Selective Inhibitor Of Protein Kinase Research Articles

Activation of the TRPV4 Ion Channel Is Enhanced by Phosphorylation

The TRPV4 (transient receptor potential vanilloid 4) ion channel, a member of the vanilloid subfamily of the transient receptor potential channels, is activated by membrane stretch, by non-noxious warm temperatures, and by a range of chemical activators. In the present study we examined the role of phosphorylation in modulating the activation of TRPV4. We expressed TRPV4 in HEK293 cells and activated the channel by cell swelling in a hypotonic solution. TRPV4 channel activation and serine phosphorylation were enhanced by exposure to the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate or by application of bradykinin, which activates PKC via a G-protein-coupled mechanism. The enhancement was inhibited by the PKC inhibitors staurosporine, bisindolylmaleimide I, and rottlerin or by mutation of the serine/threonine residues Ser(162), Thr(175), and Ser(189). The adenylate cyclase activator forskolin also enhanced activation of TRPV4, and the enhancement was antagonized by the selective cyclic AMP-dependent protein kinase (PKA) inhibitor H89 or by mutation of serine residue Ser(824). Sensitization of TRPV4 by both PKC and PKA depended on the scaffolding protein AKAP79, because channel activation and phosphorylation were enhanced by co-transfection of AKAP79 and were antagonized by removal of AKAP79 using small interfering RNA. We conclude that the serine/threonine kinases PKC and PKA enhance activation of the TRPV4 ion channel by phosphorylation at specific sites and that phosphorylation depends on assembly of PKC and PKA by AKAP79 into a signaling complex with TRPV4.

Discovery of 3-(1H-Indol-3-yl)-4-[2-(4-methylpiperazin-1-yl)quinazolin-4-yl]pyrrole-2,5-dione (AEB071), a Potent and Selective Inhibitor of Protein Kinase C Isotypes

A series of novel maleimide-based inhibitors of protein kinase C (PKC) were designed, synthesized, and evaluated. AEB071 (1) was found to be a potent, selective inhibitor of classical and novel PKC isotypes. 1 is a highly efficient immunomodulator, acting via inhibition of early T cell activation. The binding mode of maleimides to PKCs, proposed by molecular modeling, was confirmed by X-ray analysis of 1 bound in the active site of PKCalpha.

Selective Inhibition of Protein Kinase C β2 Attenuates Inducible Nitric Oxide Synthase–Mediated Cardiovascular Abnormalities in Streptozotocin-Induced Diabetic Rats

OBJECTIVEImpaired cardiovascular function in diabetes is partially attributed to pathological overexpression of inducible nitric oxide synthase (iNOS) in cardiovascular tissues. We examined whether the hyperglycemia-induced increased expression of iNOS is protein kinase C-β2 (PKCβ2) dependent and whether selective inhibition of PKCβ reduces iNOS expression and corrects abnormal hemodynamic function in streptozotocin (STZ)-induced diabetic rats.RESEARCH DESIGN AND METHODSCardiomyocytes and aortic vascular smooth muscle cells (VSMC) from nondiabetic rats were cultured in low (5.5 mmol/l) or high (25 mmol/l) glucose or mannitol (19.5 mmol/l mannitol + 5.5 mmol/l glucose) conditions in the presence of a selective PKCβ inhibitor, LY333531 (20 nmol/l). Further, the in vivo effects of PKCβ inhibition on iNOS-mediated cardiovascular abnormalities were tested in STZ-induced diabetic rats.RESULTSExposure of cardiomyocytes to high glucose activated PKCβ2 and increased iNOS expression that was prevented by LY333531. Similarly, treatment of VSMC with LY333531 prevented high glucose–induced activation of nuclear factor κB, extracellular signal–related kinase, and iNOS overexpression. Suppression of PKCβ2 expression by small interference RNA decreased high-glucose–induced nuclear factor κB and extracellular signal–related kinase activation and iNOS expression in VSMC. Administration of LY333531 (1 mg/kg/day) decreased iNOS expression and formation of peroxynitrite in the heart and superior mesenteric arteries and corrected the cardiovascular abnormalities in STZ-induced diabetic rats, an action that was also observed with a selective iNOS inhibitor, L-NIL.CONCLUSIONSCollectively, these results suggest that inhibition of PKCβ2 may be a useful approach for correcting abnormal hemodynamics in diabetes by preventing iNOS mediated nitrosative stress.

