G Protein-coupled Receptors Allosteric Modulators Research Articles

Local anesthetics inhibit muscarinic acetylcholine receptor-mediated calcium responses and the recruitment of β-arrestin in HSY human parotid cells

ObjectivesLocal anesthetics act on G protein-coupled receptors (GPCRs); thus, their potential as allosteric modulators of GPCRs has attracted attention. Intracellular signaling via GPCRs involves both G-protein- and β-arrestin-mediated pathways. To determine the effects of local anesthetics on muscarinic acetylcholine receptors (mAChR), a family of GPCRs, we analyzed the effects of local anesthetics on mAChR-mediated Ca2+ responses and formation of receptor–β-arrestin complexes in the HSY human parotid cell line. MethodsCa2+ responses were monitored by fura-2 spectrofluorimetry. Ligand-induced interactions between mAChR and β-arrestin were examined using a β-arrestin GPCR assay kit. ResultsLidocaine reduced mAChR-mediated Ca2+ responses but did not change the intracellular Ca2+ concentration in non-stimulated cells. The membrane-impermeant lidocaine analog QX314 and procaine inhibited mAChR-mediated Ca2+ responses, with EC50 values of 48.0 and 20.4 μM, respectively, for 50 μM carbachol-stimulated Ca2+ responses. In the absence of extracellular Ca2+, the pretreatment of cells with QX314 reduced carbachol-induced Ca2+ release, indicating that QX314 reduced Ca2+ release from intracellular stores. Lidocaine and QX314 did not affect store-operated Ca2+ entry as they did not alter the thapsigargin-induced Ca2+ response. QX314 and procaine reduced the carbachol-mediated recruitment of β-arrestin, and administration of procaine suppressed pilocarpine-induced salivary secretion in mice. ConclusionLocal anesthetics, including QX314, act on mAChR to reduce carbachol-induced Ca2+ release from intracellular stores and the recruitment of β-arrestin. These findings support the notion that local anesthetics and their derivatives are starting points for the development of functional allosteric modulators of mAChR.

Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics.

Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular-level understanding of the general principles that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. In this study, we rigorously quantified the affinity, efficacy, and magnitude of modulation of two different positive allosteric modulators, LY2033298 (LY298) and VU0467154 (VU154), combined with the endogenous agonist acetylcholine (ACh) or the high-affinity agonist iperoxo (Ipx), at the human M4 mAChR. By determining the cryo-electron microscopy structures of the M4 mAChR, bound to a cognate Gi1 protein and in complex with ACh, Ipx, LY298-Ipx, and VU154-Ipx, and applying molecular dynamics simulations, we determine key molecular mechanisms underlying allosteric pharmacology. In addition to delineating the contribution of spatially distinct binding sites on observed pharmacology, our findings also revealed a vital role for orthosteric and allosteric ligand-receptor-transducer complex stability, mediated by conformational dynamics between these sites, in the ultimate determination of affinity, efficacy, cooperativity, probe dependence, and species variability. There results provide a holistic framework for further GPCR mechanistic studies and can aid in the discovery and design of future allosteric drugs.

Therapeutic potential of allosteric modulators for the treatment of gastrointestinal motility disorders.

Gastrointestinal motility is tightly regulated by the enteric nervous system (ENS). Disruption of coordinated enteric nervous system activity can result in dysmotility. Pharmacological treatment options for dysmotility include targeting of G protein-coupled receptors (GPCRs) expressed by neurons of the enteric nervous system. Current GPCR-targeting drugs for motility disorders bind to the highly conserved endogenous ligand-binding site and promote indiscriminate activation or inhibition of the target receptor throughout the body. This can be associated with significant side-effect liability and a loss of physiological tone. Allosteric modulators of GPCRs bind to a distinct site from the endogenous ligand, which is typically less conserved across multiple receptor subtypes and can modulate endogenous ligand signalling. Allosteric modulation of GPCRs that are important for enteric nervous system function may provide effective relief from motility disorders while limiting side-effects. This review will focus on how allosteric modulators of GPCRs may influence gastrointestinal motility, using 5-hydroxytryptamine (5-HT), acetylcholine (ACh) and opioid receptors as examples. LINKED ARTICLES: This article is part of a themed issue Therapeutic Targeting of G Protein-Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc.

Allosteric Modulator Leads Hiding in Plain Site: Developing Peptide and Peptidomimetics as GPCR Allosteric Modulators.

