Synthesis and evaluation of a phosphonate analogue of the soluble guanylate cyclase activator YC-1

Soluble guanylate cyclase (sGC) is activated by the known benzylindazole derivative YC-1 [1-benzyl-3-(5'-hydroxymethyl-2'-furyl)-indazole]. YC-1 also acts synergistically with CO, activating sGC to a level comparable to that achieved upon binding of nitric oxide, the endogenous activator of sGC. We here describe the synthesis of a YC-1 phosphonate analogue with improved aqueous solubility as well as its effects on sGC.     

Role of mitochondrial Bax, caspases, and MAPKs for ceramide-induced apoptosis in renal proximal tubular cells

It remains elusive whether crosstalk exists among mitochondrial Bax, caspases, and mitogen-activated protein kinases (MAPKs), and whether epidermal growth factor (EGF), which may activate MAPKs, affects ceramide-induced apoptosis through the crosstalk in renal proximal tubular cells (RPTCs). Effect of ceramide on expression of mitochondrial Bax and phosphorylated (p)-ERK, p38MAPK and JNK, that of MAPKs inhibition, and of EGF in the presence or absence of MAPKs inhibition on ceramide-induced apoptosis were examined in HK-2 cells. Apoptosis and expression of mitochondrial Bax and p-MAPKs were measured by Hoechst 33258 staining and Western blotting. C2-ceramide, but not dihydroC2-ceramide, inactive C2-ceramide, induced apoptosis at 24 h. C2-ceramide enhanced the mitochondrial Bax expression at 1 h, which was peaked at 3–6 h and decreased at 24 h, but remained increased, compared to control. An inhibitor of caspases, zVAD-fmk, ameliorated ceramide-induced apoptosis, suggesting a role of caspases for ceramide-induced apoptosis. C2-ceramide enhanced the expression of p-ERK and p-p38MAPK, but not p-JNK, at 1 h, which was increased till 24 h. An inhibitor of ERK, PD98059, or of p38MAPK, SB202190, failed to affect C2-ceramide-induced apoptosis. EGF, which enhanced the expression of p-ERK and p-p38MAPK but not p-JNK, ameliorated C2-ceramide-induced apoptosis without affecting mitochondrial Bax. Inhibition of ERK or p38MAPK failed to abolish the protective effect of EGF on C2-ceramide-induced apoptosis. Mitochondrial Bax and caspases, but not MAPKs, play a role for ceramide-induced apoptosis in RPTCs. EGF ameliorates ceramide-induced apoptosis in Bax- and MAPKs-independent pathways. The mechanism of ceramide-induced apoptosis and anti-apoptotic effect of EGF deserves further investigations.     

AMP-activated protein kinase (AMPK)/Ulk1-dependent autophagic pathway contributes to C6 ceramide-induced cytotoxic effects in cultured colorectal cancer HT-29 cells

Colorectal cancer is the second leading cause of cancer-related deaths. Drug resistance and/or off-target toxicity against normal cells limit the effectiveness of current chemotherapies for the treatment of colorectal cancer. In the current study, we studied the potential cytotoxic effects of short-chain and cell-permeable C6 ceramide in cultured colorectal cancer HT-29 cells and focused on the underlying mechanisms. We observed that C6 ceramide-induced HT-29 cell death and growth inhibition in a dose- and time-dependent manner. However, no significant apoptosis was observed in C6 ceramide-treated HT-29 cells. Our data support that autophagy contributed to C6 ceramide-induced cytotoxic effects, as autophagy inhibitors, 3-methyladenine (3-MA) and hydroxychloroquine, inhibited C6 ceramide’s effect; however, autophagy activators, everolimus (RAD001) and temsirolimus, mimicked C6 ceramide effects and induced HT-29 cell death. Further, we indentified that AMP-activated protein kinase (AMPK)/Ulk1 signaling was required for autophagy induction by C6 ceramide, and AMPK silencing by a specific short hairpin RNA suppressed C6 ceramide-induced autophagy and cytotoxic effects. Reversely, forced activation of AMPK by its activator AICAR or by genetic manipulation caused autophagic death in HT-29 cells, which was inhibited by 3-MA. Our results suggest that autophagy, but not apoptosis, is a major contributor for C6 ceramide-induced cytotoxic effects in HT-29 cells, and activation of AMPK/Ulk1 is required for the process.     

