A diastereoselective three-component reaction for the assembly of succinimide and isatin hybrid molecules with potent anticancer activities.

A Rh2(OAc)4 catalyzed three-component reaction of vinyl diazosuccinimides with alcohols and isatins has been reported, which provides a practical assess to the direct assembly of succinimide and isatin hybrid molecules in good-to-high yields with excellent stereoselectivity. The antiproliferation activity of these synthesized succinimide and isatin hybrid products has been tested via the CCK8 assay in different cancer cell lines, and compounds 4g (SJSA-1, IC50 = 3.82 µM) and 4r (HCT-116, IC50 = 9.02 µM) exhibit higher anticancer potency than other tested compounds.

Three-Dimensional Reconstructed Bone Marrow Matrix Culture Improves the Viability of Primary Myeloma Cells In-Vitro via a STAT3-Dependent Mechanism.

Primary myeloma (PM) cells are short-lived in conventional culture, which limited their usefulness as a study model. Here, we evaluated if three-dimensional (3D) culture can significantly prolong the longevity of PM cells in-vitro. We employed a previously established 3D model for culture of bone marrow mononuclear cells isolated from 15 patients. We assessed the proportion of PM cells, viability and proliferation using CD38 staining, trypan blue exclusion assays and carboxy fluorescein succinimidyl ester (CFSE) staining, respectively. We observed significantly more CD38+ viable cells in 3D than in conventional culture (65% vs. 25%, p = 0.006) on day 3. CFSE staining showed no significant difference in cell proliferation between the two culture systems. Moreover, we found that PM cells in 3D culture are more STAT3 active by measure of pSTAT3 staining (66% vs. 10%, p = 0.008). Treatment of IL6, a STAT3 activator significantly increased CD38+ cell viability (41% to 68%, p = 0.021). In comparison, inhibition of STAT3 with Stattic significantly decreased PM cell viability in 3D culture (38% to 17% p = 0.010). Neither IL6 nor Stattic affected the PM cell viability in conventional culture. This study suggests that 3D culture can significantly improve the longevity of PM cells in-vitro, and STAT3 activation can further improve their viability.

Development of succinimide-based inhibitors for the mitochondrial rhomboid protease PARL.

While the biochemistry of rhomboid proteases has been extensively studied since their discovery two decades ago, efforts to define the physiological roles of these enzymes are ongoing and would benefit from chemical probes that can be used to manipulate the functions of these proteins in their native settings. Here, we describe the use of activity-based protein profiling (ABPP) technology to conduct a targeted screen for small-molecule inhibitors of the mitochondrial rhomboid protease PARL, which plays a critical role in regulating mitophagy and cell death. We synthesized a series of succinimide-containing sulfonyl esters and sulfonamides and discovered that these compounds serve as inhibitors of PARL with the most potent sulfonamides having submicromolar affinity for the enzyme. A counterscreen against the bacterial rhomboid protease GlpG demonstrates that several of these compounds display selectivity for PARL over GlpG by as much as two orders of magnitude. Both the sulfonyl ester and sulfonamide scaffolds exhibit reversible binding and are able to engage PARL in mammalian cells. Collectively, our findings provide encouraging precedent for the development of PARL-selective inhibitors and establish N-[(arylsulfonyl)oxy]succinimides and N-arylsulfonylsuccinimides as new molecular scaffolds for inhibiting members of the rhomboid protease family.

Synthesis and kinetic characterization of hyperbolic inhibitors of human cathepsins K and S based on a succinimide scaffold.

