Design, Synthesis, and Application of Fluorescent Ligands Targeting the Intracellular Allosteric Binding Site of the CXC Chemokine Receptor 2.

The inhibition of CXC chemokine receptor 2 (CXCR2), a key inflammatory mediator, is a potential strategy in the treatment of several pulmonary diseases and cancers. The complexity of endogenous chemokine interaction with the orthosteric binding site has led to the development of CXCR2 negative allosteric modulators (NAMs) targeting an intracellular pocket near the G protein binding site. Our understanding of NAM binding and mode of action has been limited by the availability of suitable tracer ligands for competition studies, allowing direct ligand binding measurements. Here, we report the rational design, synthesis, and pharmacological evaluation of a series of fluorescent NAMs, based on navarixin (2), which display high affinity and preferential binding for CXCR2 over CXCR1. We demonstrate their application in fluorescence imaging and NanoBRET binding assays, in whole cells or membranes, capable of kinetic and equilibrium analysis of NAM binding, providing a platform to screen for alternative chemophores targeting these receptors.

Mass spectrometry captures biased signalling and allosteric modulation of a G-protein-coupled receptor.

G-protein-coupled receptors signal through cognate G proteins. Despite the widespread importance of these receptors, their regulatory mechanisms for G-protein selectivity are not fully understood. Here we present a native mass spectrometry-based approach to interrogate both biased signalling and allosteric modulation of the ß1-adrenergic receptor in response to various ligands. By simultaneously capturing the effects of ligand binding and receptor coupling to different G proteins, we probed the relative importance of specific interactions with the receptor through systematic changes in 14 ligands, including isoprenaline derivatives, full and partial agonists, and antagonists. We observed enhanced dynamics of the intracellular loop 3 in the presence of isoprenaline, which is capable of acting as a biased agonist. We also show here that endogenous zinc ions augment the binding in receptor-Gs complexes and propose a zinc ion-binding hotspot at the TM5/TM6 intracellular interface of the receptor-Gs complex. Further interrogation led us to propose a mechanism in which zinc ions facilitate a structural transition of the intermediate complex towards the stable state.

Interaction With the Lipid Membrane Influences Fentanyl Pharmacology.

Overdose deaths from fentanyl have reached epidemic proportions in the USA and are increasing worldwide. Fentanyl is a potent opioid agonist that is less well reversed by naloxone than morphine. Due to fentanyl's high lipophilicity and elongated structure we hypothesised that its unusual pharmacology may be explained by its interactions with the lipid membrane on route to binding to the µ-opioid receptor (MOPr). Through coarse-grained molecular dynamics simulations, electrophysiological recordings and cell signalling assays, we determined how fentanyl and morphine access the orthosteric pocket of MOPr. Morphine accesses MOPr via the aqueous pathway; first binding to an extracellular vestibule, then diffusing into the orthosteric pocket. In contrast, fentanyl may take a novel route; first partitioning into the membrane, before accessing the orthosteric site by diffusing through a ligand-induced gap between the transmembrane helices. In electrophysiological recordings fentanyl-induced currents returned after washout, suggesting fentanyl deposits in the lipid membrane. However, mutation of residues forming the potential MOPr transmembrane access site did not alter fentanyl's pharmacological profile in vitro. A high local concentration of fentanyl in the lipid membrane, possibly in combination with a novel lipophilic binding route, may explain the high potency and lower susceptibility of fentanyl to reversal by naloxone.

Exploring the kinetic selectivity of drugs targeting the ß1 -adrenoceptor.

