The neutrophil dynamic mass redistribution assay as a medium throughput primary cell screening assay.

In a typical GPCR drug discovery campaign, an in vitro primary functional screening assay is often established in a recombinant system over-expressing 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 which has been developed in a format suitable to support medium throughput drug screening in primary human neutrophils. Neutrophils are known to express both CXCR1 and CXCR2 chemokine receptors that are thought to play significant roles in various inflammatory disorders and cancer. Using multiple relevant chemokine ligands and a range of selective and non-selective 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 utilized 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 towards 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 co-expression of related receptors confounding results.