BMS-345541 is a highly selective inhibitor of IκB kinase that binds at an allosteric site of the enzyme and blocks NF-κB-dependent transcription in mice

The signal-inducible phosphorylation of serines 32 and 36 of IkappaBalpha is critical in regulating the subsequent ubiquitination and proteolysis of IkappaBalpha, which then releases NF-kappaB to promote gene transcription. The multi-subunit licB kinase responsible for this phosphorylation contains two catalytic subunits, termed licB kinase (IKK)-1 and IKK-2. BMS-345541 (4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline) was identified as a selective inhibitor of the catalytic subunits of IKK (IKK-2 IC50 = 0.3 muM, IKK-1 IC50 = 4 muM). The compound failed to inhibit a panel of 15 other kinases and selectively inhibited the stimulated phosphorylation Of IkappaBalpha in cells (IC50 = 4 muM) while failing to affect c-Jun and STAT3 phosphorylation, as well as mitogen-activated protein kinase-activated protein kinase 2 activation in cells. Consistent with the role of IKK/NF-kappaB in the regulation of cytokine transcription, BMS-345541 inhibited lipopolysaccharide-stimulated tumor necrosis factor a, interleukin-1beta, interleukin-8, and interleukin-6 in THP-1 cells with IC50 values in the 1- to 5-muM range. Although a Dixon plot of the inhibition of IKK-2 by BMS-345541 showed a non-linear relationship indicating non-Michaelis-Menten kinetic binding, the use of multiple inhibition analyses indicated that BMS-345541 binds in a mutually exclusive manner with respect to a peptide inhibitor corresponding to amino acids 26-42 of IkappaBalpha with Ser-32 and Ser-36 changed to aspartates and in a non-mutually exclusive manner with respect to ADP. The opposite results were obtained when studying the binding to IKK-1. A binding model is proposed in which BMS-345541 binds to similar allosteric sites on IKK-1 and IKK-2, which then affects the active sites of the subunits differently. BMS-345541 was also shown to have excellent pharmacokinetics in mice, and peroral administration showed the compound to dose-dependently inhibit the production of serum tumor necrosis factor a following intraperitoneal challenge with lipopolysaccharide. Thus, the compound is effective against NF-kappaB activation in mice and represents an important tool for investigating the role of IKK in disease models.