Thematic Minireview Series: New Directions in G Protein-coupled Receptor Pharmacology

Over the past half-century, The Journal of Biological Chemistry has been the venue for many landmark publications on the topic of G protein-coupled receptors (GPCRs, also known as seven-transmembrane receptors). The GPCR superfamily in humans is composed of about 800 members, and is the target of about one-third of all pharmaceuticals. Most of these drugs target a very small subset of GPCRs, and do so by mimicking or competing with endogenous hormones and neurotransmitters. This thematic minireview series examines some emerging trends in GPCR drug discovery. The first article describes efforts to systematically interrogate the human “GPCR-ome,” including more than 150 uncharacterized “orphan” receptors. The second article describes recent efforts to target alternative receptor binding sites with drugs that act as allosteric modulators of orthosteric ligands. The third article describes how the recent expansion of GPCR structures is providing new opportunities for computer-guided drug discovery. Collectively, these three articles provide a roadmap for the most important emerging trends in GPCR pharmacology.     

"Molecular switches" on mGluR allosteric ligands that modulate modes of pharmacology

G-protein-coupled receptors (GPCRs) represent the largest class of drug targets, accounting for more than 40% of marketed drugs; however, discovery efforts for many GPCRs have failed to provide viable drug candidates. Historically, drug discovery efforts have focused on developing ligands that act at the orthosteric site of the endogenous agonist. Recently, efforts have focused on functional assay paradigms and the discovery of ligands that act at allosteric sites to modulate receptor function in either a positive, negative, or neutral manner. Allosteric modulators have numerous advantages over orthosteric ligands, including high subtype selectivity; the ability to mimic physiological conditions; the lack of densensitization, downregulation, and internalization; and reduced side effects. Despite these virtues, challenging issues have now arisen for allosteric modulators of metabotropic glutamate receptors (mGluRs): shallow SAR, ligand-directed trafficking, and the identification of subtle "molecular switches" that modulate the modes of pharmacology. Here, we will discuss the impact of modest structural changes to multiple mGluR allosteric ligands scaffolds that unexpectedly modulate pharmacology and raise concerns over metabolism and the pharmacology of metabolites.     

Allostery in GPCRs: 'MWC' revisited

G protein-coupled receptors (GPCRs) constitute the largest family of receptors in the genome and are the targets for at least 30% of current medicines. In recent years, there has been a dramatic increase in the discovery of allosteric modulators of GPCR activity and a growing appreciation of the diverse modes by which GPCRs can be regulated by both orthosteric and allosteric ligands. Interestingly, some of the contemporary views of GPCR function reflect characteristics that are shared by prototypical allosteric proteins, as encompassed in the classic Monod-Wyman-Changeux (MWC) model initially proposed for enzymes and subsequently extended to other protein families. In this review, we revisit the MWC model in the context of emerging structural, functional and operational data on GPCR allostery.     

Critical role for the second extracellular loop in the binding of both orthosteric and allosteric G protein-coupled receptor ligands

The second extracellular (E2) loop of G protein-coupled receptors (GPCRs) plays an essential but poorly understood role in the binding of non-peptidic small molecules. We have utilized both orthosteric ligands and allosteric modulators of the M2 muscarinic acetylcholine receptor, a prototypical Family A GPCR, to probe possible E2 loop binding dynamics. We developed a homology model based on the crystal structure of bovine rhodopsin and predicted novel cysteine substitutions that should dramatically reduce E2 loop flexibility via disulfide bond formation and significantly inhibit the binding of both types of ligands. This prediction was validated experimentally using radioligand binding, dissociation kinetics, and cell-based functional assays. The results argue for a flexible "gatekeeper" role of the E2 loop in the binding of both allosteric and orthosteric GPCR ligands.     