MAPK8 and MAPK11 Modulate TGFB1-Induced Changes in Epithelial Function Across Human and Porcine Vas Deferens Epithelial Cells.

Transforming growth factor 1 (TGFB1) is associated with pulmonary disease progression in cystic fibrosis (CF) patients although the impact of TGFB1 on the male reproductive duct is unknown. Cystic fibrosis, the most common recessive genetic disease in Caucasians, is almost universally (>98%) associated with congenital bilateral absence of the vas deferens (CBAVD) resulting in male infertility although pulmonary and pancreatic manifestations, which affect slightly smaller proportions of the population (~85%), are more likely to lead to mortality. Male CF patients may with present infertility or subfertility without CBAVD. In addition, a small number of CBAVD cases occur that are not accompanied by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and various cases of idiopathic human male infertility or subfertility are reported that are not of testicular-based etiology. This study aimed to determine whether TGFB1 can alter human and porcine vas deferens epithelial cell physiology and to investigate the signal transduction pathway through which TGFB1 may induce changes in vas deferens epithelial cell function. Primary porcine (1°PVD) and human (1°HVD) vas deferens epithelial cell monolayers were exposed to either vehicle or TGFB1, for 24 and/or 72 hours, and then assessed in modified Ussing chambers. Vehicle-treated 1°PVD presented basal transepithelial resistance (RTE, a sensitive measure of epithelial barrier function) of 4800 Ohms/cm2, while paired 1°PVD exposed to TGFB1 (100 ng/ml) for 24 and 72 hours had 470 and 45 Ohms/cm2, respectively. 1°HVD exposed to TGFB1 (5 ng/ml) for 72 hours also revealed significant reductions in RTE. The ability of both 1°HVD and 1°PVD to respond with increases in short-circuit current (ISC, a sensitive measure of anion secretion) upon forskolin exposure (2 μM) was also impaired in TGFB1-exposed monolayers at 72 hours. To determine if protein kinases modulate the TGFB1 effect, 1°PVD monolayers were exposed to TGFB1 (5 ng/ml) for 24 hours, either in the absence or presence of selective inhibitors of mitogen-activated protein kinases 1 and 3 (MAPK1/3, previously known as ERK1/2), MAPK8 (previously known as JNK-1), MAPK11 (previously known as p38-MAPK) and phosphoinositide-3-kinase (PIK3). Outcomes suggest that SP600125, a selective MAPK8-inhibitor (10 μM), and SKF86002 (10 μM), a selective MAPK11-inhibitor (10 μM), are able to reduce the decreases in RTE induced by TGFB1. These data suggest that both human and porcine vas deferens epithelial cells respond functionally to activation of the TGFB1 receptor in vitro with a significant reduction in epithelial barrier function. Thus, TGFB1 signaling seems to lead to a loss of cell-cell association that could lead to a change in tissue architecture that may ultimately result in destruction of the duct. (Supported by NIH HD058398 and RR017686) (platform)

Differential Effects of p38 Mitogen-Activated Protein Kinase and Cyclooxygenase 2 Inhibitors in a Model of Cardiovascular Disease

The evidence is compelling for a role of inflammation in cardiovascular diseases; however, the chronic use of anti-inflammatory drugs for these indications has been disappointing. The recent study compares the effects of two anti-inflammatory agents [cyclooxygenase 2 (COX2) and p38 inhibitors] in a model of cardiovascular disease. The vascular, renal, and cardiac effects of 4-(4-methylsulfonylphenyl)-3-phenyl-5H-furan-2-one (rofecoxib; a COX2 inhibitor) and 6-{5-[(cyclopropylamino)carbonyl]-3-fluoro-2-methylphenyl}-N-(2,2-dimethylpropyl)-3-pyridinecarboxamide [GSK-AHAB, a selective p38 mitogen-activated protein kinase (MAPK) inhibitor], were examined in the spontaneously hypertensive stroke-prone rat (SHR-SP). In SHR-SPs receiving a salt-fat diet (SFD), chronic treatment with GSK-AHAB significantly and dose-dependently improved survival, endothelial-dependent and -independent vascular relaxation, and indices of renal function, and it attenuated dyslipidemia, hypertension, cardiac remodeling, plasma renin activity (PRA), aldosterone, and interleukin-1beta (IL-1beta). In contrast, chronic treatment with a COX2-selective dose of rofecoxib exaggerated the harmful effects of the SFD, i.e., increasing vascular and renal dysfunction, dyslipidemia, hypertension, cardiac hypertrophy, PRA, aldosterone, and IL-1beta. The protective effects of a p38 MAPK inhibitor are clearly distinct from the deleterious effects of a selective COX2 inhibitor in the SHR-SP and suggest that anti-inflammatory agents can have differential effects in cardiovascular disease. The results also suggest a method for evaluating long-term cardiovascular efficacy and safety.

