Novel non-peptide small molecules preventing IKKβ/NEMO association inhibit NF-κB activation in LPS-stimulated J774 macrophages

Nuclear Factor-kappa B (NF-kappa B) is a transcription factor regulating several genes involved in important physiological and pathological processes. NF-kappa B has been found constitutively activated in many inflammatory/immune diseases. In addition, a positive correlation between persistent activation of NF-kappa B and tumor promotion has been demonstrated. Since the IKK (I kappa B kinase) activation is an indispensable component of all pro-inflammatory signaling pathways leading to NF-kappa B activation, considerable efforts have been done in order to develop novel anti-inflammatory therapeutics targeting IKK. Association of the IKK complex relies on critical interactions between the C-terminus NBD (NEMO binding domain) of the catalytic subunits IKK alpha, and IKK beta, and the regulatory subunit NEMO (NF-kappa B Essential Modulator). Thus, this IKK/NEMO interacting region provides an attractive target to prevent the IKK complex formation and NF-kappa B activation. In this regard, we have identified non-peptide small molecule disruptors of IKK beta/NEMO complex through a structure-based virtual screening (SBVS) of the NCI chemical library. Phenothiazine 22 and its close analogues (22.2, 22.4 and 22.10) were able to reduce nitrite production and iNOS mRNA expression in J774 murine macrophages stimulated with LPS for 24 h. These effects were associated with a reduced NF-kappa B/DNA binding activity as well as a decreased expression of phosphorylated IKK beta, I kappa B alpha. and NF-kappa B/p65 in these cells. These observations suggest that compound 22 and its three structural analogues by inhibiting IKK beta/NEMO association mediate the blockage of NF-kappa B signaling pathway and may prove effective in treatment of diseases in which the IKK/NF-kappa B pathway is dysregulated. (C) 2016 Elsevier Inc. All rights reserved.