A selective small molecule NF-kB inhibitor from a high-throughput cell based assay for “AP-1 hits”

A142

Environmentally induced AP-1 dependent transcription can contribute not only to normal physiological processes but also to tumorigenesis and tumor progression. Elevated AP-1 activation can be repressed by a dominant-negative c-Jun, TAM67, inhibiting tumorigenesis and tumor progression without inhibiting cell proliferation or cell survival. We sought small molecules that might recapitulate the specificity of TAM67. A high-throughput cell based assay using fluorescence resonance energy transfer (FRET) was carried out to identify compounds that inhibit transcription factor AP-1. The optimized High Throughput Screening (HTS) was validated, repeated and coupled with an XTT assay to exclude inhibitors of cell proliferation. Screening with synthetic compound libraries and the NCI natural product repository including pure natural products and natural product extracts yielded several compounds (Ruocco el al J Biomol Screen 2007). The active compounds were tested with AP-1 and NF-kB (nuclear factor kappa B) promoted luciferase reporters to confirm and quantitate inhibitory activity. NF-kB is an inducible transcription factor also targeted by TAM67 that regulates genes involved in inflammation and immunity. An SRE (Serum response element) reporter was also used as a specificity control and indicator of cell proliferation. Interestingly, one of the active compounds (NSC676914, MW = 301) has shown substantially more potent inhibitory activity toward NF-kB than AP-1 measured by IC50. Additional analysis was performed to identify the mechanism by which this compound inhibits NF-kB activity. TPA exposure increased the phosphorylation of IkBa and stimulated translocation into the nucleus of NF-kB p65 and p50 which were sequestered in the cytoplasm by IkBa. Pretreatment with active compound blocked the phosphorylation of IkBa, translocation of p65/50 to the nucleus and the processing of p52 from p100. Moreover, the phosphorylated IkBa expression was diminished by this compound even at 0.8uM. Importantly, phosphorylated IKKa/b, a major regulator of IkBa and p105 processing, was reduced by the compound without inhibition of JNK. The inhibitory activity of the compound will be tested in vitro to determine IC50 and target specificity against several kinases including IKKa and IKKb. Furthermore, a matrigel invasion assay with ER-positive (MCF-7 and T47D) and ER-negative (MDA-MB-231) breast cancer cell lines revealed that induced invasion was completely repressed by the compound. The results suggest that compound NSC676914 could be a novel inhibitor having potential therapeutic activity to target NF-kB for cancer treatment or prevention.