Trends in application of advancing computational approaches in GPCR ligand discovery

G protein-coupled receptors (GPCRs) comprise the most important superfamily of protein targets in current ligand discovery and drug development. GPCRs are integral membrane proteins that play key roles in various cellular signaling processes. Therefore, GPCR signaling pathways are closely associated with numerous diseases, including cancer and several neurological, immunological, and hematological disorders. Computer-aided drug design (CADD) can expedite the process of GPCR drug discovery and potentially reduce the actual cost of research and development. Increasing knowledge of biological structures, as well as improvements on computer power and algorithms, have led to unprecedented use of CADD for the discovery of novel GPCR modulators. Similarly, machine learning approaches are now widely applied in various fields of drug target research. This review briefly summarizes the application of rising CADD methodologies, as well as novel machine learning techniques, in GPCR structural studies and bioligand discovery in the past few years. Recent novel computational strategies and feasible workflows are updated, and representative cases addressing challenging issues on olfactory receptors, biased agonism, and drug-induced cardiotoxic effects are highlighted to provide insights into future GPCR drug discovery.     

A novel mechanism of G protein-coupled receptor functional selectivity. Muscarinic partial agonist McN-A-343 as a bitopic orthosteric/allosteric ligand

Many G protein-coupled receptors (GPCRs) possess allosteric binding sites distinct from the orthosteric site utilized by their cognate ligands, but most GPCR allosteric modulators reported to date lack signaling efficacy in their own right. McN-A-343 (4-(N-(3-chlorophenyl)carbamoyloxy)-2-butynyltrimethylammonium chloride) is a functionally selective muscarinic acetylcholine receptor (mAChR) partial agonist that can also interact allosterically at the M(2) mAChR. We hypothesized that this molecule simultaneously utilizes both an allosteric and the orthosteric site on the M(2) mAChR to mediate these effects. By synthesizing progressively truncated McN-A-343 derivatives, we identified two, which minimally contain 3-chlorophenylcarbamate, as pure allosteric modulators. These compounds were positive modulators of the orthosteric antagonist N-[(3)H]methylscopolamine, but in functional assays of M(2) mAChR-mediated ERK1/2 phosphorylation and guanosine 5'-3-O-([(35)S]thio)triphosphate binding, they were negative modulators of agonist efficacy. This negative allosteric effect was diminished upon mutation of Y177A in the second extracellular (E2) loop of the M(2) mAChR that is known to reduce prototypical allosteric modulator potency. Our results are consistent with McN-A-343 being a bitopic orthosteric/allosteric ligand with the allosteric moiety engendering partial agonism and functional selectivity. This finding suggests a novel and largely unappreciated mechanism of "directed efficacy" whereby functional selectivity may be engendered in a GPCR by utilizing an allosteric ligand to direct the signaling of an orthosteric ligand encoded within the same molecule.     

Allosteric modulation of kinases and GPCRs: design principles and structural diversity

Allosteric binding sites, as opposed to traditional orthosteric binding sites, offer unparalleled opportunities for drug discovery by providing high levels of selectivity, mimicking physiological conditions, affording fewer side effects because of desensitization/downregulation, and engendering ligands with chemotypes divergent from orthosteric ligands. For kinases, allosteric mechanisms described to date include alteration of protein kinase conformation blocking productive ATP binding which appear 'ATP competitive' or blocking kinase activation by conformational changes that are 'ATP non-competitive'. For GPCRs, allosteric mechanisms impart multiple modes of target modulation (positive allosteric modulation (PAM), negative allosteric modulation (NAM), neutral cooperativity, partial antagonism (PA), allosteric agonism and allosteric antagonism). Here, we review recent developments in the design principles and structural diversity of allosteric ligands for kinases and GPCRs.     

Learning About Allosteric Drugs and Ways to Design Them

While the accelerating quest for precision medicine requires new individually targeting and selective drugs, and the ability to work with so-called undruggable targets, the realm of allosteric drugs meeting this need remains largely uncharted. Generalizing the observations on two major drug targets with widely observed inherent allostery, GPCRs and kinases, we describe and discuss basic allosteric modes of action that are universally applicable in all types of structures and functions. Using examples of Class A GPCRs and CMGC protein kinases, we show how Allosteric Signalling and Probing Fingerprints can be used to identify potential allosteric sites and reveal effector-leads that may serve as a starting point for the development of allosteric drugs targeting these regulatory sites. A set of distinct characteristics of allosteric ligands was established, which highlights the versatility of their design and make them advantageous before their orthosteric counterparts in personalized medicine. We argue that rational design of allosteric drugs should begin with the search for latent sites or design of non-natural binding sites followed by fragment-based design of allosteric ligands and by the mutual adjustment of the site-ligand pair in order to achieve required drug efficacy. On the basis of the perturbative nature and reversibility of allosteric communication, we propose a generic protocol for computational design of allosteric effectors, enabling also the allosteric tuning of biologics, in obtaining allosteric control over protein functions.     