Milrinone inhibits hypoxia or hydrogen dioxide-induced persistent sodium current in ventricular myocytes

Much evidence indicates that increased persistent sodium current (I Na.P) is associated with cellular calcium overload and I Na.P is considered to be a potential target for therapeutic intervention in ischaemia and heart failure. By inhibiting type III phosphodiesterase, milrinone increases intracellular cyclic adenosine monophosphate (cAMP), with a positive inotropic effect. However, the effect of milrinone on increased I Na.P under pathological conditions remains unknown. Accordingly, we investigated the effect of milrinone on increased I Na.P induced by hypoxia or hydrogen dioxide in guinea pig ventricular myocytes. While milrinone (0.01 mM or 0.1 mM) or cAMP (0.1 mM) decreased I Na.P respectively in control condition, application of 1 μM H-89, a selective cAMP-dependant protein kinase inhibitor, prevented the effect of 0.1 mM milrinone in control condition. Milrinone (0.1 mM) reduced the increased I Na.P induced by hypoxia. Furthermore, 0.01 mM or 0.1 mM milrinone reduced the enhanced I Na.P induced by 0.3 mM hydrogen peroxide. In addition, 0.01 mM or 0.1 mM milrinone shortened action potential duration at 90% repolarization (APD 90). Bath application of 0.3 mM hydrogen dioxide markedly prolonged APD 90, while 2 μM tetrodotoxin (TTX) reversed the prolonged APD 90. In the other two groups, 0.01 mM or 0.1 mM milrinone shortened the prolonged APD 90 induced by 0.3 mM hydrogen peroxide, ultimately 2 μM TTX causing a further decurtation of APD 90. These findings demonstrate that milrinone inhibited I Na.P under normal condition, hypoxia or hydrogen dioxide-induced I Na.P, and the APD 90 prolonged by hydrogen dioxide-induced I Na.P in ventricular myocytes, which is associated with the mechanism of milrinone increasing intracellular cAMP.

Mode of Action of cGMP-dependent Protein Kinase-specific Inhibitors Probed by Photoaffinity Cross-linking Mass Spectrometry

The inhibitor peptide DT-2 (YGRKKRRQRRRPPLRKKKKKH) is the most potent and selective inhibitor of the cGMP-dependent protein kinase (PKG) known today. DT-2 is a construct of a PKG tight binding sequence (W45, LRKKKKKH, KI=0.8 microM) and a membrane translocating sequence (DT-6, YGRKKRRQRRRPP, KI=1.1 microM), that combined strongly inhibits PKG catalyzed phosphorylation (KI=12.5 nM) with approximately 1000-fold selectivity toward PKG over protein kinase A, the closest relative of PKG. However, the molecular mechanism behind this inhibition is not entirely understood. Using a combination of photoaffinity labeling, stable isotope labeling, and mass spectrometry, we have located the binding sites of PKG-specific substrate and inhibitor peptides. Covalent linkage of a PKG-specific substrate analogue was localized in the catalytic core on residues 356-372, also known as the glycine-rich loop, essential for ATP binding. By analogy, the individual inhibitor peptides W45 and DT-6 were also found to cross-link near the glycine-rich loop, suggesting these are both substrate competitive inhibitors. A bifunctional photoreactive analogue of DT-2 was found to generate dimers of PKG. This cross-linking induced covalent PKG dimerization was not observed for an N-terminal deletion mutant of PKG, which lacks the dimerization domain. In addition, non-covalent mass spectrometry was used to determine binding stoichiometry and binding order of the inhibitor peptides. Dimeric PKG binds two W45 and DT-6 peptides, whereas only one DT-2 molecule was observed to bind to the dimeric PKG. Taken together, these findings imply that (i) the two individual components making up DT-2 are both targeted against the substrate-binding site and (ii) binding of a single DT-2 molecule inactivates both PKG monomers simultaneously, which is an indication that (iii) in cGMP-activated PKG the catalytic centers of both subunits may be in each other's proximity.

