Necroptosis is a regulated, caspase-independent form of necrotic cell death driven by the kinases RIPK1 and RIPK3, which subsequently activate the pseudokinase MLKL as the executioner protein. Necroptosis plays a role in various human pathologies, including neurodegeneration and inflammation [1]. Recent evidence suggests that dysregulated necroptotic signaling may contribute to the development or exacerbation of inflammatory diseases, including those affecting the gut [[2], [3], [4]], skin [5], and brain [[6], [7], [8], [9]]. Necroptosis has also been implicated in cancer cell metabolism, with key regulatory genes, RIP3 [[10], [11], [12]] and MLKL [13,14], being downregulated in many cancers.

MLKL consists of three segments: (1) An N-terminal domain that forms oligomers to create membrane pores, executing necroptosis. Murine (1–125 aa), Human (1–154 aa). (2) The C-terminal kinase-like domain (KLD) of MLKL. Upon phosphorylation by RIP3, it dimerizes, exposing the N-terminal four-helix bundle (4HB) structural motif, and driving the tetramerization of full-length MLKL. Murine (182–464 aa), Human (179–471 aa). (3) The MLKL brace domain, which connects the KLD to the four-helix bundle (4HB) and provides an interface for MLKL oligomerization [[15], [16], [17], [18], [19], [20]].

MLKL undergoes activation through a series of stages, beginning with KLD dimer formation, followed by autonomous oligomerization of the intramolecular coiled-coil domains. This facilitates the formation of a functional MLKL oligomer. The dimerization of the MLKL KLD is a direct result of RIP3-mediated phosphorylation of MLKL [20,21]. Several small molecules associated with necroptosis have been identified, including the widely recognized RIP1 inhibitor Necrostatin-1 (Nec-1), the RIP3 inhibitor GSK’872, the human MLKL (hMLKL) inhibitor Necrosulfonamide (NSA), and murine MLKL inhibitors like GW806742X.

GW806742X is an ATP-competitive small molecule and a type II kinase inhibitor. Synthesized in 2005, it targets the Vascular Endothelial Growth Factor Receptor 2 (VEGFR2). In 2014, Murphy et al. screened a kinase library of 367 small molecules using protein thermal stability analysis and identified that GW806742X directly interacts with the KLD of murine MLKL. The dissociation constant (Kd) of GW806742X was determined to be 9.3 μM using SPR analysis [22].

Furthermore, GW806742X was shown to significantly inhibit TNF-induced necroptosis at a low concentration of 1 μM in dermal fibroblasts (MDFs), leading to its initial identification as a murine MLKL inhibitor [22]. Subsequently, in 2016, it was found that GW806742X binds to and inhibits the kinase activity of RIP1, an upstream signaling protein of MLKL. At the same time, a small molecule, Comp4, exhibiting similar binding affinity to MLKL as GW806742X in thermal shift assays, was identified. However, Comp4 neither binds to nor inhibits RIP1 and lacks efficacy in inhibiting necroptosis in cellular assays. This suggests that GW806742X inhibits necroptosis by suppressing RIP1 activity, although its direct inhibition of necroptosis through binding to MLKL remains controversial [23].

Necroptotic pathways differ significantly between humans and mice, with murine systems being more extensively studied [24]. The view that GW806742X is a murine MLKL inhibitor is now widely accepted and has been applied in multiple studies. This underscores the importance of clarifying the specific role of GW806742X in necroptosis, particularly regarding its function in relation to MLKL, which is the primary focus of the present study.