor or closely related GPCRs, while others are non-specific and affect the activity of a large number of GPCRs belonging to different families.…”

Computational Modeling and Characterization of Peptides Derived from Nanobody Complementary-Determining Region 2 (CDR2) Targeting Active-State Conformation of the β2-Adrenergic Receptor (β2AR)

“…These include resolving the crystal structures of β 2 AR in complex with agonist/Gs [6][7][8]. As highlighted in various reviews [9][10][11][12][13], this methodology has also proved effective in resolving the active-state crystal structures of other GPCRs for imaging and modulating GPCR function and as a new class of potential GPCR-targeted therapeutics.…”