The receptor-interacting serine-threonine kinase 3 (RIP3) is a key signaling molecule in the programmed necrosis (necroptosis) pathway. This pathway plays important roles in a variety of physiological and pathological conditions, including development, tissue damage response, and antiviral immunity. Here, we report the identification of a small molecule called (E)-N-(4-(N-(3-methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide--hereafter referred to as necrosulfonamide--that specifically blocks necrosis downstream of RIP3 activation. An affinity probe derived from necrosulfonamide and coimmunoprecipitation using anti-RIP3 antibodies both identified the mixed lineage kinase domain-like protein (MLKL) as the interacting target. MLKL was phosphorylated by RIP3 at the threonine 357 and serine 358 residues, and these phosphorylation events were critical for necrosis. Treating cells with necrosulfonamide or knocking down MLKL expression arrested necrosis at a specific step at which RIP3 formed discrete punctae in cells. These findings implicate MLKL as a key mediator of necrosis signaling downstream of the kinase RIP3.
Recent studies have unequivocally associated the fat mass and obesity-associated (FTO) gene with the risk of obesity. In vitro FTO protein is an AlkB-like DNA/RNA demethylase with a strong preference for 3-methylthymidine (3-meT) in single-stranded DNA or 3-methyluracil (3-meU) in single-stranded RNA. Here we report the crystal structure of FTO in complex with the mononucleotide 3-meT. FTO comprises an amino-terminal AlkB-like domain and a carboxy-terminal domain with a novel fold. Biochemical assays show that these two domains interact with each other, which is required for FTO catalytic activity. In contrast with the structures of other AlkB members, FTO possesses an extra loop covering one side of the conserved jelly-roll motif. Structural comparison shows that this loop selectively competes with the unmethylated strand of the DNA duplex for binding to FTO, suggesting that it has an important role in FTO selection against double-stranded nucleic acids. The ability of FTO to distinguish 3-meT or 3-meU from other nucleotides is conferred by its hydrogen-bonding interaction with the two carbonyl oxygen atoms in 3-meT or 3-meU. Taken together, these results provide a structural basis for understanding FTO substrate-specificity, and serve as a foundation for the rational design of FTO inhibitors.
In a single step, from [Cp*RuCl 2 ] 2 (Cp* ) η 5 -C 5 Me 5 ) and Li [BH 4 ], nido-1,2-(Cp*Ru) 2 (µ-H) 2 B 3 H 7 , 1, is produced in high yield. Addition of BH 3 ‚THF to 1 results in conversion to nido-1,2-(Cp*Ru) 2 (µ-H)B 4 H 9 , 2. Reaction of BH 3 ‚THF directly with [Cp*RuCl 2 ] 2 yields a mixture of 1 and 2. In two steps, a rhodium analogue, nido-2,3-(Cp*Rh) 2 B 3 H 7 , 9, is accessible by the reaction of [Cp*RhCl 2 ] 2 and Li [BH 4 ] to exclusively produce (Cp*Rh) 2 B 2 H 6 , 8, which adds BH 3 ‚THF to give 9 as the major product in a mixture. Reaction of BH 3 ‚THF directly with [Cp*RhCl 2 ] 2 yields the chloro derivative of 9, nido-1-Cl-2,3-(Cp*Rh) 2 B 3 H 6 , 11, in high yield via the intermediate positional isomer, nido-3-Cl-1,2-(Cp*Rh) 2 B 3 H 6 , 10. With high concentrations of Co 2 (CO) 8 , 1 reacts with Co 2 (CO) 8 to give nido-1-(Cp*Ru)-2-(Cp*RuCO)-3-Co(CO) 2 (µ 3 -CO)B 3 H 6 , 3, whereas low concentrations permit competitive degradation of 1 to yield arachno-(Cp*Ru)(CO)(µ-H)B 3 H 7 , 4. On the other hand, reaction of 11 with Co 2 (CO) 8 gives closo-1-Cl-6-{Co(CO) 2 }-2,3-(Cp*Rh) 2 (µ 3 -CO)B 3 H 3 , 12. Mild thermolysis of 3 results in loss of hydrogen and the formation of closo-6-Co(CO) 2 -2,3-(Cp*Ru) 2 (µ-CO)(µ 3 -CO)B 3 H 4 , 5, whereas thermolysis of 2 results in loss of hydrogen and formation of pileo-2,3-(Cp*Ru) 2 B 4 H 8 , 6, with a BH-capped square pyramidal structure. Finally, 6 reacts with Fe 2 (CO) 9 to yield pileo-6-Fe(CO) 3 -2,3-(Cp*Ru) 2 (µ 3 -CO)B 4 H 4 , 7, with a BH-capped octahedral cluster structure. The overall isolated yield of 7, formed in four steps from [Cp*RuCl 2 ] 2 , is ≈50% and evidences good control of reactivity.
Late-stage diversification of natural products is an efficient way to generate natural product derivatives for drug discovery and chemical biology. Benefiting from the development of site-selective synthetic methodologies, late-stage diversification of natural products has achieved notable success. This outlook will outline selected examples of novel methodologies for site-selective transformations of reactive functional groups and inert C–H bonds that enable late-stage diversification of complex natural products. Accordingly, late-stage diversification provides an opportunity to rapidly access various derivatives for modifying lead compounds, identifying cellular targets, probing protein–protein interactions, and elucidating natural product biosynthetic relationships.
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