Identification of known drugs that act as inhibitors of NF-kappaB signaling and their mechanism of action

Abstract

Nuclear factor-kappa B (NF-kappaB) is a transcription factor that plays a critical role across many cellular processes including embryonic and neuronal development, cell proliferation, apoptosis, and immune responses to infection and inflammation. Dysregulation of NF-kappaB signaling is associated with inflammatory diseases and certain cancers. Constitutive activation of NF-kappaB signaling has been found in some types of tumors including breast, colon, prostate, skin and lymphoid, hence therapeutic blockade of NF-kappaB signaling in cancer cells provides an attractive strategy for the development of anticancer drugs. To identify small molecule inhibitors of NF-kappaB signaling, we screened approximately 2800 clinically approved drugs and bioactive compounds from the NIH Chemical Genomics Center Pharmaceutical Collection (NPC) in a NF-kappaB mediated beta-lactamase reporter gene assay. Each compound was tested at fifteen different concentrations in a quantitative high throughput screening format. We identified nineteen drugs that inhibited NF-kappaB signaling, with potencies as low as 20 nM. Many of these drugs, including emetine, fluorosalan, sunitinib malate, bithionol, narasin, tribromsalan, and lestaurtinib, inhibited NF-kappaB signaling via inhibition of IkappaBalpha phosphorylation. Others, such as ectinascidin 743, chromomycin A3 and bortezomib utilized other mechanisms. Furthermore, many of these drugs induced caspase 3/7 activity and had an inhibitory effect on cervical cancer cell growth. Our results indicate that many currently approved pharmaceuticals have previously unappreciated effects on NF-kappaB signaling, which may contribute to anticancer therapeutic effects. Comprehensive profiling of approved drugs provides insight into their molecular mechanisms, thus providing a basis for drug repurposing.

Fig. 1

Principle of the β-lactamase reporter gene assay. Binding of TNF-α to its receptor activates the IκB kinase (IKK), which phosphorylates IκB. The phosphorylated IκB dissociates from the NF-κB dimmer through proteasome degradation. NF-κB translocates across cytoplasmic/nuclear boundary into the nucleus, and then enhances the transcription of the β-lactamase reporter gene by binding to the κB binding site. β-Lactamase expression cleaves the fluorescent substrate (CCF4) containing the two fluorophores, coumarin and fluorescein. This reaction disrupts the fluorescence resonance energy transfer (FRET) from coumarin to flourescein, which produces a blue fluorescent signal at 460 nm. In the absence of β-Lactamase expression, the substrate molecule remains intact and the excitation of the coumarin enables FRET to the fluorescein moiety, producing a green signal with an emission peak at 530 nm.

Fig 2

(A) TNFα stimulated β-lactamase activity in NF-kB bla ME180 cells; (B) MG132 concentration-dependent inhibition of β-lactamase activity in the presence of 1ng/ml TNFα in NF-kB bla ME180 cells.

Fig 3

Inhibitory effect of compounds on TNFα-induced IκBα phosphorylation. Cells were treated with DMSO, bortezomib (10 μM), digitoxin (10 μM), emetine (10 μM), narasin (10 μM) and fluorosalan (10 μM) in the absence or presence of TNF-α (1ng/ml) for 1 h, followed by Western blotting analysis.

Fig 4

Concentration response curves of NF-κB antagonists in NF-κB bla, IκB phosphorylation and caspase 3/7 assays. NF-κB bla activity (■) and IκB phosphorylation (▲) were measured after the cells were incubated with various concentrations of ectinascidin 743 (A), bortezomib (B), chromomycin A3 (C), emetine (D), daunorubicinum (E), and lestaurtinib (F) in the presence of 1ng/ml TNFα for 5 h (NF-κB bla assay) or 30 min (IκB phosphorylation assay). Caspase 3/7 activity (▼) was also measured in these cells after the treatment of above drugs for either 5 h or 24 h.

Fig 5

Chemical structures of NF-kB inhibitors. (A) Ectinascidin 743; (B) Bortezomib; (C) Chromomycin A3; (D) Emetine; (E) Daunorubicinum; (F) Lestaurtinib.

Fig 6

Potency correlation of 19 NF-kB inhibitors between NF-kB bla assay after 5 h treatment and cell viability assays of ME180 cells (A) and HeLa cells (B) after 72 h treatment.