Oleate prevents palmitate-induced mitochondrial dysfunction,insulin resistance and inflammatory signaling in neuronal cells

Elevated circulating levels of saturated free fatty acids (sFFAs; e.g. palmitate) are known to provoke inflammatory responses and cause insulin resistance in peripheral tissue. By contrast, mono- or poly-unsaturated FFAs are protective against sFFAs. An excess of sFFAs in the brain circulation may also trigger neuroinflammation and insulin resistance, however the underlying signaling changes have not been clarified in neuronal cells. In the present study, we examined the effects of palmitate on mitochondrial function and viability as well as on intracellular insulin and nuclear factor-κB (NF-κB) signaling pathways in Neuro-2a and primary rat cortical neurons. We next tested whether oleate preconditioning has a protective effect against palmitate-induced toxicity. Palmitate induced both mitochondrial dysfunction and insulin resistance while promoting the phosphorylation of mitogen-activated protein kinases and nuclear translocation of NF-κB p65. Oleate pre-exposure and then removal was sufficient to completely block subsequent palmitate-induced intracellular signaling and metabolic derangements. Oleate also prevented ceramide-induced insulin resistance. Moreover, oleate stimulated ATP while decreasing mitochondrial superoxide productions. The latter were associated with increased levels of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Inhibition of protein kinase A (PKA) attenuated the protective effect of oleate against palmitate, implicating PKA in the mechanism of oleate action. Oleate increased triglyceride and blocked palmitate-induced diacylglycerol accumulations. Oleate preconditioning was superior to docosahexaenoic acid (DHA) or linoleate in the protection of neuronal cells against palmitate- or ceramide-induced cytotoxicity. We conclude that oleate has beneficial properties against sFFA and ceramide models of insulin resistance-associated damage to neuronal cells.     

Synthesis of OSW saponin analogs with modified sugar residues and their antiproliferative activities

Eight monosaccharide analogs of the potent antitumor OSW saponins (2-9) were synthesized. One analog, 2-O-acetyl-alpha-l-arabinopyranoside 3, showed antiproliferative activity against the Jurkat cells (IC(50)=0.078microM) comparable to that of the disaccharide derivative (1).     

Inhibition of Neuronal Apoptosis by a Metalloporphyrin Superoxide Dismutase Mimic

Abstract: The objective of this study was to determine whether free radicals play a pathogenic role in neuronal apoptosis. The ability of Mn(III) tetrakis(benzoic acid) porphyrin (MnTBAP), a superoxide dismutase mimic, to inhibit staurosporine-induced neuronal apoptosis was tested in mixed cerebrocortical cultures. Staurosporine produced concentration-dependent cell death that was markedly inhibited by MnTBAP. Immunocytochemical analyses of cultures for neuron- and astrocyte-specific markers revealed that high concentrations of staurosporine induced the death of both neurons and astrocytes; both cell types were protected by MnTBAP. A less active congener of MnTBAP failed to protect cells against staurosporine-induced apoptosis. MnTBAP also protected cortical cultures against ceramide-induced apoptosis. These results support a role for oxidative stress in neuronal apoptosis.     

Synthesis and in vitro antibiotic activity of 16-membered 9-O-arylalkyloxime macrolides