In this study, we report the ß1 -adrenoceptor binding kinetics of several clinically relevant ß1/2 -adrenoceptor (ß1/2 AR) agonists and antagonists. [3 H]-DHA was used to label CHO-ß1 AR for binding studies. The kinetics of ligand binding was assessed using a competition association binding method. Ligand physicochemical properties, including logD7.4 and the immobilized artificial membrane partition coefficient (KIAM ), were assessed using column-based methods. Protein Data Bank (PDB) structures and hydrophobic and electrostatic surface maps were constructed in PyMOL. We demonstrate that the hydrophobic properties of a molecule directly affect its kinetic association rate (kon ) and affinity for the ß1 AR. In contrast to our findings at the ß2 -adrenoceptor, KIAM , reflecting both hydrophobic and electrostatic interactions of the drug with the charged surface of biological membranes, was no better predictor than simple hydrophobicity measurements such as clogP or logD7.4 , at predicting association rate. Bisoprolol proved kinetically selective for the ß1 AR subtype, dissociating 50 times slower and partly explaining its higher measured affinity for the ß1 AR. We speculate that the association of positively charged ligands at the ß1 AR is curtailed somewhat by its predominantly neutral/positive charged extracellular surface. Consequently, hydrophobic interactions in the ligand-binding pocket dominate the kinetics of ligand binding. In comparison at the ß2 AR, a combination of hydrophobicity and negative charge attracts basic, positively charged ligands to the receptor's surface promoting the kinetics of ligand binding. Additionally, we reveal the potential role kinetics plays in the on-target and off-target pharmacology of clinically used ß-blockers.

Inhibition of the Proliferation of Human Lung Fibroblasts by Prostacyclin Receptor Agonists is Linked to a Sustained cAMP Signal in the Nucleus.

Idiopathic pulmonary fibrosis is a chronic and progressive fibrotic lung disease, and current treatments are limited by their side effects. Proliferation of human lung fibroblasts in the pulmonary interstitial tissue is a hallmark of this disease and is driven by prolonged ERK signalling in the nucleus in response to growth factors such as platelet-derived growth factor (PDGF). Agents that increase cAMP have been suggested as alternative therapies, as this second messenger can inhibit the ERK cascade. We previously examined a panel of eight Gαs-cAMP-coupled G protein-coupled receptors (GPCRs) endogenously expressed in human lung fibroblasts. Although the cAMP response was important for the anti-fibrotic effects of GPCR agonists, the magnitude of the acute cAMP response was not predictive of anti-fibrotic efficacy. Here we examined the reason for this apparent disconnect by stimulating the Gαs-coupled prostacyclin receptor and measuring downstream signalling at a sub-cellular level. MRE-269 and treprostinil caused sustained cAMP signalling in the nucleus and complete inhibition of PDGF-induced nuclear ERK and fibroblast proliferation. In contrast, iloprost caused a transient increase in nuclear cAMP, there was no effect of iloprost on PDGF-induced ERK in the nucleus, and this agonist was much less effective at reversing PDGF-induced proliferation. This suggests that sustained elevation of cAMP in the nucleus is necessary for efficient inhibition of PDGF-induced nuclear ERK and fibroblast proliferation. This is an important first step towards understanding of the signalling events that drive GPCR inhibition of fibrosis.

Structure-Kinetic Profiling of Haloperidol Analogues at the Human Dopamine D2 Receptor.

Haloperidol is a typical antipsychotic drug (APD) associated with an increased risk of extrapyramidal side effects (EPSs) and hyperprolactinemia relative to atypical APDs such as clozapine. Both drugs are dopamine D2 receptor (D2R) antagonists, with contrasting kinetic profiles. Haloperidol displays fast association/slow dissociation at the D2R, whereas clozapine exhibits relatively slow association/fast dissociation. Recently, we have provided evidence that slow dissociation from the D2R predicts hyperprolactinemia, whereas fast association predicts EPS. Unfortunately, clozapine can cause severe side effects independent of its D2R action. Our results suggest an optimal kinetic profile for D2R antagonist APDs that avoids EPS. To begin exploring this hypothesis, we conducted a structure-kinetic relationship study of haloperidol and revealed that subtle structural modifications dramatically change binding kinetic rate constants, affording compounds with a clozapine-like kinetic profile. Thus, optimization of these kinetic parameters may allow development of novel APDs based on the haloperidol scaffold with improved side-effect profiles.

Investigating the Influence of Tracer Kinetics on Competition-Kinetic Association Binding Assays: Identifying the Optimal Conditions for Assessing the Kinetics of Low-Affinity Compounds.

