Harnessing dual-mode RIPK1 ligands for cross-species anti-necroptosis inhibitor compounds.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) has a crucial role in cell death and inflammation. A promising approach to develop novel inhibitors of RIPK1 mediated necroptosis is to mix the different binding modes of the known RIPK1 inhibitors into one molecule. Herein we report the synthesis and biological evaluation of novel mixed type inhibitors. Using Eclitasertib as a starting point, and applying our previous, published knowledge regarding cyclic malonamides, we successfully identified a library of active compounds. The active enantiomer of the most balanced and promising compound was subjected to pharmacokinetics and in vivo hypothermia study in mice.

Design, synthesis and biological evaluation of novel cyclic malonamide derivatives as selective RIPK1 inhibitors.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) plays a key role in cell death and inflammation. RIPK1 is a well-established therapeutic target, due to the presence of a unique kinase-regulating allosteric pocket, which enables selective inhibition. Herein we used GSK2982772 as our starting point in our discovery campaign. Applying isosteric replacement, we successfully identified the malonamide scaffold, instead of the well-established serine template. Further structural optimization led to the design and synthesis of a series of analog inhibitors. The enantiomers of the most promising compound were tested on 97 different kinases. The active enantiomer proved to be kinase selective.

Immediate insulin treatment prevents gut motility alterations and loss of nitrergic neurons in the ileum and colon of rats with streptozotocin-induced diabetes.

The streptozotocin-induced diabetic rat model was used to investigate the relation between the deranged gut motility and the segment-specific quantitative changes in the nitrergic myenteric neurons. Additionally, we studied the effectiveness of early insulin replacement to prevent the diabetes-induced changes. Rats were divided into three groups: controls, diabetics and insulin-treated diabetics. Ten weeks after the onset of diabetes, animals were chosen from each group for intestinal transit measurements. The remainder were killed and gut segments were processed for NADPH-diaphorase histochemistry and HuC/HuD immunohistochemistry. The diabetic rats displayed faster transit than that for the controls. In the insulin-treated group, the transit time was the same as that in the controls. In the duodenum of the diabetic rats, the number of nitrergic neurons was decreased, while the total neuronal number was not altered. In the jejunum, ileum and colon, both the total and the nitrergic neuronal cell number decreased significantly. Insulin treatment did not prevent the nitrergic cell loss significantly in the duodenum and jejunum, but it did prevent it significantly in the ileum and colon. These findings comprise the first evidence that the nitrergic neurons located in different intestinal segments exhibit different susceptibilities to a diabetic state and to insulin treatment.

Chronic ethanol exposure impairs neuronal nitric oxide synthase in the rat intestine.

BACKGROUND: Nitric oxide (NO), synthesized by neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) nitric oxide synthases, plays an essential role in the physiological functions of the gastrointestinal (GI) tract. Chronic ethanol intake has been shown to interfere with several of these physiological functions, leading to the pathological alterations observed in alcoholic individuals. Our aim therefore was to investigate the effects of chronic ethanol consumption on NOS isoforms in different GI segments. METHODS: Rats received either 20% aqueous ethanol solution or water for 8 weeks. Tissue samples of the duodenum, jejunum, ileum, and colon of the rats were used for measurement of the NOS activity, protein content, and nNOS immunohistochemistry. Anti-HuC/D immunohistochemistry was used to determine the total number of neurons. RESULTS: Measurement of the physiological constitutive NOS (cNOS) activity revealed a 20 times higher activity in the colon than in the small intestine and after chronic ethanol treatment demonstrated a significant decrease in the jejunum, ileum, and colon, while in the duodenum it remained unchanged compared with the control group. The physiological iNOS activity was higher in the ileum and colon than in the duodenum and jejunum, and these levels were not significantly affected by ethanol. Neuronal nitric oxide synthase immunohistochemistry revealed a significant decrease in the numbers of immunostained cells in all investigated intestinal segments, while the total number of myenteric neurons remained constant. The nNOS protein content measured by Western blotting indicated a significant decrease in the colon after ethanol consumption, while in other intestinal segments change was not detectable. CONCLUSIONS: This study has demonstrated for the first time that chronic ethanol consumption has a differential effect on NOS activity, NOS protein content, and the number of nitrergic neurons in different intestinal segments, suggesting that chronic ethanol administration affects the NO pathways in the enteric nervous system.