*6.1. Chemical Inhibitors of LUBAC*

To date, BAY11-7082 [147], gliotoxin [148], and bendamustine [149] have been reported as chemical inhibitors of LUBAC. However, using non-toxic concentrations, we recently showed that these inhibitors lack the selectivity and inhibitory effects on LUBAC-induced linear ubiquitination and NF-κB activation in HEK293T cells [73].

Recently, Rittinger's group developed <sup>α</sup>,β-unsaturated methyl ester-containing compounds, such as compound [5], as LUBAC inhibitors [150,151]. Among them, compound [11a] reportedly inhibited the overexpressed LUBAC-induced NF-κB activity in HEK293T cells (IC50 = 37 μM). Importantly, they showed that compound [5] was covalently attached to the catalytic Cys885 of HOIP via Michael addition, by accommodation in a hydrophobic pocket formed by Tyr878, Leu880, and Phe888, and stabilization by hydrogen bonds with the main-chain CO and NH groups of His889 and the Oγ atom of Ser899. These compound [5]-interacting residues of HOIP are located in the RING2 domain [150,151], but are not conserved in other RBR E3s. These sequence variations of RING2 may be beneficial for the HOIP specificity of compound [5].