Phase 1 study of LY317615 (enzastaurin) and temozolomide in patients with gliomas - EORTC trial 26054

e13005 Background: Enzastaurin (Enz) is a small selective inhibitor of protein kinase C (PKC). Synergy with temozolomide (TMZ) has been demonstrated in vitro. This phase I trial investigated optimal dosing and toxicity when Enz was combined with a standard TMZ regimen. Methods: Patients (pts) with recurrent high-grade glioma or newly diagnosed disease not amenable to radiotherapy (RT) and WHO PS 0–1 were eligible. Pts on EIAEDs were excluded. Two dose levels (DL) of Enz were explored. DL1: Loading dose 500 mg followed by 250 mg daily. DL2: Loading dose 1,125 mg followed by 500 mg daily. TMZ was given at 150 mg/m2 d1-d5 every 28 days in cycle 1, escalating to 200 mg/m2 in subsequent cycles. Dose-limiting toxicity (DLT) was defined by CTCAE 3.0 criteria in the first two cycles as (1) any non haematological toxicity grade 3/4 (2) ANC < 500/mm3 (3) thrombocytopenia grade 3/4 (4) any toxicity reducing drug dose intensity to <80%. Results: 15 pts were included, three at DL1, 12 at DL2. 13 pts had received prior RT, seven prior chemotherapy, and two were newly diagnosed. Median number of cycles was four (range 2–8).Treatment is ongoing in five pts. TMZ dose was escalated in all pts with no DLT. Modest and reversible thrombocytopenia was the most frequent toxicity, occurring in seven pts (3 @ Gd 1, 3 @ Gd2, 1 @ Gd 3) and led to TMZ dose reduction in two pts and treatment delays in four pts (multiple in 2). One pt discontinued after three cycles due to persistent thrombocytopenia. Five pts developed neutropenia not requiring dose modification. Two pts developed hyperbilirubinemia. Six SAE's (3 at each DL) were recorded, and one death (DL2). None was considered treatment related. Two pts showed a partial response, six pts stable disease. Conclusions: Enz can be safely combined with standard TMZ regimen. Results of the just completed phase I part with Enz 250 mg twice daily dosing will also be presented at the meeting. [Table: see text]

A Phase I Trial of Enzastaurin in Patients with Recurrent Gliomas

Enzastaurin is a selective inhibitor of protein kinase C beta. Prior phase I studies did not show increased drug exposures with escalating once daily administration. Limits from gastrointestinal absorption may be overcome by twice daily dosing, potentially improving antitumor effects. We conducted a phase I dose escalation study in 26 patients with recurrent malignant glioma, stratified by use of enzyme-inducing antiepileptic drugs, to investigate whether divided twice daily dosing results in higher exposures compared with once daily dosing. Phosphorylated glycogen synthase 3 beta was analyzed as a potential biomarker of enzastaurin activity. Enzastaurin was poorly tolerated at all dose levels evaluated (500, 800, and 1,000 mg total daily), with thrombocytopenia and prolonged QTc as dose-limiting toxicities. The average drug concentration of enzastaurin under steady-state conditions was doubled by twice daily dosing compared with daily dosing [1.990; 90% confidence interval (CI), 1.450-2.730]. Additionally, geometric mean ratios doubled with 800 versus 500 mg dosing for both daily (2.687; 90% CI, 1.232-5.860) and twice daily regimens (1.852; 90% CI, 0.799-4.292). Two patients achieved long-term benefit (over 150 weeks progression free). Higher and more frequent dosing of enzastaurin resulted in improved drug exposure but with unacceptable toxicity at the doses tested. Phosphorylated glycogen synthase 3 beta may be a useful biomarker of the biological activity of enzastaurin. Enzastaurin has activity in a subset of malignant glioma patients and warrants continued study in combination with other agents using a maximal once daily dose of 500 mg.

Structural Characterization of Proline-rich Tyrosine Kinase 2 (PYK2) Reveals a Unique (DFG-out) Conformation and Enables Inhibitor Design

Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptor tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase inhibitors.

