Ethyl Pyruvate Inhibits Nuclear Factor-κB-Dependent Signaling by Directly Targeting p65

Abstract

Ethyl pyruvate has been shown to have anti-inflammatory properties in numerous cell culture and animal studies. In this series of experiments, we tested the hypothesis that ethyl pyruvate inhibits signaling by the pro-inflammatory transcription factor, NF-B. Ethyl pyruvate inhibited luciferase expression in lipopolysaccharide-stimulated murine macrophage-like RAW 264.7 cells transfected with an NF-B-dependent luciferase reporter vector. Ethyl pyruvate also decreased NF-B DNA-binding activity in lipopolysaccharide-stimulated RAW 264.7 cells and decreased lipopolysaccharide-induced expression of an NF-B-dependent gene, inducible nitric oxide synthase. Ethyl pyruvate had no effect on the degradation of IB␣ or IB␤ in lipopolysaccharide-stimulated RAW 264.7 cells, suggesting that ethyl pyruvate acts distally to this step in the activation of NF-B. In a cell-free system, binding of p50 homodimers to an NF-B consensus oligonucleotide sequence was unaffected by ethyl pyruvate over a wide range of concentrations, indicating that ethyl pyruvate probably does not modify or interact with the p50 subunit of NF-B. In contrast, ethyl pyruvate inhibited DNA binding by ectopically overexpressed wild-type p65 homodimers. However, ethyl pyruvate failed to inhibit the DNAbinding activity of homodimers of an overexpressed mutant form of a p65 with substitution of serine for cysteine 38. Taken together, these results suggest that ethyl pyruvate inhibits DNA-binding by covalently modifying p65 at Cys 38 . We conclude that some of the beneficial anti-inflammatory effects of ethyl pyruvate may be due to modification of p65, thereby inhibiting signaling via the NF-B pathway.

Microglial Activation in Stroke: Therapeutic Targets

“…At the in vitro level, ethyl pyruvate (a prodrug form of pyruvate with improved brain penetrance) was found to inhibit NF-B activation in both cultured microglia and RAW 264.7 cells through a modification of the p65 subunit. [201][202][203] Ethyl pyruvate also reduced mortality and reduced circulating levels of HMGB1 in a model of lethal sepsis. 202 Further, pyruvate was also shown to inhibit microglial NF-B activation in rats given bacterial lipopolysaccharide, which causes transient microglial activation independent of any cell death.…”

Hydrogen Sulfide Suppresses Oxidized Low-density Lipoprotein (Ox-LDL)-stimulated Monocyte Chemoattractant Protein 1 generation from Macrophages via the Nuclear Factor κB (NF-κB) Pathway

“…Cysteine 38 in NF-B p65 is highly sensitive to various agents (43)(44)(45)(46)(47)(48)(49). The importance of cysteine 38 in p65 has been appreciated for many years.…”

Nuclear Factor κB Inhibitors Alleviate and the Proteasome Inhibitor PS-341 Exacerbates Radiation Toxicity in Zebrafish Embryos

“…First, we tested EP, a ROS scavenger and inhibitor of NF-κBp65 38 . EP inhibits the DNA binding activity of NF-κBp65 by binding to a reactive cysteine in the DNA binding site (Cys 38) of NF-κBp65 13 , which is shared between humans and zebrafish ( Supplementary Fig. S2C).…”

“…12 Among other molecular targets, both drugs inhibit activation of NF-κB. EP inhibits NF-κB signaling through direct molecular interaction with a reactive cysteine of the p65 subunit of NF-κB 13 whereas CDDO-TFEA binds to a reactive cysteine (Cys179) of IKKα, thus inhibiting its kinase activity. 14 However, these drugs also target other signaling molecules and pathways of potential relevance to the radiation response, including signal transducers and activators of transcription 3 and Jaks.…”