Synthesis and biological activity of pyridopyridazin-6-one p38 MAP kinase inhibitors. Part 1

The development and synthesis of potent p38α MAP kinase inhibitors containing a pyridazinone platform is described. Evolution of the p38α selective pyridopyridazin-6-one series from the p38α/β dual inhibitor 2H-quinolizin-2-one series will be discussed in full detail.     

Translation inhibition during cell cycle arrest and apoptosis: Mcl-1 is a novel target for RNA binding protein CUGBP2

CUGBP2, a translation inhibitor, induces colon cancer cells to undergo apoptosis. Mcl-1, an antiapoptotic Bcl-2 family protein, interferes with mitochondrial activation to inhibit apoptosis. Here, we have determined the effect of CUGBP2 on Mcl-1 expression. We developed a HCUG2 cell line by stably expressing CUGBP2 in the HCT-116 colon cancer cells. HCUG2 cells demonstrate decreased levels of proliferation and increased apoptosis, compared with HCT-116 cells. Flow cytometry analysis demonstrated higher levels of cells in the G(2)-M phase. Western blot analyses demonstrated that there was decreased Bcl-2 and Mcl-1 protein but increased expression of Bax, cyclin B1, and Cdc2. Immunocytochemistry also demonstrated increased levels of cyclin B1 and Cdc2 in the nucleus of HCUG2 cells. However, there was colocalization of phosphorylated histone H3 with transferase-mediated dUTP nick-end labeling (TUNEL). Furthermore, immunostaining for alpha-tubulin demonstrated that there was disorganization of microtubules. These data suggest that CUGBP2 expression in HCUG2 cells induces the cells to undergo apoptosis during the G(2)-M phase of the cell cycle. We next determined the mechanism of CUGBP2-mediated reduction in Mcl-1 expression. Mcl-1 protein, but not Mcl-1 mRNA, was lower in HCUG2 cells, suggesting translation inhibition. CUGBP2 binds to Mcl-1 3'-untranslated region (3'-UTR) both in vitro and in HCUG2 cells. Furthermore, CUGBP2 increased the stability of both endogenous Mcl-1 and luciferase mRNA containing the Mcl-1 3'-UTR. However, luciferase protein expression from the luciferase-Mcl-1 3'-UTR mRNA was suppressed. Taken together, these data demonstrate that CUGBP2 inhibits Mcl-1 expression by inhibiting Mcl-1 mRNA translation, resulting in driving the cells to apoptosis during the G(2) phase of the cell cycle.     

Rethinking pseudokinases

Pseudokinases lack conservation of one or more of the catalytic residues in the kinase core and as a consequence are typically thought to be catalytically inactive. New work by Mukherjee et al. (2008) challenges this assumption. They show that the pseudokinase domain of CASK (Ca2+/calmodulin activated serine-threonine kinase) adopts an active conformation and displays catalytic activity in vivo.     

Fluid shear stress inhibits TNF-mediated JNK activation via MEK5-BMK1 in endothelial cells

Steady laminar blood flow protects vessels from atherosclerosis. We showed that flow decreased tumor necrosis factor-α (TNF)-mediated VCAM1 expression in endothelial cells (EC) by inhibiting JNK. Here, we determined the relative roles of MEK1, MEK5 and their downstream kinases ERK1/2 and BMK1 (ERK5) in flow-mediated inhibition of JNK activation. Steady laminar flow (shear stress = 12 dyn/cm²) increased BMK1 and ERK1/2 activity in EC. Pre-exposing EC for 10 min to flow inhibited TNF activation of JNK by 58%. A key role for BMK1, but not ERK1/2 was shown. (1) Incubation of EC with PD184352, at concentrations that blocked ERK1/2, but not BMK1, had no effect on flow inhibition of TNF-mediated JNK activation. (2) BIX02188, a MEK5-selective inhibitor, completely reversed the inhibitory effects of flow. These findings indicate that flow inhibits TNF-mediated signaling events in EC by a mechanism dependent on activation of MEK5-BMK1, but not MEK1-ERK1/2. These results support a key role for the MEK5-BMK1 signaling pathway in the atheroprotective effects of blood flow.     

In vivo measurement of flow-mediated vasodilation in living rats using high-resolution ultrasound

In humans, endothelial vasodilator function serves as a surrogate marker for cardiovascular health and is measured as changes in conduit artery diameter after temporary ischemia [flow-mediated dilation (FMD)]. Here we present an FMD-related approach to study femoral artery (FA) vasodilation in anesthetized rats. Diameter and Doppler flow were monitored in the FA. Using high-resolution ultrasound (35 MHz) and automated analysis software, we detected dose-dependent vasodilation using established endothelium-independent [intravenous nitroglycerin EC(50) = 3.3 x 10(-6) mol/l, peak 21Delta% (SD 4)] and endothelium-dependent [intra-arterial acetylcholine EC(50) = 1.3 x 10(-6) mol/l, peak 27Delta% (SD 4)] pharmacological vasodilators. Wall shear stress induced by intra-aortic injection of adenosine and infusion of saline at increasing rates (1.5-4.5 ml/min) led to vasodilation at 1 to 2 min. Transient hindlimb ischemia by common iliac occlusion (5 min) led to reactive hyperemia with flow velocity and wall shear stress increase and was followed by FA dilation [16Delta% (SD 2)], the latter of which was completely abolished by nitric oxide synthase (NOS) inhibition with N(G)-monomethyl-L-arginine [1Delta% (SD 2)]. FMD was significantly reduced in adult 20-24-wk-old animals compared with 9- to 10-wk-old animals, consistent with age-dependent endothelial dysfunction [16Delta% (SD 3) vs. 10Delta% (SD 3), P < 0.05]. Whereas FMD was completely NOS dependent in 9- to 10-wk-old animals, NOS-dependent mechanisms accounted for only half of the FMD in 20-24-wk-old animals, with the remainder being blocked by charybdotoxin and apamin, suggesting a contribution of endothelium-derived hyperpolarizing factor. To our knowledge, this is the first integrative physiological model to reproducibly study FMD of conduit arteries in living rats.     

