A Quantitative High‐Throughput Screen to Identify Small Molecules for the Induction of Tristetraprolin Expression

Abstract

The rapid production of cytokines and chemokines from circulating leukocytes is one of the body's most powerful responses to acute inflammation. More persistent production of these pro‐inflammatory mediators can result in the development of common chronic diseases, such as rheumatoid arthritis, Crohn's disease, and many others. One of the most prominent pro‐inflammatory cytokines is tumor necrosis factor alpha (TNF), which is released from macrophages and other inflammatory cells as part of the innate immune response. Anti‐TNF “biologic” therapies have been very effective in the treatment of many of these diseases, but these recombinant protein‐based therapies are expensive, must be injected, and can cause side effects. There is a strong need for alternative ways to inhibit TNF expression, preferably using orally available small molecular drugs.Tristetraprolin (TTP) is an inducible zinc finger protein that binds to AU‐rich elements in 3′‐untranslated regions of its target mRNAs and promotes the decay of those mRNAs, many of which encode pro‐inflammatory cytokines like TNF. TTP thereby acts as an intracellular inhibitor of TNF production, and mice in which TTP is overexpressed exhibit resistance to several models of immune/inflammatory disease. We hope to achieve the same goal of increasing “whole body” expression of TTP by modifying its expression with small molecules, representing potential anti‐inflammatory drugs.We, therefore, conducted a quantitative high‐throughput screen for small molecules that stimulated TTP biosynthesis in intact macrophages, using a cell‐based assay involving cells expressing human TTP fused to green fluorescence protein (GFP) (hTTP‐GFP RAW264.7). Following optimization of the fluorescence assay in a 1536‐well format by using lipopolysaccharide as a positive control stimulator of TTP biosynthesis, we screened approximately 314,000 compounds. We identified 30 active and 21 inconclusive compounds that promoted TTP expression while not stimulating TNF secretion. Secondary validation assays, using western blotting and fluorescence‐activated cell sorting, yielded 8 compounds that could reliably stimulate TTP expression in cultured RAW264.7 macrophages at concentrations of 10−6 M or less. Some of these were FDA approved drugs with known microtubule or topoisomerase inhibitor activity, whereas two compounds had no known functions. Further analyses showed no significant impact of these 8 substances on macrophage survival, although some substances led to the expected cell cycle changes.These results suggest that the hTTP‐GFP RAW264.7 cell line can be used productively for cell‐based screens for TTP‐inducing compounds. We identified anti‐microtubule drugs as novel regulators of TTP biosynthesis, and are currently exploring the mechanism of this effect. Compounds identified in this and future screens may represent lead compounds for the future development of a novel class of anti‐inflammatory agents.Support or Funding InformationThis research was supported by the Intramural Research Program of the National Institute of Environmental Health Sciences, NIH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.