*6.2. HOIPINs*

To search for novel inhibitors of LUBAC, we constructed a FRET-based assay system to quantify the linear ubiquitination level, using a recombinant LUBAC fragment [152]. After screening 250,000 small molecular chemicals, we identified a thiol-reactive, <sup>α</sup>,β-unsaturated carbonyl-containing compound, sodium (*E*)-2-(3-(2-methoxyphenyl)-3-oxoprop-1-en-1-yl)benzoate, named HOIPIN-1 from HOIP inhibitor-1, as a LUBAC inhibitor (Figure 5). We developed derivatives of HOIPIN-1, and found that sodium (*E*)-2-(3-(2,6-difluoro-4-(1*H*-pyrazol-4-yl)phenyl)-3-oxoprop-1-en-1-yl)- 4-(1-methyl-1*<sup>H</sup>*-pyrazol-4-yl)benzoate, designated as HOIPIN-8, is the most potent LUBAC inhibitor (Figure 5) [153]. HOIPIN-1 and HOIPIN-8 inhibited the in vitro linear ubiquitination activity of recombinant LUBAC, with IC50 values of 2.8 μM and 11 nM, respectively. Furthermore, HOIPIN-1 and HOIPIN-8 suppressed the overexpressed LUBAC-induced NF-κB activity in HEK293T cells, with IC50 values of 4.0 μM and 0.42 μM, respectively, indicating that HOIPIN-8 is the most potent among the reported LUBAC inhibitors.

**Figure 5.** LUBAC inhibitors, HOIPIN-1 and HOIPIN-8. Chemical structures of HOIPIN-1 and -8, and crystal structure of the HOIPIN-8-bound RING2-LDD domain of HOIP are shown.

We recently determined that HOIPINs are conjugated to the active site Cys885 in the RING2 domain of HOIP through Michael addition, and interrupt the RING-HECT-hybrid reaction in HOIP [73]. The benzoate, 2,6-difluorophenyl, and <sup>1</sup>*H*-pyrazol-4-yl moieties of HOIPIN-8 interact with Arg935 and Asp936, which are located in the LDD domain of human HOIP (Figure 5). Arg935 and Asp936 in the LDD domain reportedly interact with Glu16 and Thr14 in the acceptor ubiquitin, and the Ala mutations of these residues abrogated the linear ubiquitination activity [19–22]. Thus, HOIPIN-8 not only interacts with the active Cys885, but also masks the critical residues for acceptor ubiquitin-binding by the benzoate, 2,6-difluorophenyl, and <sup>1</sup>*H*-pyrazol-4-yl moieties. In addition, the 1-methyl-1*<sup>H</sup>*-pyrazol-4-yl moiety of HOIPIN-8 interacts with His887, Phe905, and Leu922 of HOIP (Figure 5). These sequence variations of RING2 may be beneficial for the HOIP specificity of compound [5]. On the other hand, the LDD domain of HOIP may be a key determinant for the HOIP specificity of HOIPINs. Therefore, the mechanisms underlying the HOIP specificity are completely different between HOIPINs and compound [5].

Although the <sup>α</sup>,β-unsaturated carbonyl-containing chemicals seemed to react with various SH-groups, HOIPINs did not inhibit the E1-mediated ubiquitin transfer to E2, or the activities of the HECT-, RING-, and other RBR-type E3s, and specifically suppressed the intracellular linear ubiquitin level induced by TNF-<sup>α</sup>, IL-1β, and poly(I:C) [73]. As shown in Section 5.2, the enhanced LUBAC activity is associated with the progression of ABC-DLBCL, and we found that HOIPINs effectively suppressed the cell viability of human ABC-DLBCL cells, but not GCB-DLBCL cells, by inhibiting the linear ubiquitination-mediated NF-κB activation [73]. Therefore, the cryptic peptide targeting LUBAC and the HOIPINs may serve as drug seeds to treat ABC-DLBCL patients with a poor prognosis. Moreover, we showed the HOIPIN-1-mediated alleviation of imiquimod-induced psoriasis in model mice. To date, dysfunctions in skin barrier production, IL-23/IL17-mediated lymphocyte signaling, and the NF-κB pathway are reportedly involved in the pathogenesis of psoriasis [154,155]. Since LUBAC a ffects not only NF-κB but also the production of IL-17 [156], HOIPINs may be e ffective to treat psoriasis.