An increased appreciation of the importance of optimizing drug-binding kinetics has lead to the development of various techniques for measuring the kinetics of unlabeled compounds. One approach is the competition-association kinetic binding method first described in the 1980s. The kinetic characteristics of the tracer employed greatly affects the reliability of estimated kinetic parameters, a barrier to successfully introducing these kinetic assays earlier in the drug discovery process. Using a modeling and Monte Carlo simulation approach, we identify the optimal tracer characteristics for determining the kinetics of the range of unlabeled ligands typically encountered during the different stages of a drug discovery program (i.e., rapidly dissociating, e.g., k off = 10 minute-1 low-affinity "hits" through to slowly dissociating e.g., k off = 0.01 minute-1 high-affinity "candidates"). For more rapidly dissociating ligands (e.g., k off = 10 minute-1), the key to obtaining accurate kinetic parameters was to employ a tracer with a relatively fast off-rate (e.g., k off = 1 minute-1) or, alternatively, to increase the tracer concentration. Reductions in assay start-time ≤1second and read frequency ≤5 seconds significantly improved the reliability of curve fitting. Timing constraints are largely dictated by the method of detection, its inherent sensitivity (e.g., TR-FRET versus radiometric detection), and the ability to inject samples online. Furthermore, we include data from TR-FRET experiments that validate this simulation approach, confirming its practical utility. These insights into the optimal experimental parameters for development of competition-association assays provide a framework for identifying and testing novel tracers necessary for profiling unlabeled competitors, particularly rapidly dissociating low-affinity competitors.

Pharmacological Characterization of a Novel 5-Hydroxybenzothiazolone-Derived ß 2-Adrenoceptor Agonist with Functional Selectivity for Anabolic Effects on Skeletal Muscle Resulting in a Wider Cardiovascular Safety Window in Preclinical Studies.

The anabolic effects of ß 2-adrenoceptor (ß 2-AR) agonists on skeletal muscle have been demonstrated in various species. However, the clinical use of ß 2-AR agonists for skeletal muscle wasting conditions has been limited by their undesired cardiovascular effects. Here, we describe the preclinical pharmacological profile of a novel 5-hydroxybenzothiazolone (5-HOB) derived ß 2-AR agonist in comparison with formoterol as a representative ß 2-AR agonist that have been well characterized. In vitro, 5-HOB has nanomolar affinity for the human ß 2-AR and selectivity over the ß 1-AR and ß 3-AR. 5-HOB also shows potent agonistic activity at the ß 2-AR in primary skeletal muscle myotubes and induces hypertrophy of skeletal muscle myotubes. Compared with formoterol, 5-HOB demonstrates comparable full-agonist activity on cAMP production in skeletal muscle cells and skeletal muscle tissue-derived membranes. In contrast, a greatly reduced intrinsic activity was determined in cardiomyocytes and cell membranes prepared from the rat heart. In addition, 5-HOB shows weak effects on chronotropy, inotropy, and vascular relaxation compared with formoterol. In vivo, 5-HOB significantly increases hind limb muscle weight in rats with attenuated effects on heart weight and ejection fraction, unlike formoterol. Furthermore, changes in cardiovascular parameters after bolus subcutaneous treatment in rats and rhesus monkeys are significantly lower with 5-HOB compared with formoterol. In conclusion, the pharmacological profile of 5-HOB indicates superior tissue selectivity compared with the conventional ß 2-AR agonist formoterol in preclinical studies and supports the notion that such tissue-selective agonists should be investigated for the safe treatment of muscle-wasting conditions without cardiovascular limiting effects.

Airway remodeling disease: primary human structural cells and phenotypic and pathway assays to identify targets with potential to prevent or reverse remodeling.

Airway remodeling is a characteristic of many chronic respiratory diseases and occurs when there are significant changes to the architecture of the small and large airways leading to progressive loss of lung function. Some common features include airway smooth muscle and goblet cell hyperplasia, basement membrane thickening and subepithelial fibrosis. To explore the mechanisms driving airway remodeling and identify novel targets to treat this aspect of respiratory disease, appropriate models must be used that will accurately predict the pathology of disease. Phenotypic assays can be used in primary human lung cells to measure changes in cell behavior that are associated with particular disease pathology. This is becoming increasingly popular when targeting chronic pathologies such as airway remodeling, where phenotypic assays are likely to model disease in vitro more accurately than traditional second messenger assays. Here we review the use of primary human lung structural cells in a range of disease-relevant chronic phenotypic assays, and how they may be used in target identification/validation and drug discovery.

Preassembled GPCR signaling complexes mediate distinct cellular responses to ultralow ligand concentrations.

G protein-coupled receptors (GPCRs) are the largest class of cell surface signaling proteins, participate in nearly all physiological processes, and are the targets of 30% of marketed drugs. Typically, nanomolar to micromolar concentrations of ligand are used to activate GPCRs in experimental systems. We detected GPCR responses to a wide range of ligand concentrations, from attomolar to millimolar, by measuring GPCR-stimulated production of cyclic adenosine monophosphate (cAMP) with high spatial and temporal resolution. Mathematical modeling showed that femtomolar concentrations of ligand activated, on average, 40% of the cells in a population provided that a cell was activated by one to two binding events. Furthermore, activation of the endogenous ß2-adrenergic receptor (ß2AR) and muscarinic acetylcholine M3 receptor (M3R) by femtomolar concentrations of ligand in cell lines and human cardiac fibroblasts caused sustained increases in nuclear translocation of extracellular signal-regulated kinase (ERK) and cytosolic protein kinase C (PKC) activity, respectively. These responses were spatially and temporally distinct from those that occurred in response to higher concentrations of ligand and resulted in a distinct cellular proteomic profile. This highly sensitive signaling depended on the GPCRs forming preassembled, higher-order signaling complexes at the plasma membrane. Recognizing that GPCRs respond to ultralow concentrations of neurotransmitters and hormones challenges established paradigms of drug action and provides a previously unappreciated aspect of GPCR activation that is quite distinct from that typically observed with higher ligand concentrations.

Single Step Determination of Unlabeled Compound Kinetics Using a Competition Association Binding Method Employing Time-Resolved FRET.

The competition association binding method allows the characterization of the kinetics of unlabeled compounds and the calculation of receptor-drug affinity (K d). The K d value is defined as the ratio of the dissociation constant (or k off) of the receptor-bound ligand to its association rate constant (or k on) for a system at equilibrium. Traditionally, competition association binding experiments have been carried out using radiometric detection methods with limited assay throughput. Here we describe a novel method for the determination of unlabeled compound kinetics using the technique of time-resolved fluorescence resonance energy transfer (TR-FRET) performed at physiological temperature and sodium ion concentration. Based on a traditional screening format (10-point curves), up to 28 compounds can be tested on a single 384-well plate by this method.

The inhibition of human lung fibroblast proliferation and differentiation by Gs-coupled receptors is not predicted by the magnitude of cAMP response.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive fibrotic lung disease for which there is no cure. Current therapeutics are only able to slow disease progression, therefore there is a need to explore alternative, novel treatment options. There is increasing evidence that the 3', 5' cyclic adenosine monophosphate (cAMP) pathway is an important modulator in the development of fibrosis, with increasing levels of cAMP able to inhibit cellular processes associated with IPF. In this study we investigate the expression of Gs-coupled G protein-coupled receptors (GPCR) on human lung fibroblasts (HLF), and explore which can increase cAMP levels, and are most efficacious at inhibiting proliferation and differentiation. METHODS: Using TaqMan arrays we determined that fibroblasts express a range of Gs-coupled GPCR. The function of selected agonists at expressed receptors was then tested in a cAMP assay, and for their ability to inhibit fibroblast proliferation and differentiation. RESULTS: Expression analysis of GPCR showed that the prostacyclin, prostaglandin E2 (PGE2) receptor 2 and 4, melanocortin-1, ß2 adrenoceptor, adenosine 2B, dopamine-1, and adenosine 2A receptors were expressed in HLF. Measuring cAMP accumulation in the presence of selected Gs-coupled receptor ligands as well as an adenylyl cyclase activator and inhibitors of phosphodiesterase showed formoterol, PGE2, treprostinil and forskolin elicited maximal cAMP responses. The agonists that fully inhibited both fibroblast proliferation and differentiation, BAY60-6583 and MRE-269, were partial agonists in the cAMP accumulation assay. CONCLUSIONS: In this study we identified a number of ligands that act at a range of GPCR that increase cAMP and inhibit fibroblast proliferation and differentiation, suggesting that they may provide novel targets to develop new IPF treatments. From these results it appears that although the cAMP response is important in driving the anti-fibrotic effects we have observed, the magnitude of the acute cAMP response is not a good predictor of the extent of the inhibitory effect. This highlights the importance of monitoring the kinetics and localisation of intracellular signals, as well as multiple pathways when profiling novel compounds, as population second messenger assays may not always predict phenotypic outcomes.

