6E11, a highly selective inhibitor of Receptor-Interacting Protein Kinase 1, protects cells against cold hypoxia-reoxygenation injury.

Necroptosis is a programmed cell death pathway that has been shown to be of central pathophysiological relevance in multiple disorders (hepatitis, brain and cardiac ischemia, pancreatitis, viral infection and inflammatory diseases). Necroptosis is driven by two serine threonine kinases, RIPK1 (Receptor Interacting Protein Kinase 1) and RIPK3, and a pseudo-kinase MLKL (Mixed Lineage Kinase domain-Like) associated in a multi-protein complex called necrosome. In order to find new inhibitors for use in human therapy, a chemical library containing highly diverse chemical structures was screened using a cell-based assay. The compound 6E11, a natural product derivative, was characterized as a positive hit. Interestingly, this flavanone compound: inhibits necroptosis induced by death receptors ligands TNF-α (Tumor Necrosis Factor) or TRAIL (TNF-Related Apoptosis-Inducing Ligand); is an extremely selective inhibitor, among kinases, of human RIPK1 enzymatic activity with a nM Kd; has a non-ATP competitive mode of action and a novel putative binding site; is weakly cytotoxic towards human primary blood leukocytes or retinal pigment epithelial cells at effective concentrations; protects human aortic endothelial cells (HAEC) from cold hypoxia/reoxygenation injury more effectively than necrostatin-1 (Nec-1) and Nec-1s. Altogether, these data demonstrate that 6E11 is a novel potent small molecular inhibitor of RIPK1-driven necroptosis.

A new bacterial dehydrogenase oxidizing the lignin model compound guaiacylglycerol beta-O-4-guaiacyl ether.

A lignin model compound, named in short guaiagylglycerol beta-guaiacyl ether (GGE), contains the beta-0-4 ether linkage that is common in the chemical structure of lignin. A Pseudomonas sp. (GU5) had been isolated as an organism able to grow with GGE as the sole source of carbon and energy. When grown on vanillate, the bacteria contained a NAD+ -dependent dehydrogenase converting GGE to a 355 nm absorbing product. The enzyme, named GGE-dehydrogenase, was purified about 160-fold using gel permeation, ion exchange on DEAE-Sephadex, and dye-ligand affinity chromatography. The new protein was about 52 kDa in apparent size with but one polypeptide chain after denaturation and reduction. According to several criteria, the product of GGE oxidation (Km = 12 microM) was identified as the corresponding conjugated ketone at the alpha-carbon of the C3 side-chain. The secondary alcohol function in GGE was apparently the sole target of the enzyme action. However the conversion of GGE into ketone catalyzed by the enzyme was only partial, and did not exceed 50%, probably because only one of the alpha-enantiomers was susceptible to enzyme attack. In contrast the ketone, either made by organic synthesis or by enzymic oxidation of GGE, could be totally reduced back to GGE (Km = 13 microM at pH 8.4, 8 microM at neutral pH), with NADH as the reductant, as confirmed by UV absorption and NMR spectra. Other model compounds with no primary alcoholic function, ether linkage or phenolic group were also substrates for the enzyme, confirming the specificity of GGE-dehydrogenase for the alpha-carbon position. Conjugation of the alpha-ketone with an adjacent phenolic nucleus interfered strongly with equilibrium constants and redox potentials of the system according to pH, and the enzyme displayed widely different optima with pH over 9 when oxidizing GGE, below 7 when reducing the ketone. Equilibrium studies showed that the ketone/GGE potential was -0.37 volt at pH 8.7, -0.23 volt at pH 7 (30 degrees C). The significance of this new dehydrogenase and its properties are discussed, especially in the general concern of lignin biodegradation.

Selective inhibition of rat heart cAMP phosphodiesterases by lipophilic C-methyl-2-phenyl-4H-1-benzopyran-4-ones (C-methylflavones).

A series of eight methoxylated C-methyl-2-phenyl-4H-1-benzopyran-4-ones 3, 6, 10-15 was evaluated as inhibitors of rat heart cytosolic cyclic nucleotide phosphodiesterase (PDE). The 2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-3,8-dimethyl-4H-1-benzopyran-4-one (3) and the 2-(4-methoxyphenyl)-5,7-dimethoxy-3,8-dimethyl-4H-1-benzopyran-4-one (10) have never been previously described. Inhibition was performed on the whole cytosolic preparation and on the four PDE isoforms after HPLC purification. The flavones 3, 6, 10, 13 and 14 were selective and potent inhibitors of the isoforms, namely ROI (rolipram-sensitive) and CGI (cGMP-sensitive) PDEs specifically hydrolyzing cAMP. The di-C-methylflavones 3 and 13 have been shown to be potent inhibitors of these two isoforms, with IC50 values in the micromolar range.