Repurposing of Ibrutinib and Quizartinib as potent inhibitors of necroptosis

Abstract

Necroptosis is a form of regulated cell death that has been implicated in multiple diseases. TNF-induced necroptosis is regulated by necrosomes, complexes consisting of RIPK1, RIPK3 and MLKL. In this study, by screening of a small-compound library, we identified dozens of compounds that inhibited TNF-induced necroptosis. According to the mechanisms by which they inhibited necroptosis, these compounds were classified into different groups. We then identified Ibrutinib as an inhibitor of RIPK3 and found that Quizartinib protected against the TNF-induced systemic inflammatory response syndrome in mice by inhibiting the activation of RIPK1. Altogether, our work revealed dozens of necroptosis inhibitors, suggesting new potential approaches for treating necroptosis-related diseases.

Pervasive aggregation and depletion of host and viral proteins in response to cysteine-reactive electrophilic compounds

“…More broadly, we observe formation of stress granules in response to nearly every cysteine-reactive compound evaluated, including the drugs afatinib and auranofin, with the latter recently implicated in increasing nuclear H2O2 levels 147 . Notably, afatinibinduced stress granules were only observed to occur at 10 µM, which, while a dosage that is reported in a number of prior studies [158][159][160][161][162][163] , is >50-fold above 175 the dose require to effect full covalent modification of EGFR 84 . The parallel recruitment of proteasome subunits into stress granules, which aligns with prior reports of stress-induced phase separation of the proteasome 167 , allows us to put forth a model whereby covalent modifiers cause widespread protein aggregation and subsequent proteasome-mediated degradation, driven by elevated local concentrations of hyper-active proteasomes.…”

“…Thus, we further broadened the scope of compounds assessed to include RA190, which, like other cysteinereactive electrophiles, increased proteasome activity in our proteasome activation assays (Figure S12A), bardoxolone, a widely utilized NRF2 activator 154 , sulforaphane, a covalent modulator of the ubiquitin-proteasome and autophagic pathways with anticancer activity 155 , as well as chemotherapeutic agents such as FDA-approved ibrutinib and afatinib, which are covalent kinase inhibitors, auranofin, which targets TXNRD1/2 156,157 and was recently reported to increase the activity of the DNA damage checkpoint kinase CHK1 via oxidation of an allosteric cysteine residue 147 . We subjected cells to compound dosing consistent with prior reports-for afatinib and ibrutinib we chose to treat with 10 µM compound, which, while a dosage used widely [158][159][160][161][162][163] are in substantial excess of those required to achieve full target engagement 84 . With the exception of ibrutinib, all cysteine-reactive compounds induced G3BP puncta consistent with SG formation (Figure 6B, S31A).…”

Die hard: necroptosis and its impact on age-dependent neuroinflammatory diseases

“…Though GSK'872 and GSK'840 inhibit RIPK3 and necroptosis, once bound to RIPK3, they have been shown to elicit a conformational change that promotes the recruitment of RIPK1 via the RHIM domain leading to the activation of casape 8 resulting in apoptosis (Mandal et al, 2014). However, the FDAapproved cancer treatment drug Ibrutinib was recently identified as a RIPK3 inhibitor that acts similarly to GSK'872 (Huang et al, 2023). Ibrutinib inhibits the phosphorylation and autophosphorylation of RIPK3 independent of RIPK1 (Huang et al, 2023).…”

“…However, the FDAapproved cancer treatment drug Ibrutinib was recently identified as a RIPK3 inhibitor that acts similarly to GSK'872 (Huang et al, 2023). Ibrutinib inhibits the phosphorylation and autophosphorylation of RIPK3 independent of RIPK1 (Huang et al, 2023). Unlike GSK'872 or GSK'840, Ibrutinib inhibits necroptosis without enforcing the apoptosis-inducing RIPK3 conformational change, making it a more ideal inhibitor of RIPK3 and TNF⍺-induced necroptotic cell death.…”

“…Additionally, Quizartinib, another FDA-approved anti-cancer drug, was identified as an inhibitor of RIPK1, RIPK3, and MLKL phosphorylation (Huang et al, 2023). While the specific target and mechanism of action for quizartinib is still unknown, it is a promising inhibitor of TNF⍺-induced necroptosis.…”