Micro-pharmacokinetics: Quantifying local drug concentration at live cell membranes.

Fundamental equations for determining pharmacological parameters, such as the binding affinity of a ligand for its target receptor, assume a homogeneous distribution of ligand, with concentrations in the immediate vicinity of the receptor being the same as those in the bulk aqueous phase. It is, however, known that drugs are able to interact directly with the plasma membrane, potentially increasing local ligand concentrations around the receptor. We have previously reported an influence of ligand-phospholipid interactions on ligand binding kinetics at the ß2-adrenoceptor, which resulted in distinct "micro-pharmacokinetic" ligand profiles. Here, we directly quantified the local concentration of BODIPY630/650-PEG8-S-propranolol (BY-propranolol), a fluorescent derivative of the classical ß-blocker propranolol, at various distances above membranes of single living cells using fluorescence correlation spectroscopy. We show for the first time a significantly increased ligand concentration immediately adjacent to the cell membrane compared to the bulk aqueous phase. We further show a clear role of both the cell membrane and the ß2-adrenoceptor in determining high local BY-propranolol concentrations at the cell surface. These data suggest that the true binding affinity of BY-propranolol for the ß2-adrenoceptor is likely far lower than previously reported and highlights the critical importance of understanding the "micro-pharmacokinetic" profiles of ligands for membrane-associated proteins.

Biased Agonism in Drug Discovery-Is It Too Soon to Choose a Path?

A single receptor can activate multiple signaling pathways that have distinct or even opposite effects on cell function. Biased agonists stabilize receptor conformations preferentially stimulating one of these pathways, and therefore allow a more targeted modulation of cell function and treatment of disease. Dedicated development of biased agonists has led to promising drug candidates in clinical development, such as the G protein-biased µ opioid receptor agonist oliceridine. However, leveraging the theoretical potential of biased agonism for drug discovery faces several challenges. Some of these challenges are technical, such as techniques for quantitative analysis of bias and development of suitable screening assays; others are more fundamental, such as the need to robustly identify in a very early phase which cell type harbors the cellular target of the drug candidate, which signaling pathway leads to the desired therapeutic effect, and how these pathways may be modulated in the disease to be treated. We conclude that biased agonism has potential mainly in the treatment of conditions with a well-understood pathophysiology; in contrast, it may increase effort and commercial risk under circumstances where the pathophysiology has been less well defined, as is the case with many highly innovative treatments.

Extrapyramidal side effects of antipsychotics are linked to their association kinetics at dopamine D2 receptors.

Atypical antipsychotic drugs (APDs) have been hypothesized to show reduced extrapyramidal side effects (EPS) due to their rapid dissociation from the dopamine D2 receptor. However, support for this hypothesis is limited to a relatively small number of observations made across several decades and under different experimental conditions. Here we show that association rates, but not dissociation rates, correlate with EPS. We measured the kinetic binding properties of a series of typical and atypical APDs in a novel time-resolved fluorescence resonance energy transfer assay, and correlated these properties with their EPS and prolactin-elevating liabilities at therapeutic doses. EPS are robustly predicted by a rebinding model that considers the microenvironment of postsynaptic D2 receptors and integrates association and dissociation rates to calculate the net rate of reversal of receptor blockade. Thus, optimizing binding kinetics at the D2 receptor may result in APDs with improved therapeutic profile.Atypical antipsychotics show reduced extrapyramidal side effects compared to first generation drugs. Here the authors use time-resolved FRET to measure binding kinetics, and show that side effects correlate with drug association rates to the D2 receptor, while dissociation rates correlate with prolactin elevation.

