LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation.
In this report we investigate the molecular mechanisms that contribute to tissue damage following ischemia and ischemia coupled with reperfusion (ischemia/ reperfusion) in the rat heart and kidney. We observe the activation of three stress-inducible mitogen-activated protein (MAP) kinases in these tissues: p38 MAP kinase and the 46-and 55-kDa isoforms of Jun N-terminal kinase (JNK 46 and JNK 55 ). The heart and kidney show distinct time courses in the activation of p38 MAP kinase during ischemia but no activation of either JNK 46 or JNK 55 . These two tissues also respond differently to ischemia/reperfusion. In the heart we observe activation of JNK 55 and p38 MAP kinase, whereas in the kidney all three kinases are active. We also examined the expression pattern of two stress-responsive genes, c-Jun and ATF3. Our results indicate that in the heart both genes are induced by ischemia and ischemia/reperfusion. However, in the kidney c-Jun and ATF3 expression is induced only by ischemia/reperfusion. To correlate these molecular events with tissue damage we examined DNA laddering, a common marker of apoptosis. A significant increase in DNA laddering was evident in both heart and kidney following ischemia/reperfusion and correlated with the pattern of kinase activation, supporting a link between stress kinase activation and apoptotic cell death in these tissues.
This study discovered oncogenic BRAF deletions with a distinct activation mechanism dependent on the BRAF dimer formation in tumor cells. LY3009120 is active against these cells and represents a potential treatment option for patients with cancer with these BRAF deletions, or other atypical BRAF mutations where BRAF functions as a dimer.
The hemagglutinin-tagged human trace amine-associated receptor1 (TAAR1) was stably coexpressed with rat G␣ s in the AV12-664 cell line, and receptor activation was measured as the stimulation of cAMP formation. After blockade of endogenously expressed ␣ 2 -and -adrenoceptors with 2-[2-(2-methoxy-1,4-benzodioxanyl)]-imidazoline hydrochloride (2-methoxyidazoxan, RX821002) and alprenolol, respectively, the resulting pharmacology was consistent with that of a unique receptor subtype. -Phenylethylamine (-PEA), the putative endogenous ligand, gave an EC 50 of 106 Ϯ 5 nM in the assay. For a series of -PEA analogs used to explore the pharmacophore, small substituents at ring positions 3 and/or 4 generally resulted in compounds having lower potency than -PEA, although several were as potent as -PEA. However, small substituents at ring position 2 resulted in a number of compounds having potencies as good as or better than -PEA. A number of nonselective antagonists known to share affinity for multiple monoaminergic receptors were evaluated for their ability to inhibit -PEA stimulation of the human TAAR1. None had an IC 50 Ͻ10 M. For comparison, the rat TAAR1 receptor was expressed in the AV12-664 cell line. A number of agonist compounds had significantly different relative potencies between the rat and human TAAR1, demonstrating a significant species difference between the rat and human TAAR1. The TAAR1 receptor exhibits a pharmacologic profile uniquely different from those of classic monoaminergic receptors, consistent with the structural information that places them in a distinct family of receptors. This unique pharmacologic profile suggests the potential for development of TAAR-selective agonists and antagonists to study their physiologic roles.The trace amines are congeners of the so-called classic monoamine or biogenic amine neurotransmitters, e.g., dopamine, norepinephrine and serotonin, but are found in the brain in much lower concentrations (nanograms per gram or less) than the classic neurotransmitters (Baldessarini and Fischer, 1978;Philips et al., 1978). Compounds typically discussed under the category of trace amines include (but are not limited to) -phenylethylamine, m-and p-tyramine, octopamine, and tryptamine. Hypotheses regarding the possible actions of the trace amines in normal physiology and disease states were published as early as the 1970s (Baldessarini and Fischer, 1978;Philips et al., 1978;Boulton, 1980). However, this field of study remained on the fringes of neurotransmitter research because of the lack of tools that would differentiate the actions of trace amines from those of other biogenic amine neurotransmitters. In 2001, cloning studies revealed the existence of a group of receptors described as the trace amine receptor family (Borowsky et al., 2001;Bunzow et al., 2001). These initial reports have been followed by several reviews and additional characterizations of these receptors (Branchek and Blackburn, 2003;Lewin, 2006;Navarro et al., 2006). However, given the time since ...
Nuclear factor-kappa B (NF-kappaB) is a multisubunit transcription factor that when activated induces the expression of genes encoding acute-phase proteins, cell adhesion molecules, cell surface receptors, and cytokines. NF-kappaB is composed of a variety of protein subunits of which p50-and p65-kDa (RelA) are the most widely studied. Under resting conditions, these subunits reside in the cytoplasm as an inactive complex bound by inhibitor proteins, IkappaB alpha and IkappaB beta. On activation, IkappaB is phosphorylated by IkappaB kinase and ubiquitinated and degraded by the proteasome; simultaneously, the active heterodimer translocates to the nucleus where it can initiate gene transcription. In the periphery, NF-kappaB is involved in inflammation through stimulation of the production of inflammatory mediators. The role of NF-kappaB in the brain is unclear. In vitro, NF-kappaB activation can be either protective or deleterious. The role of NF-kappaB in ischemic neuronal cell death in vivo was investigated. Adult male rats were subjected to 2 hours of focal ischemia induced by middle cerebral artery occlusion (MCAO). At 2, 6, and 12 hours after reperfusion, the expression and transactivation of NF-kappaB in ischemic versus nonischemic cortex and striatum were determined by immunocytochemistry and by electrophoretic mobility gel-shift analysis. At all time points studied, p50 and p65 immunoreactivity was found exclusively in the nuclei of cortical and striatal neurons in the ischemic hemisphere. The contralateral nonischemic hemisphere showed no evidence of nuclear NF-kappaB immunoreactivity. Double immunofluorescence confirmed expression of p50 in nuclei of neurons. Increased NF-kappaB DNA-binding activity in nuclear extracts prepared from the ischemic hemisphere was further substantiated by electrophoretic mobility gel-shift analysis. Because the activation of NF-kappaB by many stimuli can be blocked by antioxidants in vitro, the effect of the antioxidant, LY341122, previously shown to be neuroprotective, on NF-kappaB activation in the MCAO model was evaluated. No significant activation of NF-kappaB was found by electrophoretic mobility gel-shift analysis in animals treated with LY341122. These results demonstrate that transient focal cerebral ischemia results in activation of NF-kappaB in neurons and supports previous observations that neuroprotective antioxidants may inhibit neuronal death by preventing the activation of NF-kappaB.
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