INPHARMA-Based Determination of Ligand Binding Modes at α1 -Adrenergic Receptors Explains the Molecular Basis of Subtype Selectivity.

The structural poses of ligands that bind weakly to protein receptors are challenging to define. In this work we have studied ligand interactions with the adrenoreceptor (AR) subtypes, α1A -AR and α1B -AR, which belong to the G protein-coupled receptor (GPCR) superfamily, by employing the solution-based ligand-observed NMR method interligand NOEs for pharmacophore mapping (INPHARMA). A lack of receptor crystal structures and of subtype-selective drugs has hindered the definition of the physiological roles of each subtype and limited drug development. We determined the binding pose of the weakly binding α1A -AR-selective agonist A-61603 relative to an endogenous agonist, epinephrine, at both α1A -AR and α1B -AR. The NMR experimental data were quantitatively compared, by using SpINPHARMA, to the back-calculated spectra based on ligand poses obtained from all-atom molecular dynamics simulations. The results helped mechanistically explain the selectivity of (R)-A-61603 towards α1A -AR, thus demonstrating an approach for targeting subtype selectivity in ARs.

Impact of Substitution Registry on the Receptor-Activation Profiles of Backbone-Modified Glucagon-like Peptide-1 Analogues.

Family B G protein-coupled receptors play important physiological roles and possess large extracellular domains (ECDs) that aid in binding the long polypeptide hormones that are their natural agonists. We have previously shown that agonist analogues in which subsets of native α-amino acid residues are replaced with ß-amino acid residues can retain activity while avoiding proteolytic degradation. This study focuses on eight new α/ß analogues of glucagon-like peptide 1 (GLP-1) that each contain five α-to-ß replacements in the C-terminal half of the peptide. This portion of GLP-1 is known to adopt an α-helical conformation and contact the ECD. All four registries of the αααß backbone pattern were evaluated; previous work has shown that the αααß pattern supports adoption of an α-helix-like conformation. Two α-to-ß replacement formats were employed, one involving ß3 homologues of the native residues replaced and the other involving a cyclic ß residue. GLP-1R response was characterized in terms of stimulation of cAMP production and ß-arrestin recruitment. Some of the backbone-modified GLP-1 analogues display biased agonism of the GLP-1R. This study helps to establish the scope of the αâ†’ß backbone modification strategy.

Bright, highly water-soluble triazacyclononane europium complexes to detect ligand binding with time-resolved FRET microscopy.

Luminescent europium complexes are used in a broad range of applications as a result of their particular emissive properties. The synthesis and application of bright, highly water-soluble, and negatively charged sulfonic- or carboxylic acid derivatives of para-substituted aryl-alkynyl triazacyclononane complexes are described. Introduction of the charged solubilizing moieties suppresses cellular uptake or adsorption to living cells making them applicable for labeling and performing assays on membrane receptors. These europium complexes are applied to monitor fluorescent ligand binding on cell-surface proteins with time-resolved Förster resonance energy transfer (TR-FRET) assays in plate-based format and using TR-FRET microscopy.

Structure-Activity Relationship Studies of the Natural Product Gq/11 Protein Inhibitor YM-254890.

G proteins act as molecular switches in G protein-coupled receptor signaling pathways and are key mediators for numerous important physiological processes. The natural product, cyclic depsipeptide YM-254890, together with the structurally similar FR900359, is the only known selective inhibitor of the Gq/11 subfamily of G proteins. We recently reported the first total synthesis of YM-254890 and FR900359, followed by synthesizing analogues to perform structure-activity relationship studies. However, incomplete information about their structure-activity relationship prevents the further development of potent and structurally simplified analogues. Herein we report the first systematic structure-activity relationship study toward the N-methyldehydroalanine moiety in YM-254890, by designing and synthesizing seven new analogues. Pharmacological characterization of the seven compounds for Gq/11 -, Gi/o - and Gs -mediated signaling showed that the simplified analogue YM-19 is the most potent Gq/11 inhibitor among the new analogues. This study provides information for the future design of potent and simplified YM-254890 analogues.

A Deep Hydrophobic Binding Cavity is the Main Interaction for Different Y2 R Antagonists.

