Tpl-2 small molecule project

Tpl-2 is a serine/threonine kinase that has been studied extensively in monocytes. Tpl-2 is believed to phosphorylate MEK1/2, which is upstream of ERK1/2, and regulates inflammatory gene expression in response to TLR and IL-1b receptor signaling. In the course of performing proof-of-concept studies using a small molecule inhibitor (SMI) of Tpl-2, we were surprised to see the inhibitor affect cytokine production in human neutrophils. Unlike human monocytes, which respond at least some degree to both Tpl-2 and MEK inhibitors, neutrophils showed a disconnect between Tpl-2 and MEK. A panel of genes in this cell type can be fully blocked by a Tpl-2 SMI, and yet show no response to a MEK SMI, suggesting Tpl-2 mediates its effect through substrates other than MEK. The goal of this study is to compare the gene expression profiles of LPS-treated human neutrophils treated with vehicle, Tpl-2 SMI, or MEK SMI, in order to shed light on the MEK-independent arm of the Tpl-2 response. Purified neutrophils from 5 healthy donors were split into 4 groups: Unstimulated neutrophils, vehicle treated for 6.5 hrs; Neutrophils pretreated 30 min with vehicle, then stimulated 6 hrs with 10 ng/ml LPS; Neutrophils pretreated 30 min with G-432, then stimulated 6 hrs with 10 ng/ml LPS; and Neutrophils pretreated 30 min with G-573, then stimulated 6 hrs with 10 ng/ml LPS. This yields a total of 20 samples (5 donors x 4 conditions).