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 IκBα is critical in regulating the subsequent ubiquitination and proteolysis of IκBα, which then releases NF-κB to promote gene transcription. The multisubunit IκB kinase responsible for this phosphorylation contains two catalytic subunits, termed IκB 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 μm, IKK-1 IC50 = 4 μm). The compound failed to inhibit a panel of 15 other kinases and selectively inhibited the stimulated phosphorylation of IκBα in cells (IC50 = 4 μm) 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-κB in the regulation of cytokine transcription, BMS-345541 inhibited lipopolysaccharide-stimulated tumor necrosis factor α, interleukin-1β, interleukin-8, and interleukin-6 in THP-1 cells with IC50 values in the 1- to 5-μmrange. 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 IκBα 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 α following intraperitoneal challenge with lipopolysaccharide. Thus, the compound is effective against NF-κB activation in mice and represents an important tool for investigating the role of IKK in disease models.

[1]  M. Karin,et al.  IKKα controls formation of the epidermis independently of NF-κB , 2001, Nature.

[2]  T. Maniatis,et al.  Activation of the IκBα Kinase Complex by MEKK1, a Kinase of the JNK Pathway , 1997, Cell.

[3]  David Baltimore,et al.  NF-κB: Ten Years After , 1996, Cell.

[4]  G. W. Peet,et al.  Recombinant IκB Kinases α and β Are Direct Kinases of IκBα* , 1998, The Journal of Biological Chemistry.

[5]  K. Miller,et al.  The Multisubunit IκB Kinase Complex Shows Random Sequential Kinetics and Is Activated by the C-terminal Domain of IκBα* , 1998, The Journal of Biological Chemistry.

[6]  The catalytic subunits of IkappaB kinase, IKK-1 and IKK-2, contain non-equivalent active sites when expressed as homodimers. , 2002, Biochemical and biophysical research communications.

[7]  Jill K Thompson,et al.  Role of IB Ubiquitination in Signal-induced Activation of NF-B in Vivo(*) , 1996, The Journal of Biological Chemistry.

[8]  David M. Rothwarf,et al.  A cytokine-responsive IκB kinase that activates the transcription factor NF-κB , 1997, Nature.

[9]  A. Israël,et al.  Regulation of IkappaBbeta degradation. Similarities to and differences from IkappaBalpha. , 1997, The Journal of biological chemistry.

[10]  A. Israël,et al.  I kappa B epsilon, a novel member of the IκB family, controls RelA and cRel NF‐κB activity , 1997 .

[11]  C. Koboldt,et al.  Characterization of the Recombinant IKK1/IKK2 Heterodimer , 2000, The Journal of Biological Chemistry.

[12]  E. Schmidt,et al.  IKKα Provides an Essential Link between RANK Signaling and Cyclin D1 Expression during Mammary Gland Development , 2001, Cell.

[13]  Matthias Mann,et al.  IKK-1 and IKK-2: Cytokine-Activated IκB Kinases Essential for NF-κB Activation , 1997 .

[14]  S. Gerstberger,et al.  Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation , 1995, Science.

[15]  J C Lee,et al.  Novel homologues of CSBP/p38 MAP kinase: activation, substrate specificity and sensitivity to inhibition by pyridinyl imidazoles. , 1997, Biochemical and biophysical research communications.

[16]  A. Baldwin,et al.  Inducible phosphorylation of I kappa B alpha is not sufficient for its dissociation from NF-kappa B and is inhibited by protease inhibitors. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Mike Rothe,et al.  IκB Kinase-β: NF-κB Activation and Complex Formation with IκB Kinase-α and NIK , 1997 .

[18]  L. Baldi,et al.  Critical Role for Lysines 21 and 22 in Signal-induced, Ubiquitin-mediated Proteolysis of IB- (*) , 1996, The Journal of Biological Chemistry.

[19]  H. Gutfreund,et al.  Enzyme kinetics , 1975, Nature.

[20]  D. Goeddel,et al.  Identification and Characterization of an IκB Kinase , 1997, Cell.

[21]  P. Ghezzi,et al.  Lps induces IL-6 in the brain and in serum largely through TNF production. , 2000, Cytokine.

[22]  E. Zandi,et al.  The IκB Kinase Complex (IKK) Contains Two Kinase Subunits, IKKα and IKKβ, Necessary for IκB Phosphorylation and NF-κB Activation , 1997, Cell.

[23]  R. Ryseck,et al.  Peptides Corresponding to the N and C Termini of IκB-α, -β, and -ε as Probes of the Two Catalytic Subunits of IκB Kinase, IKK-1 and IKK-2* , 1999, The Journal of Biological Chemistry.

[24]  T. Maniatis,et al.  Site-Specific Phosphorylation of IκBα by a Novel Ubiquitination-Dependent Protein Kinase Activity , 1996, Cell.

[25]  A. Israël,et al.  IκB proteins: structure, function and regulation , 1997 .

[26]  G. Franzoso,et al.  Structure, regulation and function of NF-kappa B. , 1994, Annual review of cell biology.