Phosphodiesterase 5 restricts NOS3/Soluble guanylate cyclase signaling to L-type Ca 2+ current in cardiac myocytes

Endothelial nitric oxide synthase (NOS3) regulates the functional response to β-adrenergic (β-AR) stimulation via modulation of the L-type Ca 2+ current ( I Ca). However, the NOS3 signaling pathway modulating I Ca is unknown. This study investigated the contribution of soluble guanylate cyclase (sGC) and phosphodiesterase type 5 (PDE5), a cGMP-specific PDE, in the NOS3-mediated regulation of I Ca. Myocytes were isolated from NOS3 knockout ( NOS3 −/− ) and wildtype (WT) mice. We measured I Ca (whole-cell voltage-clamp), and simultaneously measured Ca 2+ transients (Fluo-4 AM) and cell shortening (edge detection). Zaprinast (selective inhibitor of PDE5), decreased β-AR stimulated (isoproterenol, ISO)- I Ca, and Ca 2+ transient and cell shortening amplitudes in WT myocytes. However, YC-1 (NO-independent activator of sGC) only reduced ISO-stimulated I Ca, but not cardiac contraction. We further investigated the NOS3/sGC/PDE5 pathway in NOS3 −/− myocytes. PDE5 is mislocalized in these myocytes and we observed dissimilar effects of PDE5 inhibition and sGC activation compared to WT. That is, zaprinast had no effect on ISO-stimulated I Ca, or Ca 2+ transient and cell shortening amplitudes. Conversely, YC-1 significantly decreased both ISO-stimulated I Ca, and cardiac contraction. Further confirming that PDE5 localizes NOS3/cGMP signaling to I Ca; YC-1, in the presence of zaprinast, now significantly decreased ISO-stimulated Ca 2+ transient and cell shortening amplitudes in WT myocytes. The effects of YC-1 on I Ca and cardiac contraction were blocked by KT5823 (a selective inhibitor of the cGMP-dependent protein kinase, PKG). Our data suggests a novel physiological role for PDE5 in restricting the effects of NOS3/sGC/PKG signaling pathway to modulating β-AR stimulated I Ca, while limiting effects on cardiac contraction.

β-adrenergic enhancement of brain kynurenic acid production mediated via cAMP-related protein kinase A signaling

The central levels of endogenous tryptophan metabolite kynurenic acid (KYNA), an antagonist of N-methyl- d-aspartate (NMDA) and α7-nicotinic receptors, affect glutamatergic and dopaminergic neurotransmission. Here, we demonstrate that selective agonists of β 1-receptors (xamoterol and denopamine), β 2-receptors (formoterol and albuterol), α- and β-receptors (epinephrine), 8pCPT-cAMP and 8-Br-cAMP (analogues of cAMP) increase the production of KYNA in rat brain cortical slices and in mixed glial cultures. Neither betaxolol, β 1-adrenergic antagonist, nor timolol, a non-selective β 1,2-adrenergic antagonist has influenced synthesis of KYNA in both paradigms. In contrast, KT5720, a selective inhibitor of protein kinase A (PKA), strongly reduced KYNA formation in cortical slices (2–10 µM) and in glial cultures (100 nM). β-adrenergic antagonists and KT5720 prevented the β-adrenoceptor agonists-induced increases of KYNA synthesis. In vivo, β-adrenergic agonist clenbuterol (0.1–1.0 mg/kg) increased the cortical endogenous level of KYNA; the effect was blocked with propranolol (10 mg/kg). β-adrenoceptors agonists, cAMP analogues and KT5720 did not affect directly the activity of KAT I or KAT II measured in partially purified cortical homogenate. In contrast, the exposure of intact cultured glial cells to pCPT-cAMP, 8-Br-cAMP and formoterol has lead to an enhanced action of KATs. These findings demonstrate that β-adrenoceptor-mediated enhancement of KYNA production is a cAMP- and PKA-dependent event. PKA activity appears to be an essential signal affecting KYNA formation. Described here novel mechanism regulating KYNA availability may be of a potential importance, considering that various stimuli, among them clinically used drugs, activate cAMP/PKA pathway, and thus could counteract the central deficits of KYNA.