Allosteric modulators (AMs) of G-protein coupled receptors (GPCRs) are desirable drug targets because they can produce fewer on-target side effects, improved selectivity, and better biological specificity (e.g., biased signaling or probe dependence) than orthosteric drugs. An underappreciated source for identifying AM leads are peptides and proteins—many of which were evolutionarily selected as AMs—derived from endogenous protein-protein interactions (e.g., transducer/accessory proteins), intramolecular receptor contacts (e.g., pepducins or extracellular domains), endogenous peptides, and exogenous libraries (e.g., nanobodies or conotoxins). Peptides offer distinct advantages over small molecules, including high affinity, good tolerability, and good bioactivity, and specific disadvantages, including relatively poor metabolic stability and bioavailability. Peptidomimetics are molecules that combine the advantages of both peptides and small molecules by mimicking the peptide’s chemical features responsible for bioactivity while improving its druggability. This review 1) discusses sources and strategies to identify peptide/peptidomimetic AMs, 2) overviews strategies to convert a peptide lead into more drug-like “peptidomimetic,” and 3) critically analyzes the advantages, disadvantages, and future directions of peptidomimetic AMs. While small molecules will and should play a vital role in AM drug discovery, peptidomimetics can complement and even exceed the advantages of small molecules, depending on the target, site, lead, and associated factors.

Integrated Multi-Class Classification and Prediction of GPCR Allosteric Modulators by Machine Learning Intelligence.

G-protein-coupled receptors (GPCRs) are the largest and most diverse group of cell surface receptors that respond to various extracellular signals. The allosteric modulation of GPCRs has emerged in recent years as a promising approach for developing target-selective therapies. Moreover, the discovery of new GPCR allosteric modulators can greatly benefit the further understanding of GPCR cell signaling mechanisms. It is critical but also challenging to make an accurate distinction of modulators for different GPCR groups in an efficient and effective manner. In this study, we focus on an 11-class classification task with 10 GPCR subtype classes and a random compounds class. We used a dataset containing 34,434 compounds with allosteric modulators collected from classical GPCR families A, B, and C, as well as random drug-like compounds. Six types of machine learning models, including support vector machine, naïve Bayes, decision tree, random forest, logistic regression, and multilayer perceptron, were trained using different combinations of features including molecular descriptors, Atom-pair fingerprints, MACCS fingerprints, and ECFP6 fingerprints. The performances of trained machine learning models with different feature combinations were closely investigated and discussed. To the best of our knowledge, this is the first work on the multi-class classification of GPCR allosteric modulators. We believe that the classification models developed in this study can be used as simple and accurate tools for the discovery and development of GPCR allosteric modulators.

Hemorphins Targeting G Protein-Coupled Receptors.

Hemorphins are short peptides produced by the proteolysis of the beta subunit of hemoglobin. These peptides have diverse physiological effects especially in the nervous and the renin-angiotensin systems. Such effects occur through the modulation of a diverse range of proteins including enzymes and receptors. In this review, we focus on pharmacological and functional targeting of G protein-coupled receptors (GPCRs) by hemorphins and their implication in physiology and pathophysiology. Among GPCRs, the opioid receptors constitute the first set of targets of hemorphins with implication in analgesia. Subsequently, several other GPCRs have been reported to be directly or indirectly involved in hemorphins’ action. This includes the receptors for angiotensin II, oxytocin, bombesin, and bradykinin, as well as the human MAS-related G protein-coupled receptor X1. Interestingly, both orthosteric activation and allosteric modulation of GPCRs by hemorphins have been reported. This review links hemorphins with GPCR pharmacology and signaling, supporting the implication of GPCRs in hemorphins’ effects. Thus, this aids a better understanding of the molecular basis of the action of hemorphins and further demonstrates that hemorphin-GPCR axis constitutes a valid target for therapeutic intervention in different systems.

Allosteric Modulation of GPCRs of Class A by Cholesterol.

G-protein coupled receptors (GPCRs) are membrane proteins that convey extracellular signals to the cellular milieu. They represent a target for more than 30% of currently marketed drugs. Here we review the effects of membrane cholesterol on the function of GPCRs of Class A. We review both the specific effects of cholesterol mediated via its direct high-affinity binding to the receptor and non-specific effects mediated by cholesterol-induced changes in the properties of the membrane. Cholesterol binds to many GPCRs at both canonical and non-canonical binding sites. It allosterically affects ligand binding to and activation of GPCRs. Additionally, it changes the oligomerization state of GPCRs. In this review, we consider a perspective of the potential for the development of new therapies that are targeted at manipulating the level of membrane cholesterol or modulating cholesterol binding sites on to GPCRs.