A crystal clear solution for determining G-protein-coupled receptor structures

G-protein-coupled receptors (GPCRs) are medically important membrane proteins that are targeted by over 30% of small molecule drugs. At the time of writing, 15 unique GPCR structures have been determined, with 77 structures deposited in the PDB database, which offers new opportunities for drug development and for understanding the molecular mechanisms of GPCR activation. Many different factors have contributed to this success, but if there is one single factor that can be singled out as the foundation for producing well-diffracting GPCR crystals, it is the stabilisation of the detergent-solubilised receptor-ligand complex. This review will focus predominantly on one of the successful strategies for the stabilisation of GPCRs, namely the thermostabilisation of GPCRs using systematic mutagenesis coupled with thermostability assays. Structures of thermostabilised GPCRs bound to a wide variety of ligands have been determined, which has led to an understanding of ligand specificity; why some ligands act as agonists as opposed to partial or inverse agonists; and the structural basis for receptor activation.     

GPCR structures in drug design,emerging opportunities with new structures

In recent years, GPCR targets from diverse regions of phylogenetic space have been determined. This effort has culminated this year in the determination of representatives of all major classes of GPCRs (A, B, C, and F). Although much of the now well established knowledge on GPCR structures has been known for some years, the new high-resolution structures allow structural insight into the causes of ligand efficacy, biased signaling, and allosteric modulation. In this digest the structural basis for GPCR signaling in the light of the new structures is reviewed and the use of the new non-class A GPCRs for drug design is discussed.     

Novel Allosteric Modulators of G Protein-coupled Receptors

G protein-coupled receptors (GPCRs) are allosteric proteins, because their signal transduction relies on interactions between topographically distinct, yet conformationally linked, domains. Much of the focus on GPCR allostery in the new millennium, however, has been on modes of targeting GPCR allosteric sites with chemical probes due to the potential for novel therapeutics. It is now apparent that some GPCRs possess more than one targetable allosteric site, in addition to a growing list of putative endogenous modulators. Advances in structural biology are also shedding new insights into mechanisms of allostery, although the complexities of candidate allosteric drugs necessitate rigorous biological characterization.     

Sequence,Structure and Ligand Binding Evolution of Rhodopsin-Like G Protein-Coupled Receptors: A Crystal Structure-Based Phylogenetic Analysis

G protein-coupled receptors (GPCRs) form the largest family of membrane receptors in the human genome. Advances in membrane protein crystallization so far resulted in the determination of 24 receptors available as high-resolution atomic structures. We performed the first phylogenetic analysis of GPCRs based on the available set of GPCR structures. We present a new phylogenetic tree of known human rhodopsin-like GPCR sequences based on this structure set. We can distinguish the three separate classes of small-ligand binding GPCRs, peptide binding GPCRs, and olfactory receptors. Analyzing different structural subdomains, we found that small molecule binding receptors most likely have evolved from peptide receptor precursors, with a rhodopsin/S1PR1 ancestor, most likely an ancestral opsin, forming the link between both classes. A light-activated receptor therefore seems to be the origin of the small molecule hormone receptors of the central nervous system. We find hints for a common evolutionary path of both ligand binding site and central sodium/water binding site. Surprisingly, opioid receptors exhibit both a binding cavity and a central sodium/water binding site similar to the one of biogenic amine receptors instead of peptide receptors, making them seemingly prone to bind small molecule ligands, e.g. opiates. Our results give new insights into the relationship and the pharmacological properties of rhodopsin-like GPCRs.     