Prostaglandin E2 couples through EP4 prostanoid receptors to induce IL‐8 production in human colonic epithelial cell lines

Prostaglandin (PG) E(2) and interleukin (IL)-8 are simultaneously increased during the inflammation that characterizes numerous pathologies such as inflammatory bowel disease. IL-8 is a potent neutrophil chemo-attractant and activator, and can initiate and/or exacerbate tissue injury. PGE(2) signals principally through prostanoid receptors of the EP(2) and/or EP(4) subtypes to promote cAMP-dependent cellular functions. The aim of this study was to identify the role of the EP(2) and EP(4) receptor subtype(s) on two human colonic epithelial cell lines (Caco-2 and T84), in regulating PGE(2)-induced IL-8 production. To identify the causative receptor, we knocked-down and over-expressed EP(2) and EP(4) receptor subtypes in colonic epithelial cells and studied the effect of several selective EP(2)/EP(4) receptor agonists and antagonists. The inductions of IL-8 and EP receptor mRNA and protein expression were determined by real-time PCR and western blot analysis. The affinity of PGE(2) and Bmax values for the EP(2) and EP(4) receptor on colonic epithelial cells were determined by radioligand-binding assays with [(3)H]PGE(2). PGE(2) had the highest affinity for the EP(4) receptor subtype and promoted a robust stimulation of cAMP-dependent IL-8 synthesis. This effect was mimicked by a selective EP(4) receptor agonist, ONO-AE1-329, and abolished by silencing the EP(4) receptor gene by using siRNA techniques, a selective EP(4) receptor antagonist (ONO-AE3-208) and a selective inhibitor (Rp-cAMP) of cAMP-dependent protein kinase. These findings suggest that initiation and progression of colonic inflammation induced by IL-8 could be mediated, at least in part, by PGE(2) acting via the EP(4) receptor subtype.

Boundary conditions for the maintenance of memory by PKMzeta in neocortex.

We report here that ZIP, a selective inhibitor of the atypical protein kinase C isoform PKMzeta, abolishes very long-term conditioned taste aversion (CTA) associations in the insular cortex of the behaving rat, at least 3 mo after encoding. The effect of ZIP is not replicated by a general serine/threonine protein kinase inhibitor that is relatively ineffective toward PKMzeta, is independent of the intensity of training and the perceptual quality of the taste saccharin (conditioned stimulus, CS), and does not affect the ability of the insular cortex to re-encode the same specific CTA association again. The memory trace is, however, insensitive to ZIP during or immediately after training. This implies that the experience-dependent cellular plasticity mechanism targeted by ZIP is established following a brief time window after encoding, consistent with the standard period of cellular consolidation, but then, once established, does not consolidate further to gain immunity to the amnesic agent. Hence, we conclude that PKMzeta is not involved in short-term CTA memory, but is a critical component of the cortical machinery that stores long- and very long-term CTA memories.

Chemical biology of natural indolocarbazole products: 30 years since the discovery of staurosporine

Staurosporine was discovered at the Kitasato Institute in 1977 while screening for microbial alkaloids using chemical detection methods. It was during the same era that protein kinase C was discovered and oncogene v-src was shown to have protein kinase activity. Staurosporine was first isolated from a culture of Actinomyces that originated in a soil sample collected in Mizusawa City, Japan. Thereafter, indolocarbazole compounds have been isolated from a variety of organisms. The biosynthesis of staurosporine and related indolocarbazoles was finally elucidated during the past decade through genetic and biochemical studies. Subsequently, several novel indolocarbazoles have been produced using combinatorial biosynthesis. In 1986, 9 years since its discovery, staurosporine and related indolocarbazoles were shown to be nanomolar inhibitors of protein kinases. They can thus be viewed as forerunners of today's crop of novel anticancer drugs. The finding led many pharmaceutical companies to search for selective protein kinase inhibitors by screening natural products and through chemical synthesis. In the 1990s, imatinib, a Bcr-Abl tyrosine kinase inhibitor, was synthesized and, following human clinical trials for chronic myelogenous leukemia, it was approved for use in the USA in 2001. In 1992, mammalian topoisomerases were shown to be targets for indolocarbazoles. This opened up new possibilities in that indolocarbazole compounds could selectively interact with ATP-binding sites of not only protein kinases but also other proteins that had slight differences in ATP-binding sites. ABCG2, an ATP-binding cassette transporter, was recently identified as an important new target for indolocarbazoles.