Correction to "Discovery of Fevipiprant (NVP-QAW039), a Potent and Selective DP2 Receptor Antagonist for Treatment of Asthma".

[This corrects the article DOI: 10.1021/acsmedchemlett.7b00157.].

Discovery of Fevipiprant (NVP-QAW039), a Potent and Selective DP2 Receptor Antagonist for Treatment of Asthma.

Further optimization of an initial DP2 receptor antagonist clinical candidate NVP-QAV680 led to the discovery of a follow-up molecule 2-(2-methyl-1-(4-(methylsulfonyl)-2-(trifluoromethyl)benzyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)acetic acid (compound 11, NVP-QAW039, fevipiprant), which exhibits improved potency on human eosinophils and Th2 cells, together with a longer receptor residence time, and is currently in clinical trials for severe asthma.

Uncoupling the Structure-Activity Relationships of ß2 Adrenergic Receptor Ligands from Membrane Binding.

Ligand binding to membrane proteins may be significantly influenced by the interaction of ligands with the membrane. In particular, the microscopic ligand concentration within the membrane surface solvation layer may exceed that in bulk solvent, resulting in overestimation of the intrinsic protein-ligand binding contribution to the apparent/measured affinity. Using published binding data for a set of small molecules with the ß2 adrenergic receptor, we demonstrate that deconvolution of membrane and protein binding contributions allows for improved structure-activity relationship analysis and structure-based drug design. Molecular dynamics simulations of ligand bound membrane protein complexes were used to validate binding poses, allowing analysis of key interactions and binding site solvation to develop structure-activity relationships of ß2 ligand binding. The resulting relationships are consistent with intrinsic binding affinity (corrected for membrane interaction). The successful structure-based design of ligands targeting membrane proteins may require an assessment of membrane affinity to uncouple protein binding from membrane interactions.

Fevipiprant (QAW039), a Slowly Dissociating CRTh2 Antagonist with the Potential for Improved Clinical Efficacy.

Here we describe the pharmacologic properties of a series of clinically relevant chemoattractant receptor-homologous molecules expressed on T-helper type 2 (CRTh2) receptor antagonists, including fevipiprant (NVP-QAW039 or QAW039), which is currently in development for the treatment of allergic diseases. [(3)H]-QAW039 displayed high affinity for the human CRTh2 receptor (1.14 ± 0.44 nM) expressed in Chinese hamster ovary cells, the binding being reversible and competitive with the native agonist prostaglandin D2(PGD2). The binding kinetics of QAW039 determined directly using [(3)H]-QAW039 revealed mean kinetic on (kon) and off (koff) values for QAW039 of 4.5 × 10(7)M(-1)min(-1)and 0.048 minute(-1), respectively. Importantly, thekoffof QAW039 (half-life = 14.4 minutes) was >7-fold slower than the slowest reference compound tested, AZD-1981. In functional studies, QAW039 behaved as an insurmountable antagonist of PGD2-stimulated [(35)S]-GTPγS activation, and its effects were not fully reversed by increasing concentrations of PGD2after an initial 15-minute incubation period. This behavior is consistent with its relatively slow dissociation from the human CRTh2 receptor. In contrast for the other ligands tested this time-dependent effect on maximal stimulation was fully reversed by the 15-minute time point, whereas QAW039's effects persisted for >180 minutes. All CRTh2 antagonists tested inhibited PGD2-stimulated human eosinophil shape change, but importantly QAW039 retained its potency in the whole-blood shape-change assay relative to the isolated shape change assay, potentially reflective of its relatively slower off rate from the CRTh2 receptor. QAW039 was also a potent inhibitor of PGD2-induced cytokine release in human Th2 cells. Slow CRTh2 antagonist dissociation could provide increased receptor coverage in the face of pathologic PGD2concentrations, which may be clinically relevant.