Communication
ChemComm
In summary, we used phenotypic screening to identify a
novel MLKL inhibitor and subsequently conducted a rational
SAR study that led to the discovery of the most potent MLKL
inhibitors ever reported; the EC50 of compound 15 reached
levels as low as 2 nM (see Fig. S8 for the potencies of different
MLKL inhibitors in the same necroptosis assay, ESI†). We used a
compound 12 affinity probe to demonstrate that these compounds
act as covalent inhibitors of MLKL. Compounds 12 and 15 were
found to inhibit the oligomerization and translocation of MLKL to
the cell membrane. The discovery of the novel and potent MLKL
inhibitors reported here will almost certainly be of benefit in
exploring the biological function of MLKL, including its role in
necroptosis-related disease pathogenesis.
This work was supported by the National Major Scientific
and Technological Special Project for ‘‘Significant New Drugs
Development’’ during the Twelfth Five-year Plan Period
2013ZX0950910 from the Chinese Ministry of Science and
Technology.
Fig. 5 Compounds 12 and 15 block the oligomerization and translocation
of MLKL from the cytoplasm to the cell membrane. (A) The effects of
different compounds on the oligomerization of MLKL. TSZ-treated HT-29
cells were incubated with 200 nM RIPA-56, 1 mM NSA, 1 mM GW806742X,
1 mM compound 12, or 100 nM compound 15 for 8 h. The samples were
analysed by western blotting. (B) Compounds 12 or 15 block the translocation
of MLKL from the cytoplasm to the cell membrane. TSZ-treated HT-29 cells
were incubated with 200 nM RIPA-56, 1 mM NSA, 1 mM compound 12, or
100 nM compound 15 for 8 h. The cell lysates was separated to the soluble
and membrane phase. The samples were analysed by western blotting with
MLKL and pMLKL antibodies. b-Actin and Cox4 were used as controls for
the soluble and membrane phases, respectively. (C) The effects of different
compounds on the cellular localization of pMLKL. HT-29 cells were treated
as indicated for 8 h. The cellular localization of pMLKL (green) was monitored
by immunofluorescence as described in the ESI† (scale bars, 10 mm).
Notes and references
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HT-29 cells were treated with TSZ in the presence of RIPA-56,
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disrupt the phosphorylation of MLKL, but did decrease the level
of MLKL in the membrane phase, demonstrating that these
MLKL inhibitors block the translocation of MLKL to the cell
membrane, thereby protecting cells from necroptosis. We also
performed immunofluorescence staining in HT-29 cells using a
monoclonal anti-pMLKL antibody. After TSZ stimulation, large,
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Chem. Commun.
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