Molecular bases for HOIPINs-mediated inhibition of LUBAC and innate immune responses

Abstract

The NF-κB and interferon antiviral signaling pathways play pivotal roles in inflammatory and innate immune responses. The LUBAC ubiquitin ligase complex, composed of the HOIP, HOIL-1L, and SHARPIN subunits, activates the canonical NF-κB pathway through Met1-linked linear ubiquitination. We identified small-molecule chemical inhibitors of LUBAC, HOIPIN-1 and HOIPIN-8. Here we show that HOIPINs down-regulate not only the proinflammatory cytokine-induced canonical NF-κB pathway, but also various pathogen-associated molecular pattern-induced antiviral pathways. Structural analyses indicated that HOIPINs inhibit the RING-HECT-hybrid reaction in HOIP by modifying the active Cys885, and residues in the Cterminal LDD domain, such as Arg935 and Asp936, facilitate the binding of HOIPINs to LUBAC. HOIPINs effectively induce cell death in activated B cell-like diffuse large B cell lymphoma cells, and alleviate imiquimod-induced psoriasis in model mice. These results reveal the molecular and cellular bases of LUBAC inhibition by HOIPINs, and demonstrate their potential therapeutic uses.

Chain reactions: molecular mechanisms of RBR ubiquitin ligases

“…There is considerable interest in the development of small molecule modulators of RBR E3 activity as chemical tools for dissecting RBR signalling pathways, and for therapeutic applications. Covalent inhibitors that irreversibly modify the active site cysteine and inhibit activity have been developed for HOIP, and in principle this approach could be extended to other RBR E3s [67][68][69][70]. Structure-guided inhibitor design permits rational optimisation of hit compounds, although crystallisation of RBRs is often hampered by inherent interdomain flexibility.…”

Linear Ubiquitin Code: Its Writer, Erasers, Decoders, Inhibitors, and Implications in Disorders

“…However, some studies suggested that the LUBAC activity is necessary for the TLR-mediated IRF3 activation [69][70][71], and LUBAC is reportedly indispensable for the TNF-induced TBK1 and IKKε activation and prevention of cell death [72]. We showed that the LPS-, poly(I:C)-, and SeV-mediated IFN production pathway was impaired in HOIP-deficient mouse embryonic fibroblasts and human Jurkat T-lymphoblasts, and LUBAC inhibitors, HOIPINs, suppressed the antiviral pathway through the reduced activation of TBK1 and IRF3, supporting the positive function of LUBAC in this pathway [73]. Further studies are required to clarify the function of LUBAC in the IFN antiviral pathway.…”

“…From these studies, we hypothesized that the aggregable proteins in wisp inclusions may be initially conjugated with K48-linked ubiquitin chains; however, they seem to be resistant to the proteasomal degradation due to misfolding (Figure 6). Concomitant with aging, maturation, and liquid-liquid phase separation (LLPS), LUBAC-mediated linear ubiquitin and/or K63-linked 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 1H-pyrazol-4-yl moieties of HOIPIN-8 interact with Arg935 and Asp936, which are located in the LDD domain of human HOIP ( Figure 5).…”

“…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].…”

“…After screening 250,000 small molecular chemicals, we identified a thiol-reactive, α,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-(1H-pyrazol-4-yl)phenyl)-3-oxoprop-1-en-1-yl)-4-(1-methyl-1H-pyrazol-4-yl)benzoate, designated as HOIPIN-8, is the most potent LUBAC inhibitor ( Figure 5 Although the α,β-unsaturated carbonyl-containing chemicals seemed to react with various SHgroups, 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-α, IL-1β, and poly(I:C) [73]. As shown in chapter 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].…”