A series of novel 9-O-arylalkyloxime analogs based on three different 16-membered macrolide scaffolds-5-O-mycaminosyltylonolide (OMT), tilmicosin, and 20-deoxy-20-(3,5-dimethyl-1-piperidin-1-yl)-OMT-was synthesized. In vitro antibiotic activities were assayed against Gram-positive Streptococcus pneumoniae and Staphylococcus aureus and Gram-negative Haemophilus influenzae bacterial strains. Analogs derived from OMT (3-15) showed similar or better antibacterial activities against macrolide-susceptible strains and enhanced activities against macrolide-resistant strains compared with erythromycin A, tylosin, or OMT. Similar results were observed for tilmicosin 9-O-arylalkyloxime analogs (18-24). In contrast, most of the 20-deoxy-20-(3,5-dimethyl-1-piperidin-1-yl)-OMT analogs (25-33) showed reduced antibacterial activities compared with OMT. Ribosome-binding studies were performed on compounds 12 (OMT derivative), 20 (tilmicosin derivative), and 29 [20-deoxy-20-(3,5-dimethyl-1-piperidin-1-yl)-OMT derivative]. It was found that these compounds interacted with both domain V and domain II of the Escherichia coli 23S rRNA.     

Death-associated protein (DAP) kinase plays a central role in ceramide-induced apoptosis in cultured hippocampal neurons.

Treatment of cultured hippocampal neurons with high concentrations of short-chain acyl ceramide derivatives, such as N-hexanoyl-D-sphingosine (C(6)-Cer), results in apoptotic cell death. We now show that death-associated protein (DAP) kinase plays an important role in mediating this effect. Upon incubation with C(6)-Cer, DAP kinase levels are elevated as early as 1 h after treatment, reaching levels 2-3-fold higher than untreated cells after 4 h. Neurons cultured from DAP kinase-deficient mice were significantly less sensitive to apoptosis induced by C(6)-Cer or by ceramide generated by high concentrations of nerve growth factor. A peptide corresponding to the 17 amino acids at the C terminus of DAP kinase protected wild type neurons from C(6)-Cer-induced death and from death induced by the addition of exogenous bacterial neutral sphingomyelinase, whereas a scrambled peptide had no protective effect, implying that the DAP kinase C-terminal tail inhibits the function of DAP kinase. Together, these data demonstrate that DAP kinase plays a central role in ceramide-induced cell death in neurons, but the pathway in which DAP kinase is involved is not the only one via which ceramide can induce apoptosis.     

Synthesis and cytotoxicity of (-)-renieramycin G analogs

(−)-Renieramycin G and fifteen C-22 analogs were prepared employing l-tyrosine as the chiral starting material. These analogs, along with (−)-renieramycin G itself, were evaluated in vitro for cytotoxicity against HCT-8, BEL-7402, A2780, MCF-7, A549, BGC-823, Ketr3, KB, Hela cells. The IC₅₀ values of most of these analogs were at the level of μM. Among these analogs, 2-thiophenecarboxylate ester derivative 17 exhibited potent cytotoxic activity against KB cell line with the IC₅₀ of 20 nM. From this study, it could be concluded that the C-22 side chain played an important role in the cytotoxic potency and specificity of this class of (−)-renieramycin G derivatives.     

Analogs of a potent maxi-K potassium channel opener with an improved inhibitory profile toward cytochrome P450 isozymes

Quinolinone 1 is a potent maxi-K potassium channel opener. In an effort to design analogs of 1 with a better inhibitory profile toward the CYP2C9 isozyme, the two acidic sites were chemically modified independently to generate a number of analogs. These analogs were evaluated as maxi-K channel openers in vitro using Xenopus laevis oocytes expressing cloned hSlo maxi-K channels. Compounds 15, 17, and 19 showed potent activity as maxi-K channel openers and were further evaluated for inhibition of the activity of the CYP2C9 isozyme. Compounds 17 and 19 showed diminished inhibitory potency against 2C9 and also against a panel of other more common CYP isozymes.     

Regulation of apoptosis during neuronal differentiation by ceramide and b-series complex gangliosides

