David A. Sykes

Exploring the mechanism of agonist efficacy: a relationship between efficacy and agonist dissociation rate at the muscarinic M3 receptor.

Although there are several empirical approaches that enable the comparison of relative agonist efficacy, the molecular basis that underlies differences in the ability of G protein-coupled receptor agonists to elicit a response is still largely unexplained. Several models have been described that incorporate the kinetics of receptor-mediated initiation of the G protein cycle, but these have not directly addressed the influence of agonist-binding kinetics. To test this, we investigated the relationship between the efficacy of seven M3 muscarinic receptor agonists and their rate of dissociation (koff) from the M3 receptor. The association and dissociation rate constants of the agonists were determined using a l-[N-methyl]-[3H]scopolamine methyl chloride competition binding assay in the presence of GTP. The agonists displayed a range of association and dissociation rates. Relative agonist efficacy was measured at two points after M3 receptor activation: the stimulation of guanosine 5′-O-(3-[35S]thio)triphosphate binding to Gα subunits, and the subsequent increase in intracellular calcium levels. These experiments revealed a range of intrinsic efficacy, from the low-efficacy pilocarpine and oxotremorine to high-efficacy acetylcholine. There was no relationship between agonist efficacy and the equilibrium binding affinity of each agonist (Kd). When efficacy was compared with the dissociation rate constant, however, the two were highly correlated, suggesting a relationship between the duration of agonist binding at the receptor and the intrinsic efficacy. These data suggest that kinetic models incorporating the mean lifetime of specific complexes will be required to fully explain the nature of agonist efficacy.

The Influence of Receptor Kinetics on the Onset and Duration of Action and the Therapeutic Index of NVA237 and Tiotropium

Studies under nonphysiological conditions suggest that long receptor residency time is responsible for the 24-h duration of action of the long-acting muscarinic antagonist (LAMA) tiotropium. Our aim was to determine how clinically relevant dissociation rates under more physiological conditions influence the differences in onset of action between tiotropium and 3-[(cyclopentylhydroxyphenylacetyl oxy]-1,1-dimethyl-pyrrolidinium bromide (NVA237), a once-daily dry-powder formulation of the LAMA glycopyrronium bromide in development for chronic obstructive pulmonary disease. In addition, we have investigated kinetic selectivity at each of the muscarinic receptor subtypes to determine whether the improved cardiovascular therapeutic index obtained with NVA237 in animal models is attributable to differences in kinetic rate constants. The binding of radioligand [3H]N-methyl-scopolamine was measured in the presence/absence of several concentrations of unlabeled competitors, and data were analyzed using a competition kinetic model to provide on/off rates for the competitor. We found shorter dissociation half-lives for NVA237 and tiotropium under physiological (11.4 and 46.2 min, respectively) versus nonphysiological conditions (173 and 462 min, respectively). NVA237 had a more rapid onset of action (3–4.8 times) versus tiotropium, determined in an vitro calcium and rat tracheal strip assay. Simulations suggested that the more rapid onset of NVA237 action could be explained by differences in kinetic parameters. NVA237 had greater equilibrium binding and kinetic selectivity for muscarinic type 3 (M3) versus muscarinic type 2 (M2) receptors, with a faster off rate from M2 versus M3 receptors than tiotropium, potentially affording it a more favorable therapeutic index. This study suggests that the 24-h duration of action of NVA237 and tiotropium is not solely the result of their slow dissociation from the M3 receptor and highlights the importance of conducting in vitro experiments in conditions reflecting those in vivo.

The Identification of Indacaterol as an Ultralong-Acting Inhaled β2-Adrenoceptor Agonist

Following a lipophilicity-based hypothesis, an 8-hydroxyquinolinone 2-aminoindan derived series of beta(2)-adrenoceptor agonists have been prepared and evaluated for their potential as inhaled ultralong-acting bronchodilators. Determination of their activities at the human beta(2)-adrenoceptor receptor showed symmetrical substitution of the 2-aminoindan moiety at the 5- and 6-positions delivered the targeted intermediate potency and intrinsic-efficacy profiles relative to a series of clinical reference beta(2)-adrenoceptor agonists. Further assessment with an in vitro superfused electrically stimulated guinea-pig tracheal-strip assay established the onset and duration of action time courses, which could be rationalized by considering the lipophilicity, potency, and intrinsic efficacy of the compounds. From these studies the 5,6-diethylindan analogue indacaterol 1c was shown to possess a unique profile of combining a rapid onset of action with a long duration of action. Further in vivo profiling of 1c supported the long duration of action and a wide therapeutic index following administration to the lung, which led to the compound being selected as a development candidate.

