The Neutrophil Dynamic Mass Redistribution Assay as a Medium throughput Primary Cell Screening Assay.

In a typical G protein coupled receptor drug discovery campaign, an in vitro primary functional screening assay is often established in a recombinant system overexpressing the target of interest, which offers advantages with respect to overall throughput and robustness of compound testing. Subsequently, compounds are then progressed into more physiologically relevant but lower throughput ex vivo primary cell assays and finally in vivo studies. Here we describe a dynamic mass redistribution (DMR) assay that has been developed in a format suitable to support medium throughput drug screening in primary human neutrophils. Neutrophils are known to express both CXC chemokine receptor (CXCR) 1 and CXCR2 that are thought to play significant roles in various inflammatory disorders and cancer. Using multiple relevant chemokine ligands and a range of selective and nonselective small and large molecule antagonists that block CXCR1 and CXCR2 responses, we demonstrate distinct pharmacological profiles in neutrophil DMR from those observed in recombinant assays but predictive of activity in neutrophil chemotaxis and CD11b upregulation, a validated target engagement marker previously used in clinical studies of CXCR2 antagonists. The primary human neutrophil DMR cell system is highly reproducible, robust, and less prone to donor variability observed in CD11b and chemotaxis assays and thus provides a unique, more physiologically relevant, and higher throughput assay to support drug discovery and translation to early clinical trials. SIGNIFICANCE STATEMENT: Neutrophil dynamic mass redistribution assays provide a higher throughput screening assay to profile compounds in primary cells earlier in the screening cascade enabling a higher level of confidence in progressing the development of compounds toward the clinic. This is particularly important for chemokine receptors where redundancy contributes to a lack of correlation between recombinant screening assays and primary cells, with the coexpression of related receptors confounding results.

Characterisation of inverse agonism of the orphan-G protein-coupled receptor GPR52 by cannabinoid ligands Cannabidiol and O-1918.

The identification of cannabinoid ligands Cannabidiol and O-1918 as inverse agonists of the orphan receptor GPR52 is reported. Detailed characterisation of GPR52 pharmacology and modelling of the proposed receptor interaction is described. The identification of a novel and further CNS pharmacology for the polypharmacological agent and marketed drug Cannabidiol is noteworthy.

Unravelling intrinsic efficacy and ligand bias at G protein coupled receptors: A practical guide to assessing functional data.

Stephenson's empirical definition of an agonist, as a ligand with binding affinity and intrinsic efficacy (the ability to activate the receptor once bound), underpins classical receptor pharmacology. Quantifying intrinsic efficacy using functional concentration response relationships has always presented an experimental challenge. The requirement for realistic determination of efficacy is emphasised by recent developments in our understanding of G protein coupled receptor (GPCR) agonists, with recognition that some ligands stabilise different active conformations of the receptor, leading to pathway-selective, or biased agonism. Biased ligands have potential as therapeutics with improved selectivity and clinical efficacy, but there are also pitfalls to the identification of pathway selective effects. Here we explore the basics of concentration response curve analysis, beginning with the need to distinguish ligand bias from other influences of the functional system under study. We consider the different approaches that have been used to quantify and compare biased ligands, many of which are based on the Black and Leff operational model of agonism. Some of the practical issues that accompany these analyses are highlighted, with opportunities to improve estimates in future, particularly in the separation of true agonist intrinsic efficacy from the contributions of system dependent coupling efficiency. Such methods are by their nature practical approaches, and all rely on Stephenson's separation of affinity and efficacy parameters, which are interdependent at the mechanistic level. Nevertheless, operational analysis methods can be justified by mechanistic models of GPCR activation, and if used wisely are key elements to biased ligand identification.