Lipid analysis of gestational day E14.5 mouse brain revealed elevation of ceramide to a tissue concentration that induced apoptosis when added to the medium of neuroprogenitor cells grown in cell culture. Elevation of ceramide was coincident with the first appearance of b-series complex gangliosides (BCGs). Expression of BCGs by stable transfection of murine neuroblastoma (F-11) cells with sialyltransferase-II (ST2) resulted in a 70% reduction of ceramide-induced apoptosis. This was most likely due to an 80% reduced expression of prostate apoptosis response-4 (PAR-4). PAR-4 expression and apoptosis were restored by preincubation of ST2-transfected cells with N-butyl deoxinojirimycin (NB-DNJ) or PD98059, two inhibitors of ganglioside biosynthesis or p42/44 mitogen-activated protein (MAPK) kinase, respectively. In sections of day E14.5 mouse brain, the intermediate zone showed intensive staining for complex gangliosides, but only low staining for apoptosis (TUNEL) and PAR-4. Apoptosis and PAR-4 expression, however, were elevated in the ventricular zone which only weakly stained for complex gangliosides. Whole cell patch clamping revealed a 2-fold increased calcium influx in ST2-transfected cells, the blocking of which with nifedipine restored apoptosis to the level of untransfected cells. In serum-free culture, supplementation of the medium with IGF-1 was required to maintain MAPK phosphorylation and the anti-apoptotic effect of BCG expression. BCG-enhanced calcium influx and the presence of insulin-like growth factor-1 may thus activate a cell survival mechanism that selectively protects developing neurons against ceramide-induced apoptosis by up-regulation of MAPK and reduction of PAR-4 expression.     

Synthesis and antitumour activity of novel diterpenequinone salvicine and the analogs.

A novel diterpenequinone named salvicine (4), structurally modified derivative of a natural product, and a series of the novel analogs have been prepared. Most of the analogs were found to be potently active against tumor cell lines in vitro. Further study on 4 in vivo demonstrated that it possessed a significant antineoplastic activity against murine S-180 Sarcoma and Lewis lung cancer, and human lung adenocarcinoma xenografts A-549 and LAX-83. The preclinical studies of 4 are now under way.     

Alkyl isocyanates as active site-directed inactivators of guinea pig liver transglutaminase.

Alkyl isocyanates are effective inactivators of guinea pig liver transglutaminase. Based on the specificity of the reaction the protection against inactivation by glutamine substrate, and the essential nature of calcium for the inactivation reaction, it is concluded that these reagents act as amide substrate analogs and, thus function in an active site-specific manner. Support for the contention that inactivation results from alkyl thiocarbamate ester formation through the single active site sulfhydryl group of the enzyme is (a) the loss of one free--SH group and the incorporation of 1 mol of reagent/mol of enzyme in the reaction, (b) similarity in chemical properties of the inactive enzyme derivative formed to those previously reported for another alkyl thiocarbamoylenzyme and an alkyl thiocarbamoylcysteine derivative, and (c) the finding that labeled peptides from digests of [methyl-14C]thiocarbamoyltransglutaminase and those from digests of iodoacetamide-inactivated enzyme occupy similar positions on peptide maps. Transglutaminase was found to be inactivated neither by urethan anlogs of its active ester substrates nor by urea analogs of its amide substrates. It is concluded on the basis of these findings that inactive carbamoylenzyme derivatives are formed only by direct addition of the transglutaminase active--SH group to the isocyanate C--N double bond, and not, like several serine active site enzymes, by nucleophilic displacement with urethan analogs of substrate, or by nucleophilic displacement with urea analogs of substrate.     

Neurotrophic Factors Prevent Ceramide-Induced Apoptosis Downstream of c-Jun N-Terminal Kinase Activation in PC12 Cells

Abstract: Neurotrophic factors prevent apoptosis of PC12 cells in serum-free medium. The present study determines whether neurotrophic factors can prevent ceramide-induced apoptosis in PC12 cells and investigates the role that c-Jun N-terminal kinase (JNK) activation may play in this system. Ceramide-induced apoptosis was inhibited by nerve growth factor, basic fibroblast growth factor, pituitary adenylyl cyclase-activating peptide, 4-(8-chlorophenylthio)cyclic AMP, and the caspase inhibitor benzyloxycarbonyl-Val-Ala- dl -Asp fluoromethyl ketone (zVAD-FMK). It was surprising that inhibition of extracellular signal-regulated kinase and/or phosphatidylinositol 3-kinase did not markedly block the protective effects exerted by neurotrophic factors against ceramide-induced apoptosis, suggesting that neurotrophic factors can promote survival independently of these signaling pathways. Treatment of PC12 cells with ceramide resulted in a time-dependent increase in JNK activity. However, neither neurotrophic factors nor zVAD-FMK attenuated ceramide-stimulated JNK activation. Further experiments indicated that ceramide-induced apoptosis in PC12 cells requires new protein synthesis, and that nerve growth factor and zVAD-FMK can prevent apoptosis after JNK activity has been detected. These results indicate that ceramide-induced JNK activation is an early event and may be required for the expression of essential components of the apoptotic machinery. It is anticipated that neurotrophic factors inhibit ceramide-induced apoptosis by affecting signaling events downstream of JNK activation.     

Treatment of experimental Pneumocystis carinii pneumonia with analogs of pentamidine

Seven analogs of pentamidine were tested for their activity against an immunosuppressed rat model of Pneumocystis carinii pneumonia. Structural alterations of the pentamidine molecule included variations of the alkyl chain linking the two p-amidino phenoxy moieties and relocation of the amidine groups from the para to the meta position on the phenoxy rings. All analogs of pentamidine were active against P. carinii pneumonia when compared to a saline-treated control group. One derivative, 1, 4-di(4'-amidinophenoxy)butane, proved to be statistically more active than the parent drug.     

Treatment of Experimental Pneumocystis carinii Pneumonia with Analogs of Pentamidine

Seven analogs of pentamidine were tested for their activity against an immunosuppressed rat model of Pneumocystis carinii pneumonia. Structural alterations of the pentamidine molecule included variations of the alkyl chain linking the two p-amidino phenoxy moieties and relocation of the amidine groups from the para to the meu position on the phenoxy rings. All analogs of pentamidine were active against P. carinii pneumonia when compared to a saline-treated control group. One derivative, 1, 4-di(4'-amidinophenoxy)butanc, proved to be statistically more active than the parent drug.     

Brassinosteroids and analogs as neuroprotectors: synthesis and structure-activity relationships

We have demonstrated previously that the brassinosteroid (BR) 24-epibrassinolide exerts neuroprotective effects deriving from its antioxidative properties. In this study, we synthesized 2 natural BRs and 5 synthetic analogs and analyzed their neuroprotective actions in neuronal PC12 cells, against 1-methyl-4-phenylpyridinium (MPP(+)), a neurotoxin known to induce oxidative stress and degenerescence of dopaminergic neurons characteristic of Parkinsonian brains. We also tested the neuroprotective potential of 2 commercially available BRs. Our results disclosed that 6 of the 9 BRs and analogs tested protected neuronal PC12 cells against MPP(+) toxicity. In addition, our structure-activity study suggests that the steroid B-ring and lateral chain play an important role for their neuroprotective action.     

Direct binding to ceramide activates protein kinase Czeta before the formation of a pro-apoptotic complex with PAR-4 in differentiating stem cells

We have reported that ceramide mediates binding of atypical protein kinase C (PKC) zeta to its inhibitor protein, PAR-4 (prostate apoptosis response-4), thereby inducing apoptosis in differentiating embryonic stem cells. Using a novel method of lipid vesicle-mediated affinity chromatography, we showed here that endogenous ceramide binds directly to the PKCzeta.PAR-4 complex. Ceramide and its analogs activated PKCzeta prior to binding to PAR-4, as determined by increased levels of phosphorylated PKCzeta and glycogen synthase kinase-3beta and emergence of a PAR-4-to-phosphorylated PKCzeta fluorescence resonance energy transfer signal that co-localizes with ceramide. Elevated expression and activation of PKCzeta increased cell survival, whereas expression of PAR-4 promoted apoptosis. This suggests that PKCzeta counteracts apoptosis, unless its ceramide-induced activation is compromised by binding to PAR-4. A luciferase reporter assay showed that ceramide analogs activate nuclear factor (NF)-kappaB unless PAR-4-dependent inhibition of PKCzeta suppresses NF-kappaB activation. Taken together, our results show that direct physical association with ceramide and PAR-4 regulates the activity of PKCzeta. They also indicate that this interaction regulates the activity of glycogen synthase kinase-3beta and NF-kappaB.