Natural Compounds as Guides for the Discovery of Drugs Targeting G-Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs), which constitute the most populous family of the human proteome, are the target of 35–45% of approved therapeutic drugs. This review focuses on natural products (excluding peptides) that target GPCRs. Natural compounds identified so far as agonists, antagonists or allosteric modulators of GPCRs have been found in all groups of existing living beings according to Whittaker’s Five Kingdom Classification, i.e., bacteria (monera), fungi, protoctists, plants and animals. Terpenoids, alkaloids and flavonoids are the most common chemical structures that target GPCRs whose endogenous ligands range from lipids to epinephrine, from molecules that activate taste receptors to molecules that activate smell receptors. Virtually all of the compounds whose formula is displayed in this review are pharmacophores with potential for drug discovery; furthermore, they are expected to help expand the number of GPCRs that can be considered as therapeutic targets.

Harnessing Ion-Binding Sites for GPCR Pharmacology.

Endogenous ions play important roles in the function and pharmacology of G-protein coupled receptors (GPCRs). Historically the evidence for ionic modulation of GPCR function dates to 1973 with studies of opioid receptors, where it was demonstrated that physiologic concentrations of sodium allosterically attenuated agonist binding. This Na+-selective effect was distinct from effects of other monovalent and divalent cations, with the latter usually counteracting sodium's negative allosteric modulation of binding. Since then, numerous studies documenting the effects of mono- and divalent ions on GPCR function have been published. While ions can act selectively and nonselectively at many sites in different receptors, the discovery of the conserved sodium ion site in class A GPCR structures in 2012 revealed the unique nature of Na+ site, which has emerged as a near-universal site for allosteric modulation of class A GPCR structure and function. In this review, we synthesize and highlight recent advances in the functional, biophysical, and structural characterization of ions bound to GPCRs. Taken together, these findings provide a molecular understanding of the unique roles of Na+ and other ions as GPCR allosteric modulators. We will also discuss how this knowledge can be applied to the redesign of receptors and ligand probes for desired functional and pharmacological profiles. SIGNIFICANCE STATEMENT: The function and pharmacology of GPCRs strongly depend on the presence of mono and divalent ions in experimental assays and in living organisms. Recent insights into the molecular mechanism of this ion-dependent allosterism from structural, biophysical, biochemical, and computational studies provide quantitative understandings of the pharmacological effects of drugs in vitro and in vivo and open new avenues for the rational design of chemical probes and drug candidates with improved properties.

State-dependent Lipid Interactions with the A2a Receptor Revealed by MD Simulations Using In Vivo-Mimetic Membranes

SummaryMembranes are known to have modulatory effects on G protein-coupled receptors (GPCRs) via specific lipid interactions. However, the mechanisms of such modulations in physiological conditions and how they influence GPCR functions remain unclear. Here we report coarse-grained molecular dynamics simulations on the Adenosine A2a receptor in different conformational states embedded in an in vivo-mimetic membrane model. Nine lipid interaction sites were revealed. The strength of lipid interactions with these sites showed a degree of dependence on the conformational states of the receptor, suggesting that these lipids may regulate the conformational dynamics of the receptor. In particular, we revealed a dual role of PIP2 on A2aR activation that involves both stabilization of the characteristic outward tilt of TM6 and enhancement of A2aR-mini-Gs association. Our results demonstrated that the bound lipids allosterically regulate the functional properties of GPCRs. These protein-lipid interactions provide a springboard for design of allosteric modulators of GPCRs.

Toward an understanding of the structural basis of allostery in muscarinic acetylcholine receptors.

Recent breakthroughs and developments in structural biology have led to a spate of crystal structures for G protein-coupled receptors (GPCRs). This is the case for the muscarinic acetylcholine receptors (mAChRs) where inactive-state structures for four of the five subtypes and two active-state structures for one subtype are available. These mAChR crystal structures have provided new insights into receptor mechanisms, dynamics, and allosteric modulation. This is highly relevant to the mAChRs given that these receptors are an exemplar model system for the study of GPCR allostery. Allosteric mechanisms of the mAChRs are predominantly consistent with a two-state model, albeit with some notable recent exceptions. Herein, we discuss the mechanisms for positive and negative allosteric modulation at the mAChRs and compare and contrast these to evidence offered by pharmacological, biochemical, and computational approaches. This analysis provides insight into the fundamental pharmacological properties exhibited by GPCR allosteric modulators, such as enhanced subtype selectivity, probe dependence, and biased modulation while highlighting the current challenges that remain. Though complex, enhanced molecular understanding of allosteric mechanisms will have considerable influence on our understanding of GPCR activation and signaling and development of therapeutic interventions.

Allosteric Modulation of Class A GPCRs: Targets, Agents, and Emerging Concepts.