Slow receptor dissociation is not a key factor in the duration of action of inhaled long‐acting β2‐adrenoceptor agonists

BACKGROUND AND PURPOSE β2‐Adrenoceptor agonists are important bronchodilators used for the treatment of chronic obstructive pulmonary disease and asthma. Clinical data on β2‐adrenoceptor agonists show a range of onset and duration of action. We have investigated whether the receptor binding kinetics of β2‐adrenoceptor agonists can explain their observed onset of action and duration of effect in the clinic.

Observed Drug-Receptor Association Rates Are Governed by Membrane Affinity: The Importance of Establishing “Micro-Pharmacokinetic/Pharmacodynamic Relationships” at the β2-Adrenoceptor

Current pharmacological models for determining affinity and kinetics of drugs for membrane receptors assume the interacting molecules are homogeneously distributed in the bulk aqueous phase. The phospholipid membrane can, however, provide a second compartment into which drugs can partition, particularly lipophilic/basic compounds. In this study we measured the phospholipid affinity and receptor binding kinetics of several clinically relevant β2-adrenoceptor agonists and antagonists and demonstrated that the degree of phospholipid interaction directly affects the observed kinetic association rate (kon) and dissociation constant (Kd), but not the dissociation rate (koff) from the target, by concentrating drug in the local environment around the receptor. When the local drug concentration was accounted for, the kon was comparable across the cohort and the corrected Kd was directly related to the koff. In conclusion, we propose a new approach to determining the pharmacology of drugs for membrane targets that accounts for differences in local drug concentration brought about by direct affinity for phospholipids, establishing “micro-pharmacokinetic/pharmacodynamic relationships” for drugs.

Observed drug-receptor association rates are governed by membrane affinity: The importance of establishing "micro PK/PD relationships" at the β2-adrenoceptor

Current pharmacological models for determining affinity and kinetics of drugs for membrane receptors assume the interacting molecules are homogeneously distributed in the bulk aqueous phase. The phospholipid membrane can, however, provide a second compartment into which drugs can partition, particularly lipophilic/basic compounds. In this study we measured the phospholipid affinity and receptor binding kinetics of several clinically relevant β2-adrenoceptor agonists and antagonists and demonstrated that the degree of phospholipid interaction directly affects the observed kinetic association rate (kon) and dissociation constant (Kd), but not the dissociation rate (koff) from the target, by concentrating drug in the local environment around the receptor. When the local drug concentration was accounted for, the kon was comparable across the cohort and the corrected Kd was directly related to the koff. In conclusion, we propose a new approach to determining the pharmacology of drugs for membrane targets that accounts for differences in local drug concentration brought about by direct affinity for phospholipids, establishing “micro-pharmacokinetic/pharmacodynamic relationships” for drugs.

Investigating the molecular mechanisms through which FTY720-P causes persistent S1P1 receptor internalization.

The molecular mechanism underlying the clinical efficacy of FTY720‐P is thought to involve persistent internalization and enhanced degradation of the S1P1 receptor subtype (S1P1R). We have investigated whether receptor binding kinetics and β‐arrestin recruitment could play a role in the persistent internalization of the S1P1R by FTY720‐P.

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. [3H]-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 [3H]-QAW039 revealed mean kinetic on (kon) and off (koff) values for QAW039 of 4.5 × 107 M-1min−1 and 0.048 minute−1, respectively. Importantly, the koff of 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 [35S]-GTPγS activation, and its effects were not fully reversed by increasing concentrations of PGD2 after 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 PGD2 concentrations, which may be clinically relevant.

Extrapyramidal side effects of antipsychotics are linked to their association kinetics at dopamine D 2 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.

Ligand Specific Efficiency (LSE) Index for PET Tracer Optimization

Ligand efficiency indices are widely used to guide chemical optimization in drug discovery, due to their predictive value in the early steps of optimization. At later stages, however, as more complex properties become critical for success, indices relying on calculated, rather than experimental, parameters become less informative. This problem is particularly acute when developing positron emission tomography (PET) imaging agents, for which nonspecific binding (NSB) to membranes and non‐target proteins is a frequent cause of failure. NSB cannot be predicted using in silico parameters. To address this gap, we explored the use of the experimentally determined chromatographic hydrophobicity index on immobilized artificial membranes, CHI(IAM), to guide the optimization of NSB. The ligand specific efficiency (LSE) index was defined as the ratio between affinity (pIC50 or pKd) and the logarithmic value of CHI(IAM). It allows for quantification of binding affinity to the target of interest, relative to NSB. Its use was illustrated by the optimization of PET tracer candidates for the prostacyclin receptor.