Discussion

Formation of neutrophil extracellular traps (NETs) has been implicated to be relevant for several diseases like autoimmune disorders (e.g. systemic lupus erythematodes), infections/sepsis and thrombosis. NET formation has been described as a non-reversible process of subsequently appearing distinct cellular morphological features which include the loss of the characteristic nucleus lubuli and the dissolution of intracellular membranes. Finally, breakup of the nuclear and cellular and membrane leads to formation of typical NETs. The signaling mechanisms that control NETs formation are poorly understood since neutrophils are not amenable to studies with conventional genetic techniques. To identify modulators of NET formation we used a chemical genomics approach and performed a high throughput screening. We employed a previously described assay principle to identify novel small molecular weight modulators of NET formation. High content analysis was used to determine the different neutrophilic phenotypes upon stimulation with phorbol 12-myristate 13-acetate. A Screen of primary human neutrophils from healthy donors of ~180.000 compounds in the 1536 well format resulted in the identification of several hit classes. The assay quality parameters within the screening campaign were good especially for an assay using primary cells. Upon hit verification the results were clustered chemically and biologically into different groups. Selected compounds have been further characterized to unravel their mode of action. In parallel, hit optimization will be pursued to generate probes for proof of concept experiments in animals. In summary, the results presented here show that screening of a very difficult phenotypic phenotype using primary cells is doable and produces meaningful results which potentially leading to an innovative drug discovery program.