G-protein-coupled receptors (GPCRs) have been tractable drug targets for decades with over one-third of currently marketed drugs targeting GPCRs. Of these, the class A GPCR superfamily is highly represented, and continued drug discovery for this family of receptors may provide novel therapeutics for a vast range of diseases. GPCR allosteric modulation is an innovative targeting approach that broadens the available small molecule toolbox and is proving to be a viable drug discovery strategy, as evidenced by recent FDA approvals and clinical trials. Numerous class A GPCR allosteric modulators have been discovered recently, and emerging trends such as the availability of GPCR crystal structures, diverse functional assays, and structure-based computational approaches are improving optimization and development. This Perspective provides an update on allosterically targeted class A GPCRs and their disease indications and the medicinal chemistry approaches toward novel allosteric modulators and highlights emerging trends and opportunities in the field.

"Selective" Class C G Protein-Coupled Receptor Modulators Are Neutral or Biased mGlu5 Allosteric Ligands.

Numerous positive and negative allosteric modulators (PAMs and NAMs) of class C G protein-coupled receptors (GPCRs) have been developed as valuable preclinical pharmacologic tools and therapeutic agents. Although many class C GPCR allosteric modulators have undergone subtype selectivity screening, most assay paradigms have failed to perform rigorous pharmacologic assessment. Using mGlu5 as a representative class C GPCR, we tested the hypothesis that allosteric modulator selectivity was based on cooperativity rather than affinity. Specifically, we aimed to identify ligands that bound to mGlu5 but exhibited neutral cooperativity with mGlu5 agonists. We additionally evaluated the potential for these ligands to exhibit biased pharmacology. Radioligand binding, intracellular calcium (iCa2+) mobilization, and inositol monophosphate (IP1) accumulation assays were undertaken in human embryonic kidney cells expressing low levels of rat mGlu5 (HEK293A-mGlu5-low) for diverse allosteric chemotypes. Numerous "non-mGlu5" class C GPCR allosteric modulators incompletely displaced allosteric mGlu5 radioligand [3H]methoxy-PEPy binding, consistent with a negative allosteric interaction. Affinity estimates for CPCCOEt (mGlu1 ligand), PHCCC (mGlu4 ligand), GS39783 (GABAB ligand), AZ12216052 (mGlu8 ligand), and CGP7930 (GABAB ligand) at mGlu5 were within 10-fold of their target receptor. Most class C GPCR allosteric modulators had neutral cooperativity with both orthosteric and allosteric mGlu5 agonists in functional assays; however, NPS2143 (calcium-sensing receptor (CaSR) NAM), cinacalcet (CaSR PAM), CGP7930, and AZ12216052 were partial mGlu5 agonists for IP1 accumulation, but not iCa2+ mobilization. By using mGlu5 as a model class C GPCR, we find that for many class C GPCR allosteric modulators, subtype selectivity is driven by cooperativity and misinterpreted owing to unappreciated bias.

Integrated In Silico Fragment-Based Drug Design: Case Study with Allosteric Modulators on Metabotropic Glutamate Receptor 5.

GPCR allosteric modulators target at the allosteric binding pockets of G protein-coupled receptors (GPCRs) with indirect influence on the effects of an orthosteric ligand. Such modulators exhibit significant advantages compared to the corresponding orthosteric ligands, including better chemical tractability or physicochemical properties, improved selectivity, and reduced risk of oversensitization towards their receptors. Metabotropic glutamate receptor 5 (mGlu5), a member of class C GPCRs, is a promising therapeutic target for treating many central nervous system diseases. The crystal structure of mGlu5 in the complex with the negative allosteric modulator mavoglurant was recently reported, providing a fundamental model for designing new allosteric modulators. Computational fragment-based drug discovery represents a powerful scaffold-hopping and lead structure-optimization tool for drug design. In the present work, a set of integrated computational methodologies was first used, such as fragment library generation and retrosynthetic combinatorial analysis procedure (RECAP) for novel compound generation. Then, the compounds generated were assessed by benchmark dataset verification, docking studies, and QSAR model simulation. Subsequently, structurally diverse compounds, with reported or unreported scaffolds, can be observed from top 20 in silico synthesized compounds, which were predicted to be potential mGlu5 modulators. In silico compounds with reported scaffolds may fill SAR holes in known, patented series of mGlu5 modulators. And the generation of compounds without reported tests on mGluR indicates that our approach is doable for exploring and designing novel compounds. Our case study of designing allosteric modulators on mGlu5 demonstrated that the established computational fragment-based approach is a useful methodology for facilitating new compound design in the future.