Protein-protein interactions and membrane localization of the human organic solute transporter

Two proteins that mediate bile acid export from the ileal enterocyte, organic solute transporter (OST)-alpha and -beta, have recently been identified. It is unclear whether these two proteins associate directly and how they interact to mediate transport function and membrane localization. In this study, the protein-protein interactions, transport functions, and membrane localization of human (h)OST-alpha and -beta proteins were examined. The results demonstrated that coexpression of hOST-alpha and -beta in transfected cells resulted in a three- to fivefold increase of the initial rate of taurocholate influx or efflux compared with cells expressing each protein individually and nontransfected cells. Confocal microscopy demonstrated plasma membrane colocalization of hOST-alpha and -beta proteins in cells cotransfected with hOST-alpha and -beta cDNAs. Protein-protein interactions between hOST-alpha and -beta were demonstrated by mammalian two-hybrid and coimmunoprecipitation analyses. Truncation of the amino-terminal 50 amino acid extracellular residues of hOST-alpha abolished its interaction with hOST-beta and led to an intracellular accumulation of the two proteins and to only background levels of taurocholate transport. In contrast, carboxyl-terminal 28 amino acid truncated hOST-alpha still interacted with hOST-beta, and majority of this cytoplasmic tail-truncated protein was expressed on the basolateral membrane when it was stably cotransfected with hOST-beta protein in Madin-Darby canine kidney cells. In summary, hOST-alpha and -beta proteins are physically associated. The intracellular carboxyl-terminal domain of hOST-alpha is not essential for this interaction with hOST-beta. The extracellular amino-terminal fragment of hOST-alpha may contain important information for the assembly of the heterodimer and trafficking to the plasma membrane.     

Photolysis of caged sphingosine-1-phosphate induces barrier enhancement and intracellular activation of lung endothelial cell signaling pathways

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates cellular functions by ligation via G protein-coupled S1P receptors. In addition to its extracellular action, S1P also has intracellular effects; however, the signaling pathways modulated by intracellular S1P remain poorly defined. We have previously demonstrated a novel pathway of intracellular S1P generation in human lung endothelial cells (ECs). In the present study, we examined the role of intracellular S1P generated by photolysis of caged S1P on EC barrier regulation and signal transduction. Intracellular S1P released from caged S1P caused mobilization of intracellular calcium, induced activation of MAPKs, redistributed cortactin, vascular endothelial cadherin, and β-catenin to cell periphery, and tightened endothelial barrier in human pulmonary artery ECs. Treatment of cells with pertussis toxin (PTx) had no effect on caged S1P-mediated effects on Ca(2+) mobilization, reorganization of cytoskeleton, cell adherens junction proteins, and barrier enhancement; however, extracellular S1P effects were significantly attenuated by PTx. Additionally, intracellular S1P also activated small GTPase Rac1 and its effector Ras GTPase-activating-like protein IQGAP1, suggesting involvement of these proteins in the S1P-mediated changes in cell-to-cell adhesion contacts. Downregulation of sphingosine kinase 1 (SphK1), but not SphK2, with siRNA or inhibition of SphK activity with an inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (CII) attenuated exogenously administrated S1P-induced EC permeability. Furthermore, S1P1 receptor inhibitor SB649164 abolished exogenous S1P-induced transendothelial resistance changes but had no effect on intracellular S1P generated by photolysis of caged S1P. These results provide evidence that intracellular S1P modulates signal transduction in lung ECs via signaling pathway(s) independent of S1P receptors.     

Can geometry control the coordination behaviour of 2-hydroxy-1-naphthaldehyde-N(4)-phenylthiosemicarbazone? A study towards its origin

2-Hydroxy-1-naphthaldehyde-N(4)-phenylthiosemicarbazone [H₂-Nap-ptsc] (H₂L) was reacted with square planar complexes of the type [MCl₂(EPh₃)₂] (where M = Ni or Pd; E = P or As) and octahedral [RuHCl(CO)(PPh₃)₃]. Square planar complexes ([Ni(Nap-ptsc)PPh₃)]; [Pd(Nap-ptsc)PPh₃)]; [Pd(Nap-ptsc)AsPh₃)]) gave products with only ONS tri coordinated thiosemicarbazone whereas octahedral ruthenium complex gave two products, one with NS and the other with ONS coordination. It is interesting to note that NS bi coordination leads to the formation of more strained four member ring. All the new complexes have been characterised by analytical, spectral, crystallographic and electrochemical methods.