An overview of the diverse roles of G-protein coupled receptors (GPCRs) in the pathophysiology of various human diseases

G-protein coupled receptors (GPCRs) modulate diverse cellular responses to the majority of neurotransmitters and hormones within the human body. They exhibit much structural and functional diversity, and are responsive to a plethora of endogenous (biogenic amines, cations, lipids, peptides, and glycoproteins) and exogenous (therapeutic drugs, photons, tastants, and odorants) ligands and stimuli. Due to the key roles of GPCRs in tissue/cell physiology and homeostasis, signaling pathways associated with GPCRs are implicated in the pathophysiology of various diseases, ranging from metabolic, immunological, and neurodegenerative disorders, to cancer and infectious diseases. Approximately 40% of clinically approved drugs mediate their effects by modulating GPCR signaling pathways, which makes them attractive targets for drug screening and discovery. The pace of discovery of new GPCR-based drugs has recently accelerated due to rapid advancements in high-resolution structure determination, high-throughput screening technology and in silico computational modeling of GPCR binding interaction with potential drug molecules. This review aims to provide an overview of the diverse roles of GPCRs in the pathophysiology of various diseases that are the major focus of biopharmaceutical research as potential drug targets.     

Real-time monitoring of binding events on a thermostabilized human A2A receptor embedded in a lipid bilayer by surface plasmon resonance

Membrane proteins (MPs) are prevalent drug discovery targets involved in many cell processes. Despite their high potential as drug targets, the study of MPs has been hindered by limitations in expression, purification and stabilization in order to acquire thermodynamic and kinetic parameters of small molecules binding. These bottlenecks are grounded on the mandatory use of detergents to isolate and extract MPs from the cell plasma membrane and the coexistence of multiple conformations, which reflects biochemical versatility and intrinsic instability of MPs. In this work ,we set out to define a new strategy to enable surface plasmon resonance (SPR) measurements on a thermostabilized and truncated version of the human adenosine (A_2A) G-protein-coupled receptor (GPCR) inserted in a lipid bilayer nanodisc in a label- and detergent-free manner by using a combination of affinity tags and GFP-based fluorescence techniques. We were able to detect and characterize small molecules binding kinetics on a GPCR fully embedded in a lipid environment. By providing a comparison between different binding assays in membranes, nanodiscs and detergent micelles, we show that nanodiscs can be used for small molecule binding studies by SPR to enhance the MP stability and to trigger a more native-like behaviour when compared to kinetics on A_2A receptors isolated in detergent. This work provides thus a new methodology in drug discovery to characterize the binding kinetics of small molecule ligands for MPs targets in a lipid environment.     

Synthesis and evaluation of homo-bivalent GnRHR ligands

G protein coupled receptors (GPCRs) are important drug targets in pharmaceutical research. Traditionally, most research efforts have been devoted towards the design of small molecule agonists and antagonists. An interesting, yet poorly investigated class of GPCR modulators comprise the bivalent ligands, in which two receptor pharmacophores are incorporated. Here, we set out to develop a general strategy for the synthesis of bivalent compounds that are projected to bind to the human gonadotropin-releasing hormone receptor (GnRHR). Our results on the dimerisation of a known GnRHR antagonist, with as key step the Huisgen 1,3-cycloaddition, and their ability to bind to and antagonize GnRH-induced GnRHR stimulation, are presented here.     

Genetically encoded photo-cross-linkers map the binding site of an allosteric drug on a G protein-coupled receptor

G protein-coupled receptors (GPCRs) are dynamic membrane proteins that bind extracellular molecules to transduce signals. Although GPCRs represent the largest class of therapeutic targets, only a small percentage of their ligand-binding sites are precisely defined. Here we describe the novel application of targeted photo-cross-linking using unnatural amino acids to obtain structural information about the allosteric binding site of a small molecule drug, the CCR5-targeted HIV-1 co-receptor blocker maraviroc.     

From G Protein-coupled Receptor Structure Resolution to Rational Drug Design

A number of recent technical solutions have led to significant advances in G protein-coupled receptor (GPCR) structural biology. Apart from a detailed mechanistic view of receptor activation, the new structures have revealed novel ligand binding sites. Together, these insights provide avenues for rational drug design to modulate the activities of these important drug targets. The application of structural data to GPCR drug discovery ushers in an exciting era with the potential to improve existing drugs and discover new ones. In this review, we focus on technical solutions that have accelerated GPCR crystallography as well as some of the salient findings from structures that are relevant to drug discovery. Finally, we outline some of the approaches used in GPCR structure based drug design.     