Cathepsins K and S are closely related papain-like cysteine peptidases and potential therapeutic targets for metabolic and inflammatory diseases such as osteoporosis and arthritis. Here we describe the reduction of a previously characterized succinimide (2,5-dioxopyrrolidine)-containing hyperbolic inhibitor of cathepsin K (methyl (RS)-N-[1-(4-methoxyphenyl)-2,5-dioxopyrrolidin-3-yl]glycinate), to obtain a better and more selective compound (compound 4a - methyl (2,5-dioxopyrrolidin-3-yl)glycinate), which acted as a hyperbolic mixed inhibitor/activator similar to already known allosteric effectors of cathepsin K. We then investigated the potential of the succinimide scaffold as inhibitors of cathepsins K and/or S and synthesized a library of such compounds by 1,4-addition of α-amino acid esters and related compounds to N-substituted maleimides. From the generated library, we identified the first small molecule hyperbolic inhibitors of cathepsin S (methyl ((R)-2,5-dioxopyrrolidin-3-yl)-l-threoninate (compound R-4c) and 3-{[(1S,2R,3'S)-2-hydroxycyclohexyl]amino}pyrrolidine-2,5-dione (compound (1S,2R,3'S-10)). The former acted via a similar mechanism to compound 4a, while the latter was a hyperbolic specific inhibitor of cathepsin S. Given the versatility of the scaffold, the identified compounds will be used as the basis for the development of high-affinity hyperbolic inhibitors of the individual peptidases and to explore the potential of hyperbolic inhibitors for the inhibition of cysteine cathepsins in in vitro models.

Research progress in biological activities of succinimide derivatives.

Succinimides are well recognized heterocyclic compounds in drug discovery which produce diverse therapeutically related applications in pharmacological practices. Researches in medicinal chemistry field have isolated and synthesized succinimide derivatives with multiple medicinal properties including anticonvulsant, anti-inflammatory, antitumor and antimicrobial agents, 5-HT receptor ligands and enzyme inhibitors. Simultaneously, SAR (Structure-Activity Relationship) analysis has been gradually possessed, along with a great deal of derivatives have been derived for potential targets. In this article, we comprehensively summarize the biological activities and SAR for succinimide derivatives, along with the featuring bioactive molecules reported in patents, wishing to provide an overall retrospect and prospect on the succinimide analogues.

Multifaceted activity of polyciclic MDR revertant agents in drug-resistant leukemic cells: Role of the spacer.

A small library of derivatives carrying a polycyclic scaffold recently identified by us as a new privileged structure in medicinal chemistry was designed and synthesized, aiming at obtaining potent MDR reverting agents also endowed with antitumor properties. In particular, as a follow-up of our previous studies, attention was focused on the role of the spacer connecting the polycyclic core with a properly selected nitrogen-containing group. A relevant increase in reverting potency was observed, going from the previously employed but-2-ynyl- to a pent-3-ynylamino moiety, as in compounds 3d and 3e, while the introduction of a triazole ring proved to differently impact on the activity of the compounds. The docking results supported the data obtained by biological tests, showing, for the most active compounds, the ability to establish specific bonds with P-glycoprotein. Moreover, a multifaceted anticancer profile and dual in vitro activity was observed for all compounds, showing both revertant and antitumor effects on leukemic cells. In this respect, 3c emerged as a "triple-target" agent, endowed with a relevant reverting potency, a considerable antiproliferative activity and a collateral sensitivity profile.

The underlying molecular mechanism and drugs for treatment in adrenal cortical carcinoma.

Purpose: The study aimed to predict and explore the possible clinical value and mechanism of genetic markers in adrenal cortical carcinoma using a bioinformatics analysis method. Methods: The RNA-seqs and miRNAs data were downloaded from TCGA database to identify the differentially expressed genes and differentially expressed miRNAs. The hub-genes were screened by building protein-protein interaction sub-networks with 12 topological analysis methods. We conducted the receiver operating characteristic curve to elevate the diagnostic value of hub-genes in distinguishing the death and alive groups. The survival analysis of hub-genes and key miRNAs were conducted using Kaplan-Meier curves. Furthermore, most significant small molecules were identified as therapeutic candidates for adrenal cortical carcinoma by the CMap analysis. Results: Compared to survival group, we found 475 up-regulated genes and 354 genes and the key pathways leading to the death of different ACC individual patients. Then we used 12 topological analysis methods to found the most possible 22 hub-genes. Among these hub-genes, nine hub-genes (C3, CXCL5, CX3CR1, GRM8, HCAR2, HTR1B, SUCNR1, PTGER3 and SSTR1) could be used to distinguish the death and survival groups for patients. We also revealed that mRNA expressions of 12 genes (C3, CXCL8, CX3CR1, GNAT3, GNGT1, GRM8, HCAR2, HTR1B, HTR1D, PTGER3, SSTR1 and SUCNR1) and four key miRNAs (hsa-mir-330, hsa-mir-489, hsa-mir-508 and hsa-mir-513b) were related to survival. Three most small molecules were identified (H-9, AZ-628 and phensuximide) as potential therapeutic drugs for adrenal cortical carcinoma. Conclusion: The hub-genes expression was significant useful in adrenal cortical carcinoma, provide new diagnostic, prognosis and therapeutic approaches for adrenal cortical carcinoma. Furthermore, we also explore the possible miRNAs involved in regulation of hub-genes.

Role of ASM/Cer/TXNIP signaling module in the NLRP3 inflammasome activation.

BACKGROUND: This study aimed to explore the effects of ceramide (Cer) on NLRP3 inflammasome activation and their underlying mechanisms. METHODS: Lipopolysaccharide (LPS)/adenosine triphosphate (ATP)-induced NLRP3 inflammasome activation in J774A.1 cells and THP-1 macrophages was used as an in vitro model of inflammation. Western blotting and real-time PCR (RT-PCR) were used to detect the protein and mRNA levels, respectively. IL-1ß and IL-18 levels were measured by ELISA. ASM assay kit and immunofluorescence were used to detect ASM activity and Cer content. RESULTS: Imipramine, a well-known inhibitor of ASM, significantly inhibited LPS/ATP-induced activity of ASM and the consequent accumulation of Cer. Additionally, imipramine suppressed the LPS/ATP-induced expression of thioredoxin interacting protein (TXNIP), NLRP3, caspase-1, IL-1ß, and IL-18 at the protein and mRNA level. Interestingly verapamil, a TXNIP inhibitor, suppressed LPS/ATP-induced activation of TXNIP/NLRP3 inflammasome but did not affect LPS/ATP-induced ASM activation and Cer formation. TXNIP siRNA and verapamil inhibited C2-Cer-induced upregulation of TXNIP and activation of the NLRP3 inflammasome. In addition, the pretreatment of cells with sulfo-N-succinimidyl oleate (SSO), an irreversible inhibitor of the scavenger receptor CD36, blocked Cer-induced upregulation of nuclear factor-κB (NF-κB) activity, TXNIP expression, and NLRP3 inflammasome activation. Inhibition of NF-κB activation by SN50 prevented Cer-induced upregulation of TXNIP and activation of the NLRP3 inflammasome but did not affect CD36 expression. CONCLUSION: This study demonstrated that the ASM/Cer/TXNIP signaling pathway is involved in NLRP3 inflammasome activation. The results documented that the CD36-dependent NF-κB-TXNIP signaling pathway plays an essential role in the Cer-induced activation of NLRP3 inflammasomes in macrophages.

New Succinimides with Potent Anticancer Activity: Synthesis, Activation of Stress Signaling Pathways and Characterization of Apoptosis in Leukemia and Cervical Cancer Cells.

Based on previously identified dicarboximides with significant anticancer and immunomodulatory activities, a series of 26 new derivatives were designed and synthesized by the Diels-Alder reaction between appropriate diene and maleimide or hydroxymaleimide moieties. The resulting imides were functionalized with alkanolamine or alkylamine side chains and subsequently converted to their hydrochlorides. The structures of the obtained compounds were confirmed by 1H and 13C NMR and by ESI MS spectral analysis. Their cytotoxicity was evaluated in human leukemia (K562, MOLT4), cervical cancer (HeLa), and normal endothelial cells (HUVEC). The majority of derivatives exhibited high to moderate cytotoxicity and induced apoptosis in K562 cells. Microarray gene profiling demonstrated upregulation of proapoptotic genes involved in receptor-mediated and mitochondrial cell death pathways as well as antiapoptotic genes involved in NF-kB signaling. Selected dicarboximides activated JNK and p38 kinases in leukemia cells, suggesting that MAPKs may be involved in the regulation of apoptosis. The tested dicarboximides bind to DNA as assessed by a plasmid DNA cleavage protection assay. The selected dicarboximides offer new scaffolds for further development as anticancer drugs.

BF3-OEt2 Catalyzed C3-Alkylation of Indole: Synthesis of Indolylsuccinimidesand Their Cytotoxicity Studies.

A simple and efficient BF3-OEt2 promoted C3-alkylation of indole has been developed to obtain3-indolylsuccinimidesfrom commercially available indoles and maleimides, with excellent yields under mild reaction conditions. Furthermore, anti-proliferative activity of these conjugates was evaluated against HT-29 (Colorectal), Hepg2 (Liver) and A549 (Lung) human cancer cell lines. One of the compounds, 3w, having N,N-Dimethylatedindolylsuccinimide is a potent congener amongst the series with IC50 value 0.02 µM and 0.8 µM against HT-29 and Hepg2 cell lines, respectively, and compound 3i was most active amongst the series with IC50 value 1.5 µM against A549 cells. Molecular docking study and mechanism of reaction have briefly beendiscussed. This method is better than previous reports in view of yield and substrate scope including electron deficient indoles.

Endocannabinoid degradation inhibitors ameliorate neuronal and synaptic alterations following traumatic brain injury.

Our previous work showed that lateral fluid percussion injury to the sensorimotor cortex (SMC) of anesthetized rats increased neuronal synaptic hyperexcitability in layer 5 (L5) neurons in ex vivo brain slices 10 days postinjury. Furthermore, endocannabinoid (EC) degradation inhibition via intraperitoneal JZL184 injection 30 min postinjury attenuated synaptic hyperexcitability. This study tested the hypothesis that traumatic brain injury (TBI) induces synaptic and intrinsic neuronal alterations of L5 SMC pyramidal neurons and that these alterations are significantly attenuated by in vivo post-TBI treatment with EC degradation inhibitors. We tested the effects of systemically administered EC degradation enzyme inhibitors (JZL184, MJN110, URB597, or JZL195) with differential selectivity for fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) on electrophysiological parameters in SMC neurons of TBI- and sham-treated rats 10 days post-TBI. We recorded intrinsic neuronal properties, including resting membrane voltage, input resistance, spike threshold, spiking responses to current input, voltage "sag" (rebound response to hyperpolarization-activated inward current), and burst firing. We also measured the frequency and amplitude of spontaneous excitatory postsynaptic currents. We then used the aggregate parameter sets (intrinsic + synaptic properties) to apply a machine learning classification algorithm to quantitatively compare neural population responses from each experimental group. Collectively, our electrophysiological and computational results indicate that sham neurons are the most distinguishable from TBI neurons. Administration of EC degradation inhibitors post-TBI exerted varying degrees of rescue, approximating the neuronal phenotype of sham neurons, with neurons from TBI/JZL195 (a dual MAGL/FAAH inhibitor) being most similar to neurons from sham rats.NEW & NOTEWORTHY This study elucidates neuronal properties altered by traumatic brain injury (TBI) in layer 5 of sensorimotor cortex, which may be implicated in post-TBI circuit dysfunction. We compared effects of systemic administration of four different endocannabinoid degradation inhibitors within a clinically relevant window postinjury. Electrophysiological measures and using a machine learning classification algorithm collectively suggest that pharmacological inhibitors targeting both monoacylglycerol lipase and fatty acid amide hydrolase (e.g., JZL195) may be most efficacious in attenuating TBI-induced neuronal dysfunction at site of injury.

