Inhibition of Canonical NF-κB Signaling by a Small Molecule Targeting NEMO-Ubiquitin Interaction

Abstract

The IκB kinase (IKK) complex acts as the gatekeeper of canonical NF-κB signaling, thereby regulating immunity, inflammation and cancer. It consists of the catalytic subunits IKKα and IKKβ and the regulatory subunit NEMO/IKKγ. Here, we show that the ubiquitin binding domain (UBAN) in NEMO is essential for IKK/NF-κB activation in response to TNFα, but not IL-1β stimulation. By screening a natural compound library we identified an anthraquinone derivative that acts as an inhibitor of NEMO-ubiquitin binding (iNUB). Using biochemical and NMR experiments we demonstrate that iNUB binds to NEMOUBAN and competes for interaction with methionine-1-linked linear ubiquitin chains. iNUB inhibited NF-κB activation upon UBAN-dependent TNFα and TCR/CD28, but not UBAN-independent IL-1β stimulation. Moreover, iNUB was selectively killing lymphoma cells that are addicted to chronic B-cell receptor triggered IKK/NF-κB activation. Thus, iNUB disrupts the NEMO-ubiquitin protein-protein interaction interface and thereby inhibits physiological and pathological NF-κB signaling.

Molecular recognition and dynamics of linear poly-ubiquitins: integrating coarse-grain simulations and experiments

“…S11), in particular residues around the hydrophobic patch I44 and the previously mentioned E182 surface. The observed interaction sites are in agreement with observed NMR chemical shifts perturbations reported in Vincendeau et al 23 . The ensemble also provides a possible explanation for the different binding stoichiometry observed in solution (NEMO:Ub2 2:1) and crystalline state (NEMO:Ub2 2:2).…”

“…In order to study the dynamics and changes of linear poly-ubiquitin dynamics upon binding to cognate proteins, we used simulations and experiments to characterize the interaction of the NEMO UBAN domain (NEMO258-350) to the linear poly-ubiquitins Ub2, Ub3 and Ub4. The NEMO UBAN domain is a dimer in solution 23 . Previous studies have shown two different binding stoichiometries in solution and crystalline state for Ub2: with either two NEMO monomers bound to one Ub2(2:1) or two NEMO monomers bound to two Ub2 (2:2) 23-24 .…”

“…LUBAC preferentially recognizes and conjugates linear ubiquitin chains on NEMO. NEMO also possesses a specific linear di-ubiquitin-binding region referred to as the "ubiquitin binding in ABIN and NEMO" (UBAN) motif 24 , which forms a helical coiled-coil dimer in solution 23 . Recognition of a linear poly-ubiquitin conjugated to NEMO by the UBAN domain of another NEMO may trigger the clustering of the IKK complex as well as conformational changes that subsequently activate IKK [25][26] .…”

“…Once active IKK can phosphorylate and inactivate the IkBs (inhibitor of the NF-kB proteins) leading to the release of NF-kB 27 . Indeed, it was recently shown that it is possible to inhibit NF-kB activation upon UBAN-dependent TNFα and TCR/CD28 stimulation by small-molecules that inhibit the binding of linear polyubiquitins to the NEMOUBAN domain 23 . While the NEMOUBAN domain can bind linear di-ubiquitin, it has been observed that full-length NEMO can only bind Ub4 or longer suggesting a length-dependent activation mechanism 21 .…”

Linear ubiquitin chain‐binding domains

“…The interaction of linear ubiquitin chains with NEMO‐UBAN alters the conformation of NEMO , which may have an allosteric effect, regulating IKK kinase activity and downstream signaling. Genetic and pharmacological inhibition of the interaction between NEMO‐UBAN and linear ubiquitin chains preclude the full activation of NF‐κB, revealing an important role for the NEMO‐linear ubiquitin chain interaction in regulating the NF‐κB pathway. Whether NEMO‐UBAN binds to linear ubiquitination formed on NEMO itself, and whether this interaction affects the oligomeric state of the IKK complex, remain unclear.…”