The neuropeptide Y2 receptor (Y2 R) is involved in various pathophysiological processes such as epilepsy, mood disorders, angiogenesis, and tumor growth. Therefore, the Y2 R is an interesting target for drug development. A detailed understanding of the binding pocket could facilitate the development of highly selective antagonists to study the role of Y2 R in vitro and in vivo. In this study, several residues crucial to the interaction of BIIE0246 and SF-11 derivatives with Y2 R were investigated by signal transduction assays. Using the experimental results as constraints, the antagonists were docked into a comparative structural model of the Y2 R. Despite differences in size and structure, all three antagonists display a similar binding site, including a deep hydrophobic cavity formed by transmembrane helices (TM) 4, 5, and 6, as well as a hydrophobic patch at the top of TM2 and 7. Additionally, we suggest that the antagonists block Q3.32 , a position that has been shown to be crucial for binding of the amidated C terminus of NPY and thus for receptor activation.

A Time-Resolved FRET Cell-Based Binding Assay for the Apelin Receptor.

Analogues of apelin-13 carrying diverse spacers and an ad hoc DY647-derived fluorophore were designed and synthesized by chemoselective acylation of α-hydrazinopeptides. The resulting probes retain very high affinity and efficacy for both the wild-type and SNAP-tagged apelin receptor (ApelinR). They give a time-resolved FRET (TR-FRET) signal with rare-earth lanthanides used as donor fluorophores grafted onto the SNAP-tagged receptor. This specific signal allowed the validation of a binding assay with a high signal-to-noise ratio. In such an assay, the most potent sub-nanomolar fluorescent probe was found to be competitively displaced by the endogenous apelin peptides with binding constants similar to those obtained in a classical radioligand assay. We have thus validated the first TR-FRET cell-based binding assay for ApelinR with potential high-throughput screening applications.

Structure-Activity Relationship Studies of the Cyclic Depsipeptide Natural Product YM-254890, Targeting the Gq Protein.

Extracellular signals perceived by G protein-coupled receptors are transmitted via G proteins, and subsequent intracellular signaling cascades result in a plethora of physiological responses. The natural product cyclic depsipeptides YM-254890 and FR900359 are the only known compounds that specifically inhibit signaling mediated by the Gq subfamily. In this study we exploit a newly developed synthetic strategy for this compound class in the design, synthesis, and pharmacological evaluation of eight new analogues of YM-254890. These structure-activity relationship studies led to the discovery of three new analogues, YM-13, YM-14, and YM-18, which displayed potent and selective Gq inhibitory activity. This provides pertinent information for the understanding of the Gq inhibitory mechanism by this class of compounds and importantly provides a pathway for the development of labeled YM-254890 analogues.

The G†Protein-Coupled Receptor GPR17: Overview and Update.

The GPR17 receptor is a G protein-coupled receptor (GPCR) that seems to respond to two unrelated families of endogenous ligands: nucleotide sugars (UDP, UDP-galactose, and UDP-glucose) and cysteinyl leukotrienes (LTD4 , LTC4 , and LTE4 ), with significant affinity at micromolar and nanomolar concentrations, respectively. This receptor has a broad distribution at the level of the central nervous system (CNS) and is found in neurons and in a subset of oligodendrocyte precursor cells (OPCs). Unfortunately, disparate results emerging from different laboratories have resulted in a lack of clarity with regard to the role of GPR17-targeting ligands in OPC differentiation and in myelination. GPR17 is also highly expressed in organs typically undergoing ischemic damage and has various roles in specific phases of adaptations that follow a stroke. Under such conditions, GPR17 plays a crucial role; in fact, its inhibition decreases the progression of ischemic damage. This review summarizes some important features of this receptor that could be a novel therapeutic target for the treatment of demyelinating diseases and for repairing traumatic injury.

Development of Potent and Metabolically Stable APJ Ligands with High Therapeutic Potential.

The apelin ligand receptor system is an important target to develop treatment strategies for cardiovascular diseases. Although apelin exhibits strong inotropic effects, its pharmaceutical application is limited because no agonist with suitable properties is available. On the one hand, peptide ligands are too instable, and on the other hand, small-molecule agonists show only low potency. This study describes the development of apelin (APJ) receptor agonists with not only high activity but also metabolic stability. Several strategies including capping of termini, insertion of unnatural amino acids, cyclization, and lipidation were analyzed. Peptide activity was tested using a Ca2+ -mobilization assay and the degradation of selected analogues was analyzed in rat plasma. The best results were obtained by N-terminal lipidation of a 13-mer apelin derivative. This analogue displayed a half-life of 29 h in rat plasma, compared with 0.025 h for the wild-type peptide. Furthermore, in vivo pharmacokinetics revealed a clearance of 0.049 L h-1 kg-1 and a half-life of 0.36 h. In summary, amino acid substitution and fatty acid modification resulted in a potent and 1000-fold more stable peptide that exhibits high pharmaceutical potential.

