GW806742X can induce mouse MLKL activation by directly promoting MLKL kinase like domain dimerization.

The dimerization of the MLKL kinase-like domain (KLD) is a crucial step for MLKL activation in necroptosis. In 2014, it was discovered that GW806742X can directly bind to the mouse MLKL KLD via surface plasmon resonance (SPR) (Kd = 9.3 μM), inhibiting TNF-induced membrane translocation of MLKL and necroptosis. Consequently, GW806742X is considered a mouse MLKL inhibitor. In this study, we found that GW806742X blocks TNF-induced RIP1-dependent necroptosis but promotes necroptosis triggered by either RIP3 or MLKL self-oligomerization in the FKBPv chemical dimerizer system. In addition, higher doses of GW806742X can directly induce MLKL-dependent necroptosis and promote MLKL oligomerization, as detected by non-reducing Western blot. Through chemical cross-linking assays, we observed that GW806742X induces dimerization of recombinant mouse MLKL KLD proteins. The dimerization of the MLKL KLD is a direct consequence of RIP3 phosphorylation, a crucial step in RIP3-induced MLKL activation and necroptosis. Therefore, GW806742X exerts a dual effect on necroptosis: it inhibits necroptosis, likely by interfering with RIP1 function, while promoting necroptosis by facilitating MLKL activation.