In vitro activity: TEPP-46 is a novel potent and selective small molecule activator of pyruvate kinase M2 (PKM2) with an EC50 of 92 nM, it showed little or no effect on PKM1, PKL and PKR. Macrophages activated by the TLR4 agonist LPS undergo dramatic changes in their metabolic activity. LPS induces expression of the key metabolic regulator Pyruvate Kinase M2 (PKM2). Activation of PKM2 using the well-characterized small molecules, TEPP-46, inhibited LPS-induced Hif-1α and IL-1β, as well as the expression of a range of other Hif-1α-dependent genes. Activation of PKM2 attenuated an LPS-induced proinflammatory M1 macrophage phenotype while promoting traits typical of an M2 macrophage. LPS-induced PKM2 enters into a complex with Hif-1α, which can directly bind to the IL-1β promoter, an event that is inhibited by activation of PKM2. TEPP-46 inhibited LPS-induced glycolytic reprogramming and succinate production. Finally, activation of PKM2 by TEPP-46 in vivo inhibited LPS and Salmonella typhimurium-induced IL-1β production, while boosting production of IL-10. PKM2 is therefore a critical determinant of macrophage activation by LPS, promoting the inflammatory response.

Kinase Assay: Pyruvate kinase activity is measured by monitoring pyruvate-dependent conversion of NADH to NAD+ by lactate dehydrogenase (LDH) as described previously. Briefly, for cell line experiments, the medium is replaced with fresh medium 1 hour prior to the start of treatment with DMSO or activator. Also, where indicates, 100 μM pervanadate is added 10 minutes prior to cell lysis. Cells are lysed on ice with NP-40 buffer containing 2 mM DTT and protease inhibitors and clarified by centrifugation at 21,000 x g. 5 μL of the supernatant is used to assess pyruvate kinase activity. Pyruvate kinase activity is subsequently normalized for total protein content.

Cell Assay: 2,000 cells are seeded in 96-well plates 24 h prior to treatment start. CellTiter96(R) AQueous is used to assess cell viability following oxidant and PKM2 activator combination treatments. MTS: (3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium).