Steven J. Charlton

Recently, the first small-molecule positive allosteric modulators (PAMs) of the dopamine D₂ receptor (D₂R) were identified. The more potent PAM potentiated the effects of D₂R signaling in vitro and in an in vivo model predictive of anti-Parkinson's efficacy. We reveal, based on the results of our site-directed mutagenesis and molecular dynamics experiments, that this scaffold binds to a hitherto unexploited lipid-exposed extrahelical allosteric site in the D₂R that lies in a cleft toward the intracellular aspect of the D₂R defined by residues in transmembrane domains 1 and 7 and helix 8. By binding to this site, the PAM acts to potentiate the binding affinity of efficacious agonists, such as dopamine. Our simulations suggest that the PAM achieves this effect by stabilizing an active-like conformation of the receptor, similar to the G protein-bound state with TM5 and the tyrosine toggle switch playing the major role.

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.

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 β₁-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–G_s complexes and propose a zinc ion-binding hotspot at the TM5/TM6 intracellular interface of the receptor–G_s 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.

In this study, we report the β₁‐adrenoceptor binding kinetics of several clinically relevant β_1/2‐adrenoceptor (β_1/2AR) agonists and antagonists. [³H]‐DHA was used to label CHO‐β₁AR for binding studies. The kinetics of ligand binding was assessed using a competition association binding method. Ligand physicochemical properties, including logD_7.4 and the immobilized artificial membrane partition coefficient (K_IAM), 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 (k_on) and affinity for the β₁AR. In contrast to our findings at the β₂‐adrenoceptor, K_IAM, 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 logD_7.4, at predicting association rate. Bisoprolol proved kinetically selective for the β₁AR subtype, dissociating 50 times slower and partly explaining its higher measured affinity for the β₁AR. We speculate that the association of positively charged ligands at the β₁AR 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 β₂AR, 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 β‐blockers.

Certain atypical antipsychotic drugs (APDs) used in the treatment of schizophrenia have been hypothesized to show reduced extrapyramidal side effects (EPS), due to their ability to promote nigrostriatal dopamine release through 5-HT _2A receptor (5-HT _2A R) blockade. The strength of this hypothesis is currently limited to a consideration of the relative receptor affinities of APDs for the 5-HT _2A R and dopamine D ₂ receptor (D ₂ R). Here we measure the 5-HT _2A R kinetic binding properties of a series of typical and atypical APDs in a novel time-resolved fluorescence resonance energy transfer assay and correlate these properties with their observed EPS at therapeutic doses. For compounds with negligible affinity for 5-HT _2A R, EPS is robustly predicted by a D ₂ R specific rebinding model that integrates D ₂ R association and dissociation rates to calculate the net rate of reversal of receptor blockade ( k _r ). However, we show that for compounds with significant affinity for the 5-HT _2A R, such as sertindole, higher relative 5-HT _2A occupancy over time is an indicator for a reduced propensity to cause EPS. This study suggests that there is room for the development of novel kinetically optimised antipsychotic agents that modulate both serotonergic and dopamine function in a manner beneficial in the treatment of this chronic and debilitating disease.

Haloperidol is a typical antipsychotic drug (APD) associated with an increased risk of extrapyramidal side-effects (EPS) 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 reveal that subtle structural modifications dramatically change binding kinetic rate constants, affording compounds with a clozapine-like kinetic profile. Thus, optimisation of these kinetic parameters may allow development of novel APDs based on the haloperidol scaffold with improved side-effect profiles.

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.

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.