Biased agonism describes the ability of ligands to stabilize different conformations of a GPCR linked to distinct functional outcomes and offers the prospect of designing pathway-specific drugs that avoid on-target side effects. This mechanism is usually inferred from pharmacological data with the assumption that the confounding influences of observational (that is, assay dependent) and system (that is, cell background dependent) bias are excluded by experimental design and analysis. Here we reveal that ‘kinetic context', as determined by ligand-binding kinetics and the temporal pattern of receptor-signalling processes, can have a profound influence on the apparent bias of a series of agonists for the dopamine D2 receptor and can even lead to reversals in the direction of bias. We propose that kinetic context must be acknowledged in the design and interpretation of studies of biased agonism.

The phenazine pigments pyocyanin and 1-hydroxyphenazine were resolved by high-pressure liquid chromatography from the sputum sol phase from 9 of 13 patients with cystic fibrosis or bronchiectasis colonized by Pseudomonas aeruginosa. The concentrations measured were each sufficient to inhibit ciliary beating in vitro and contributed a significant proportion of sol phase toxicity for respiratory epithelium.Pseudomonas aeruginosa colonizes the lungs of patients with cystic fibrosis (CF) and other forms of severe bronchiectasis. We have recently demonstrated that pyocyanin and 1-hydroxyphenazine, phenazine redox pigments generated by P. aeruginosa, disrupt human ciliary beating in vitro (15). Pyocyanin also inhibits epidermal cell growth (4) and lymphocyte proliferation (7), has antibiotic properties against other microorganisms (9), and influences the acquisition of iron by pseudomonads (3). 1-Hydroxyphenazine inhibits mammalian cell respiration (1).Sputum was collected from 12 patients who were colonized by P. aeruginosa; 8 had bronchiectasis and 4 had CF. Pulmonary secretions, from which P. aeruginosa was cultured, were aspirated directly from the large airways of a CF lung removed at transplantation. Sputum was also collected from five patients who had bronchiectasis and who had never been colonized by P. aeruginosa. Each sputum sample was centrifuged (50,000 x g for 90 min at 4°C), the watery sol phase was retained, and the gel phase was discarded. Each sol phase sample (1 ml) was loaded in 5% acetic acid on a C18 Sep Pak column (Waters Associates, Inc.) and eluted with 40% propan-2-ol in aqueous acetic acid. The eluant was fractionated by high-pressure liquid chromatography (HPLC) at 2 ml/min on a p,Bondapak Cl8 column (30 by 0.8 cm) with a 20-min linear gradient from 0 to 40% propan-2-ol in 5% aqueous acetic acid, with monitoring at 280 and 254 nm in UV light (14). Pyocyanin and 1-hydroxyphenazine were identified by their HPLC-UV profiles (14). All pyocyanin concentrations in each sol sample and 1hydroxyphenazine concentrations in sol samples 1, 2, and 7 were calculated after extraction and full UV analysis; 1hydroxyphenazine concentrations in other sol samples were calculated from the HPLC-UV peak height.Human nasal ciliated epithelium was obtained from the inferior turbinate of normal volunteers (8). The sol phase pH was variable (5.5 to 9.5) and was adjusted to 7.4 to ensure that changes in ciliary beat frequency (CBF) were not pH related. A sol phase sample was added to ciliated epithelium, a sealed microscope cover slip slide preparation was constructed, and controls were suspended in phosphate-buffered saline (PBS). Ten epithelium strips were identified, and their positions were marked. CBF was measured in each * Corresponding author. epithelial strip by a photometric technique at 1-h intervals for 4 h (8, 15), and a mean was calculated. Any change in beating pattern, ciliary stasis (previously beating cilia subsequently observed to be static), or change in epithelium structure was noted. Results are sho...

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.

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 b 2 -adrenoceptor agonists and antagonists and demonstrated that the degree of phospholipid interaction directly affects the observed kinetic association rate (k on ) and dissociation constant (K d ), but not the dissociation rate (k off ) from the target, by concentrating drug in the local environment around the receptor. When the local drug concentration was accounted for, the k on was comparable across the cohort and the corrected K d was directly related to the k off . 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.

Pseudomonas aeruginosa culture filtrates varied in their ability to slow human ciliary beat frequency (7-71%). This activity did not correlate with known virulence factors. However, a close correlation (r = 0.97) existed between ciliary slowing and pigment content. In a prolonged culture, the increase in activity correlated (r = 0.94) with pigment accumulation. Gel filtration of lyophilized filtrate yielded a single peak of activity corresponding to the pigment fraction. Pyocyanin extracted from an active strain, and 1-hydroxyphenazine were purified by high performance liquid chromatography, and characterized by ultraviolet absorbance spectra and mass spectrometry. Both slowed cilia in a dose-dependent manner, and were synthesized and shown to be indistinguishable from the biological compounds. Pyocyanin caused gradual onset of slowing and ultimate widespread ciliostasis with epithelial disruption. 1-hydroxyphenazine caused rapid onset of ciliary slowing associated with dyskinesia and ciliostasis. Pyocyanin assayed within filtrates accounted for a significant proportion of the bioactivity present.

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 agonistbinding kinetics. To test this, we investigated the relationship between the efficacy of seven M 3 muscarinic receptor agonists and their rate of dissociation (k off ) from the M 3 receptor. The association and dissociation rate constants of the agonists were determined using a l- [N-methyl] -[3 H]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 M 3 receptor activation: the stimulation of guanosine 5Ј-O-(3-[35 S]thio)triphosphate binding to G␣ subunits, and the subsequent increase in intracellular calcium levels. These experiments revealed a range of intrinsic efficacy, from the lowefficacy pilocarpine and oxotremorine to high-efficacy acetylcholine. There was no relationship between agonist efficacy and the equilibrium binding affinity of each agonist (K d ). 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.Efficacy is defined as the ability of a ligand to elicit a response upon binding to a receptor (Stephenson, 1956) and is arguably the most important parameter for optimization in novel agonist drugs. To date, efficacy has largely been treated as an empirical term, and several approaches comparing equilibrium binding to functional potency have been described for its measurement (Black and Leff, 1983;Ehlert, 1985). Although they provide a pragmatic approach to ranking ligands, these methods make no attempt to explain the molecular mechanism behind efficacy. One of the first attempts to provide a mechanistic explanation for agonist efficacy was by Paton (1961), termed "rate theory." This model considers that excitation by a stimulant drug is proportional to the rate of drug-receptor combination rather than to the proportion of receptors occupied by the drug. In this case, once the receptor has been activated and the signal transduced, it must be reset by dissociation of the agonist before another activation event can be initiated by binding of another agonist molecule. Thus, a high-efficacy ligand would dissociate rapidly from the receptor, allowing another agonist to bind rapidly, whereas a low-efficacy agonist would dissociated more slowly from the receptor, acting effectively as a competitive ant...

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 [ 3 H]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 (M 3 ) versus muscarinic type 2 (M 2 ) receptors, with a faster off rate from M 2 versus M 3 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 M 3 receptor and highlights the importance of conducting in vitro experiments in conditions reflecting those in vivo.

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