Novobiocin and peptide analogs of α-factor are positive allosteric modulators of the yeast G protein-coupled receptor Ste2p

G protein-coupled receptors (GPCRs) are the target of many drugs prescribed for human medicine and are therefore the subject of intense study. It has been recognized that compounds called allosteric modulators can regulate GPCR activity by binding to the receptor at sites distinct from, or overlapping with, that occupied by the orthosteric ligand. The purpose of this study was to investigate the nature of the interaction between putative allosteric modulators and Ste2p, a model GPCR expressed in the yeast Saccharomyces cerevisiae that binds the tridecapeptide mating pheromone α-factor. Biological assays demonstrated that an eleven amino acid α-factor analog and the antibiotic novobiocin were positive allosteric modulators of Ste2p. Both compounds enhanced the biological activity of α-factor, but did not compete with α-factor binding to Ste2p. To determine if novobiocin and the 11-mer shared a common allosteric binding site, a biologically-active analog of the 11-mer peptide ([Bio-DOPA]11-mer) was chemically cross-linked to Ste2p in the presence and absence of novobiocin. Immunoblots probing for the Ste2p–[Bio-DOPA]11-mer complex revealed that novobiocin markedly decreased cross-linking of the [Bio-DOPA]11-mer to the receptor, but cross-linking of the α-factor analog [Bio-DOPA]13-mer, which interacts with the orthosteric binding site of the receptor, was minimally altered. This finding suggests that both novobiocin and [Bio-DOPA]11-mer compete for an allosteric binding site on the receptor. These results indicate that Ste2p may provide an excellent model system for studying allostery in a GPCR.     

Oral delivery of G protein-coupled receptor modulators: an explanation for the observed class difference

G protein-coupled receptors (GPCRs) represent important targets for drug intervention. However, analysis of GPCR modulator drugs exhibits an important class difference, with many drugs available against aminergic GPCR targets, but relatively few against non-aminergic targets. The reason for this is that commonly drugs mimic the physicochemistry of the receptor ligand. Aminergic ligands generally exhibit physicochemical properties (molecular weight, lipophilicity and hydrogen bonding potential) that are consistent with extensive oral absorption. In contrast, non-aminergic ligands generally exhibit physicochemical properties that are at odds with oral delivery. Thus, combining required potency versus the receptor, with oral delivery potential is a significant challenge, and drug discovery becomes a question of finding the exceptional compound that lies at the edge of ADME space.     

Improving the apo-state detergent stability of NTS1 with CHESS for pharmacological and structural studies

The largest single class of drug targets is the G protein-coupled receptor (GPCR) family. Modern high-throughput methods for drug discovery require working with pure protein, but this has been a challenge for GPCRs, and thus the success of screening campaigns targeting soluble, catalytic protein domains has not yet been realized for GPCRs. Therefore, most GPCR drug screening has been cell-based, whereas the strategy of choice for drug discovery against soluble proteins is HTS using purified proteins coupled to structure-based drug design. While recent developments are increasing the chances of obtaining GPCR crystal structures, the feasibility of screening directly against purified GPCRs in the unbound state (apo-state) remains low. GPCRs exhibit low stability in detergent micelles, especially in the apo-state, over the time periods required for performing large screens. Recent methods for generating detergent-stable GPCRs, however, offer the potential for researchers to manipulate GPCRs almost like soluble enzymes, opening up new avenues for drug discovery. Here we apply cellular high-throughput encapsulation, solubilization and screening (CHESS) to the neurotensin receptor 1 (NTS₁) to generate a variant that is stable in the apo-state when solubilized in detergents. This high stability facilitated the crystal structure determination of this receptor and also allowed us to probe the pharmacology of detergent-solubilized, apo-state NTS₁ using robotic ligand binding assays. NTS₁ is a target for the development of novel antipsychotics, and thus CHESS-stabilized receptors represent exciting tools for drug discovery.