Effects of Enzastaurin, Alone or in Combination, on Signalling Pathway Controlling Growth and Survival of B-Cell Lymphoma Cell Lines

Background. Although, the recent introduction of Rituximab in combination with chemotherapy has improved outcome of patients with indolent lymphomas, in particular FL, these diseases are still considered incurable and the majority of patients still have a fatal course. Enzastaurin, an acyclic bisindolylmaleimide, is a potent and selective competitive inhibitor of protein kinase C beta, which has been shown to inhibit cell proliferation and angiogenesis in human cancer cell lines. The purpose of the present study was to test enzastaurin for its effects on proliferation and survival of B-cell lines established from NHL patients.Methods. The WSU-NHL and Karpas 422 were kindly provided Dr M Introna ( Division of Haematology, Ospedali Riuniti, Bergamo, Italy); the RL was purchased by DSMZ. All three lines are carrying the t(14; 18) and Karpas 422 is EBV+. We decided to conduct all the experiments under the optimal culture conditions with 10% FCS to avoid subjecting the cells to further stress stimuli. IC50 values were calculated from curves based on enzastaurin concentration ranging from 1 to 10μM using MTT assay and cell viability assessment by Trypan Blue exclusion. Cell apoptosis was assessed by flow cytometer after staining with Annexin V-FITC and propidium iodide. The effects of enzastaurin on caspases activation as well as on AKT phosphorilation were evaluated by Western blotting. We also investigated the interaction of enzastaurin with chlorambucil and fludarabine. Results using MTT assay were expressed as fraction of cells killed by the individual drug or the combination in the drug-treated versus untreated cells. The interaction between drugs was analyzed by isobologram analysis using the StaCorp8.2 software program based upon the Chou-Talalay method to determine if the combinations were additive or synergistic.Results. We found that enzastaurin alone inhibits the proliferation of B-cell lymphomas cell lines at IC50 values ranging from 5 to 7.5 μM after 48 h and from 2.5 to 3.2 μM after 72 h. Induction of apoptosis by enzastaurin evaluated on WSU-NHL and RL, by flow cytometry analysis of membrane permeability, showed that enzastaurin induces an increase in the percentage of apoptotic cells compared with untreated controls in a time-dependent fashion. Furthermore, enzastaurin induces the appearance of the cleaved caspase-3 fragment in the same cell lines in a time dependent fashion. Activation of the apoptotic pathway was confirmed by cleavage of the PARP enzyme. The apoptosis is partially prevented by the ZVAD–fmk broad caspase inhibitor. Phosphorilation status analysis of AKT up to 72h of treatment showed a decrease of AKT phosphorilation starting from 48h after treatment. We tested the effect of enzastaurin combined with chlorambucil and fludarabine,drugs that are active against B-cell lymphoma and we demonstrated that these agents enhanced the cytotoxicity triggered by enzastaurin in a dose-dependent fashion. A clear synergistic interaction (CI=0.87) appeared using low concentrations of the drugs and increased (CI=0.1) at high concentrations.Conclusions. Our data suggest that in B lymphoma cell lines carrying t(14; 18), enzastaurin elicits its antitumor effect suppressing AKT phosphorilation, inducing apoptosis and inhibiting proliferation. Furthermore, enzastaurin synergizes with chlorambucil and fludarabine. These results support the potential use of enzastaurin in patients with NHL, and in particular provide a rationale for combination with chlorambucil and fludarabine.

A role of p38 mitogen-activated protein kinase in adenosine A1 receptor-mediated synaptic depotentiation in area CA1 of the rat hippocampus

BackgroundAlthough long-term potentiation (LTP) of synaptic strength is very persistent, current studies have provided evidence that various manipulations or pharmacological treatment when applied shortly after LTP induction can reverse it. This kind of reversal of synaptic strength is termed as depotentiation and may have a function to increase the flexibility and storage capacity of neuronal networks. Our previous studies have demonstrated that an increase in extracellular levels of adenosine and subsequent activation of adenosine A1 receptors are important for the induction of depotentiation; however, the signaling downstream of adenosine A1 receptors to mediate depotentiation induction remains elusive.ResultsWe confirm that depotentiation induced by low-frequency stimulation (LFS) (2 Hz, 10 min, 1200 pulses) was dependent on adenosine A1 receptor activation, because it was mimicked by bath-applied adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and was inhibited by the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Pretreatment of the hippocampal slices with the selective p38 mitogen-activated protein kinase (MAPK) inhibitors, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl]-5-(4-pyrudyl)-1H-imidazole (SB203580) or trans-1-(4-hydroxycyclohexyl)-4-(fluorophenyl)-5-(2-methoxypyrimidin-4-yl)imidazole (SB239063), prevented the induction of depotentiation by LFS and CPA. In agreement with electrophysiological observation, both LFS- and CPA-induced depotentiation are associated with an increase in p38 MAPK activation, which are blocked by DPCPX or SB203580 application.ConclusionThese results suggest that activation of adenosine A1 receptor and in turn triggering p38 MAPK signaling may contribute to the LFS-induced depotentiation at hippocampal CA1 synapses.

The p38 MAPK signaling pathway regulates neuronal apoptosis through the phosphorylation of the retinoblastoma protein

We investigated the role of SB202190, a selective p38 mitogen-activated protein kinase (MAPK) inhibitor in cerebellar granule neurons (CGC) in response to serum potassium deprivation (S/K deprivation), an apoptotic stimulus. CGC apoptosis after S/K deprivation was shown to be mediated through cell cycle re-entry and the induction of transcription factor E2F-1. We found that SB 202190 (10 μM) inhibits retinoblastoma protein (pRb) phosphorylation, in response to S/K deprivation. Moreover, the expression of cyclin E and E2F-1 were also significantly decreased. Interestingly, SB202190 did not affect or modulate the increase in the protein expression levels of cyclin D1. Similarly, p-Akt and p-GSK3 protein levels, measured after 12 h S/K deprivation, did not appear to be regulated by SB 202190 (10 μM). These data indicate that the neuroprotective effects of the p38 inhibitor were not mediated via Akt activation. In conclusion, these results suggest that p38MAPK converged with the cell cycle in S/K deprivation-induced apoptosis through pRb phosphorylation.

Ghrelin signalling in guinea‐pig femoral artery smooth muscle cells

Our aim was to study the new signalling pathway of ghrelin in the guinea-pig femoral artery using the outward I(K) as a sensor. Whole-cell patch-clamp experiments were performed on single smooth muscle cells, freshly isolated from the guinea-pig femoral artery. The contractile force of isometric preparations of the same artery was measured using a wire-myograph. In a Ca2+- and nicardipine-containing external solution, 1 mmol L(-1) tetraethylammonium reduced the net I(K) by 49 +/- 7%. This effect was similar and not additive to the effect of the specific BK(Ca) channel inhibitor iberiotoxin. Ghrelin (10(-7) mol L(-1)) quickly and significantly reduced the amplitudes of tetraethylammonium- and iberiotoxin-sensitive currents through BK(Ca) channels. The application of 5 x 10(-6) mol L(-1) desacyl ghrelin did not affect the amplitude of the control I(K) but it successfully prevented the ghrelin-induced I(K) decrease. The effect of ghrelin on I(K) was insensitive to selective inhibitors of cAMP-dependent protein kinase, soluble guanylyl cyclase, cGMP-dependent protein kinase or a calmodulin antagonist, but was effectively antagonized by blockers of BK(Ca) channels, phosphatidylinositol-phospholipase C, phosphatidylcholine-phospholipase C, protein kinase C, SERCA, IP(3)-induced Ca2+ release and by pertussis toxin. The ghrelin-induced increase in the force of contractions was blocked when iberiotoxin (10(-7) mol L(-1)) was present in the bath solution. Ghrelin reduces I(K(Ca)) in femoral artery myocytes by a mechanism that requires activation of Galpha(i/o)-proteins, phosphatidylinositol phospholipase C, phosphatidylcholine phospholipase C, protein kinase C and IP(3)-induced Ca2+ release.

Injection of the Neuropeptide CNP into Dopaminergic Rat Brain Areas Decreases Alcohol Intake

Alcohol administration is known to alter several brain functions and behaviors in humans and in laboratory animals. One of the targets of ethanol is the mesocorticolimbic dopaminergic reward pathway. We used the "alcohol deprivation effect" test as a rat model of alcohol craving and relapse. The effect is characterized by increased alcohol intake and preference after several weeks of voluntary alcohol consumption followed by a withdrawal phase. The alcohol deprivation effect was found to be considerably reduced by the injection in dopaminergic brain structures of the neuropeptide CNP. This peptide is the most abundant natriuretic peptide in the brain, and signals via an intracellular rise in cyclic GMP. The effect of CNP was observed whether the peptide was injected in situ into the ventral tegmental area or into the prefrontal cortex. It was partially reversed by the injection in the same structures of KT5823, a selective inhibitor of the cGMP-dependent protein kinase. The results indicate that changes of cyclic GMP levels in dopaminergic rat brain areas participate in the neurobiological mechanisms underlying alcohol craving after withdrawal and/or alcohol dependence.