New approaches in the design and development of cannabinoid receptor ligands: multifunctional and bivalent compounds.

Since the identification of the endocannabinoid system, two G protein-coupled receptors (GPCRs) of this complex system were identified and characterized: cannabinoid receptors type 1 (CB1R) and type 2 (CB2R). In addition to orthosteric and subsequently allosteric ligands, new strategies have been used to target CBRs. Bivalent ligands and multifunctional ligands acting at diverse biological targets have been designed, synthesized, and characterized for both CBRs. Due to their altered receptor binding and pharmacological profiles, they are interesting tools to explore CBR functions and their interactions with other physiological systems. Moreover, this approach may bear therapeutic advantages in the therapy of CBR-related disorders, especially multifactorial diseases. Promising prospects include anorectics with fewer side effects, analgesics with decreased tolerance, and therapeutics with multiple pharmacological activities for the treatment of cancer, inflammation, multiple sclerosis, Huntington's and Alzheimer's diseases.

The Role of Target Binding Kinetics in Drug Discovery.

Traditionally structure-activity/affinity relationships (SAR) have dominated research in medicinal chemistry. However, structure-kinetics relationships (SKR) can be very informative too. In this viewpoint we explore the molecular determinants of binding kinetics and discuss challenges for future binding kinetics studies. A scheme for future kinetics-directed drug design and discovery is also proposed.

The predicted ensemble of low-energy conformations of human somatostatin receptor subtype 5 and the binding of antagonists.

Human somatostatin receptor subtype 5 (hSSTR5) regulates cell proliferation and hormone secretion. However, the identification of effective therapeutic small-molecule ligands is impeded because experimental structures are not available for any SSTR subtypes. Here, we predict the ensemble of low-energy 3D structures of hSSTR5 using a modified GPCR Ensemble of Structures in Membrane BiLayer Environment (GEnSeMBLE) complete sampling computational method. We find that this conformational ensemble displays most interhelical interactions conserved in class A G protein-coupled receptors (GPCRs) plus seven additional interactions (e.g., Y2.43-D3.49, T3.38-S4.53, K5.64-Y3.51) likely conserved among SSTRs. We then predicted the binding sites for a series of five known antagonists, leading to predicted binding energies consistent with experimental results reported in the literature. Molecular dynamics (MD) simulation of 50 ns in explicit water and lipid retained the predicted ligand-bound structure and formed new interaction patterns (e.g. R3.50-T6.34) consistent with the inactive µ-opioid receptor X-ray structure. We suggest more than six mutations for experimental validation of our prediction. The final predicted receptor conformations and antagonist binding sites provide valuable insights for designing new small-molecule drugs targeting SSTRs.

Binding kinetics of ZM241385 derivatives at the human adenosine A2A receptor.

Classical drug design and development rely mostly on affinity- or potency-driven structure-activity relationships (SAR). Thus far, a given compound's binding kinetics have been largely ignored, the importance of which is now being increasingly recognized. In the present study, we performed an extensive structure-kinetics relationship (SKR) study in addition to a traditional SAR analysis at the adenosine A2A receptor (A2A R). The ensemble of 24 A2A R compounds, all triazolotriazine derivatives resembling the prototypic antagonist ZM241385 (4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino)ethyl)phenol), displayed only minor differences in affinity, although they varied substantially in their dissociation rates from the receptor. We believe that such a combination of SKR and SAR analyses, as we have done with the A2A R, will have general importance for the superfamily of G protein-coupled receptors, as it can serve as a new strategy to tailor the interaction between ligand and receptor.

Structure-based and fragment-based GPCR drug discovery.

G protein-coupled receptors (GPCRs) are an important family of membrane proteins; historically, drug discovery in this target class has been fruitful, with many of the world's top-selling drugs being GPCR modulators. Until recently, the modern techniques of structure- and fragment-based drug discovery had not been fully applied to GPCRs, primarily because of the instability of these proteins when isolated from their cell membrane environments. Recent advances in receptor stabilisation have facilitated major advances in GPCR structural biology over the past six years, with 21 new receptor targets successfully crystallised with one or more ligands. The dramatic increase in GPCR structural information has yielded an increased use of structure-based methods for hit identification and progression, which are reviewed herein. Additionally, a number of fragment-based drug discovery techniques have been validated for use with GPCRs in recent years; these approaches and their use in hit identification are reviewed.