Endogenous allosteric modulators of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are the largest superfamily of receptors encoded by the human genome, and represent the largest class of current drug targets. Over the last decade and a half, it has become widely accepted that most, if not all, GPCRs possess spatially distinct allosteric sites that can be targeted by exogenous substances to modulate the receptors' biologic state. Although many of these allosteric sites are likely to serve other (e.g., structural) roles, they nonetheless possess appropriate properties to be serendipitously targeted by synthetic molecules. However, there are also examples of endogenous substances that can act as allosteric modulators of GPCRs. These include not only the obvious example, i.e., the G protein, but also a variety of ions, lipids, amino acids, peptides, and accessory proteins that display different degrees of receptor-specific modulatory effects. This also suggests that some GPCRs may possess true "orphan" allosteric sites for hitherto unappreciated endogenous modulators. Of note, the increasing identification of allosteric modulator lipids, inflammatory peptides, and GPCR-targeted autoantibodies indicates that disease context plays an important role in the generation of putative endogenous GPCR modulators. If an endogenous allosteric substance can be shown to play a role in disease, this could also serve as an impetus to pursue synthetic neutral allosteric ligands as novel therapeutic agents.

Opportunities and challenges in the discovery of allosteric modulators of GPCRs for treating CNS disorders.

Novel allosteric modulators of G protein-coupled receptors (GPCRs) are providing fundamental advances in the development of GPCR ligands with high subtype selectivity and novel modes of efficacy that have not been possible with traditional approaches. As new allosteric modulators are advancing as drug candidates, we are developing an increased understanding of the major advantages and broad range of activities that can be achieved with these agents through selective modulation of specific signalling pathways, differential effects on GPCR homodimers versus heterodimers, and other properties. This understanding creates exciting opportunities, as well as unique challenges, in the optimization of novel therapeutic agents for disorders of the central nervous system.

Strategies for the identification of allosteric modulators of G-protein-coupled receptors

Once considered a pharmacological curiosity, allosteric modulation of seven transmembrane domain G-protein-coupled receptors (GPCRs) has emerged as a potentially powerful means to affect receptor function for therapeutic purposes. Allosteric modulators, which interact with binding sites topologically distinct from the orthosteric ligand binding sites, can potentially provide improved selectivity and safety, along with maintenance of spatial and temporal aspects of GPCR signaling. Accordingly, drug discovery efforts for GPCRs have increasingly focused on the identification of allosteric modulators. This review is devoted to an examination of the strategies, challenges, and opportunities for high-throughput screening for allosteric modulators of GPCRs, with particular focus on the identification of positive allosteric modulators.

Allosteric Enhancers of A1 Adenosine Receptors: State of the Art and New Horizons for Drug Development

Adenosine is an important autocoid, exerting its physiological effects on the human body by activation of four different G-protein-coupled-receptors (GPCRs) classified as A(1), A(2A), A(2B), and A(3). These receptors are coupled to secondary messenger systems including adenylate cyclase, inositol phosphate metabolism, and K(+), K(ATP) and Ca(2+) channels. Pharmacological agents that increase the activation of A(1) adenosine receptors in response to adenosine would be useful for treatment of cardiovascular, central nervous system, and inflammatory pathologies. Compounds that are able to enhance the activity of the A(1)-adenosine receptors by the endogenous ligand within specific tissues may have potential therapeutic advantages over non-endogenous agonists. Such an opportunity for intervention is provided by the concept of allosteric modulation of GPCRs. Therefore the use of allosteric enhancers to increase the responsiveness of the A(1) receptors to endogenous adenosine at sites of its production is an appealing alternative to activation by exogenous agonists. This approach minimizes side effects because allosteric enhancers amplify the action of the agonist by stabilizing the agonist-A(1)-receptor-G protein ternary complex. The allosteric enhancement of the GABA(A) receptor by benzodiazepines is the most famous and successful example of this strategy. The aim of this article is to give an overview of the results obtained in this field and discuss the opportunities and challenges that this class of ligands might offer for medicinal chemistry and pharmacology.

Allosteric modulators of GPCRs: a novel approach for the treatment of CNS disorders.

Despite G-protein-coupled receptors (GPCRs) being among the most fruitful targets for marketed drugs, intense discovery efforts for several GPCR subtypes have failed to deliver selective drug candidates. Historically, drug discovery programmes for GPCR ligands have been dominated by efforts to develop agonists and antagonists that act at orthosteric sites for endogenous ligands. However, in recent years, there have been tremendous advances in the discovery of novel ligands for GPCRs that act at allosteric sites to regulate receptor function. These compounds provide high selectivity, novel modes of efficacy and may lead to novel therapeutic agents for the treatment of multiple psychiatric and neurological human disorders.

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.