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  • A High Content Screen in Macrophages Identifies Small Molecule Modulators of STING-IRF3 and NFkB Signaling.

A High Content Screen in Macrophages Identifies Small Molecule Modulators of STING-IRF3 and NFkB Signaling.

ACS chemical biology (2018-03-20)
Peter D Koch, Howard R Miller, Gary Yu, John A Tallarico, Peter K Sorger, Yuan Wang, Yan Feng, Jason R Thomas, Nathan T Ross, Timothy Mitchison
ABSTRACT

We screened a library of bioactive small molecules for activators and inhibitors of innate immune signaling through IRF3 and NFkB pathways with the goals of advancing pathway understanding and discovering probes for immunology research. We used high content screening to measure the translocation from the cytoplasm to nucleus of IRF3 and NFkB in primary human macrophages; these transcription factors play a critical role in the activation of STING and other pro-inflammatory pathways. Our pathway activator screen yielded a diverse set of hits that promoted nuclear translocation of IRF3 and/or NFkB, but the majority of these compounds did not cause activation of downstream pathways. Screening for antagonists of the STING pathway yielded multiple kinase inhibitors, some of which inhibit kinases not previously known to regulate the activity of this pathway. Structure-activity relationships (SARs) and subsequent chemical proteomics experiments suggested that MAPKAPK5 (PRAK) is a kinase that regulates IRF3 translocation in human macrophages. Our work establishes a high content screening approach for measuring pro-inflammatory pathways in human macrophages and identifies novel ways to inhibit such pathways; among the targets of the screen are several molecules that may merit further development as anti-inflammatory drugs.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal Anti-α-Tubulin antibody produced in mouse, clone DM1A, ascites fluid
Sigma-Aldrich
Human IP-10 / CXCL10 ELISA Kit, for serum, plasma, cell culture supernatant and urine