Discovery of a New Class of Uracil Derivatives as Potential Mixed Lineage Kinase Domain-like Protein (MLKL) Inhibitors

Abstract

Necroptosis is a form of programmed cell death. Mixed lineage kinase domain-like protein (MLKL) is the necroptosis executor, and it is involved in various diseases such as tissue damage and neurodegeneration-related diseases. Here, we report the development of novel MLKL inhibitors with a uracil nucleus through scaffold morphing from our previously reported xanthine MLKL inhibitor TC13172. After a rational structure–activity relationship study, we obtained the highly potent compounds 56 and 66. Mechanism studies revealed that these compounds partially inhibited MLKL oligomerization and significantly inhibited MLKL translocation to the membrane. Compared with TC13172, 56 and 66 have a different mode of action and, importantly, their reaction rate with glutathione is more than 150-fold lower. This reduction in potential off-target effects and cell toxicity makes this series an attractive starting point for further drug development for MLKL-related disease treatments.

From (Tool)Bench to Bedside: The Potential of Necroptosis Inhibitors

“…It was observed that these uracils did not inhibit necroptosis in MEF cells (stimulated with TNF, a SMAC mimetic, and zVAD.fmk), suggesting that they targeted the Cys86 in human MLKL that is not present in mouse MLKL. Furthermore, the binding of both uracil 38 and 39 to MLKL was shown to outcompete that of TC13172 35 in a pull-down assay . The binding of 38 to Cys86 was then confirmed by incubation with the MLKL protein and analysis via MS/MS, as well as mutagenesis studies wherein 38 and 39 failed to inhibit necroptosis in HT29 cells transfected to express a C86S mutant .…”

“…A scaffold-morphing strategy identified the hit compound 37 , with EC 50 = 3380 nM in HT29 cells treated with necroptotic stimulus (TNF, a SMAC mimetic, and zVAD.fmk). Elaboration of the SAR around this scaffold resulted in the development of uracils 38 and 39 (Figure ) with markedly improved potencies (EC 50 = 82 and 31 nM, respectively) . Importantly, these optimized uracils showed little-to-no inhibition of RIPK1 (18 and 0% at 10 μM, respectively) and no inhibition of RIPK3 in ADP-Glo enzymatic assays, as well as no effects on the level of MLKL phosphorylation.…”

“…Importantly, these optimized uracils showed little-to-no inhibition of RIPK1 (18 and 0% at 10 μM, respectively) and no inhibition of RIPK3 in ADP-Glo enzymatic assays, as well as no effects on the level of MLKL phosphorylation. Furthermore, no inhibition of cell proliferation or cell survival was observed when HT29 cells were treated with 5 μM 38 or 39 , and the reactive 6-chloro group showed reduced reactivity toward glutathione (GSH) compared to xanthine 35 ( t 1/2 = 48 or 160 h, respectively, compared to 20 min) when incubated together in DPBS buffer, suggesting a reduced propensity for off-target toxicity …”

“…A series of uracil compounds was recently developed ( Figure 17 ) to overcome the liabilities of the xanthine-based small molecules targeting MLKL, namely, their high levels of reactivity toward nucleophiles under physiological conditions and the potential for off-target toxicity. 168 A scaffold-morphing strategy identified the hit compound 37 , with EC 50 = 3380 nM in HT29 cells treated with necroptotic stimulus (TNF, a SMAC mimetic, and zVAD.fmk). Elaboration of the SAR around this scaffold resulted in the development of uracils 38 and 39 ( Figure 17 ) with markedly improved potencies (EC 50 = 82 and 31 nM, respectively).…”

“…Furthermore, no inhibition of cell proliferation or cell survival was observed when HT29 cells were treated with 5 μM 38 or 39 , and the reactive 6-chloro group showed reduced reactivity toward glutathione (GSH) compared to xanthine 35 ( t 1/2 = 48 or 160 h, respectively, compared to 20 min) when incubated together in DPBS buffer, suggesting a reduced propensity for off-target toxicity. 168 …”