The Effect of Nitroxyalkyl Succinimides on Activity of Cyclic Guanosine Monophosphate Phosphodiesterase.

The effect of N-nitroxymethyl succinimide (1), N-(2-nitroxyethyl) succinimide (2) and N-(3-nitroxypropyl) succinimide (3) on enzymatic activity of cyclic guanosine monophosphate (cGMP) phosphodiesterase was studied and crystal structure of compound (2) was determined. It was shown that all studied N-nitroxy succinimides inhibited cGMP phosphodiesterase in a concentration range of 0.1-0.001 mM. Compound (2) noncompetitively and reversibly inhibited hydrolytic function of enzyme with Ki=1.7×10-5 М. Inhibition constant for the reference compound N-(2-nitroethyl) nicotinamide (nicorandil) was 3×10-5 М.

Inhibition of monoacylglycerol lipase, an anti-inflammatory and antifibrogenic strategy in the liver.

OBJECTIVE: Sustained inflammation originating from macrophages is a driving force of fibrosis progression and resolution. Monoacylglycerol lipase (MAGL) is the rate-limiting enzyme in the degradation of monoacylglycerols. It is a proinflammatory enzyme that metabolises 2-arachidonoylglycerol, an endocannabinoid receptor ligand, into arachidonic acid. Here, we investigated the impact of MAGL on inflammation and fibrosis during chronic liver injury. DESIGN: C57BL/6J mice and mice with global invalidation of MAGL (MAGL -/- ), or myeloid-specific deletion of either MAGL (MAGLMye-/-), ATG5 (ATGMye-/-) or CB2 (CB2Mye-/-), were used. Fibrosis was induced by repeated carbon tetrachloride (CCl4) injections or bile duct ligation (BDL). Studies were performed on peritoneal or bone marrow-derived macrophages and Kupffer cells. RESULTS: MAGL -/- or MAGLMye-/- mice exposed to CCl4 or subjected to BDL were more resistant to inflammation and fibrosis than wild-type counterparts. Therapeutic intervention with MJN110, an MAGL inhibitor, reduced hepatic macrophage number and inflammatory gene expression and slowed down fibrosis progression. MAGL inhibitors also accelerated fibrosis regression and increased Ly-6Clow macrophage number. Antifibrogenic effects exclusively relied on MAGL inhibition in macrophages, since MJN110 treatment of MAGLMye-/- BDL mice did not further decrease liver fibrosis. Cultured macrophages exposed to MJN110 or from MAGLMye-/- mice displayed reduced cytokine secretion. These effects were independent of the cannabinoid receptor 2, as they were preserved in CB2Mye-/- mice. They relied on macrophage autophagy, since anti-inflammatory and antifibrogenic effects of MJN110 were lost in ATG5Mye-/- BDL mice, and were associated with increased autophagic flux and autophagosome biosynthesis in macrophages when MAGL was pharmacologically or genetically inhibited. CONCLUSION: MAGL is an immunometabolic target in the liver. MAGL inhibitors may show promising antifibrogenic effects during chronic liver injury.

Hormetic Effects of Mixtures of Dimethachlone and Prochloraz on Sclerotinia sclerotiorum.

Previous studies showed that dimethachlone has significant hormetic effects on phytopathogenic fungus Sclerotinia sclerotiorum. The present study investigated hormetic effects of mixtures of dimethachlone and prochloraz on mycelial growth and virulence of two dimethachlone-resistant isolates of S. sclerotiorum. The stimulatory dimethachlone dosage range was around 1 to 100 µg/ml in potato dextrose agar (PDA) medium for mycelial growth of the two isolates, and dimethachlone at 10 and 50 µg/ml had the maximum percent stimulations of 80.6 and 19.3% for isolates JMS14 and HLJ4, respectively. Prochloraz at 0.0003 and 0.002 µg/ml had the maximum percent stimulations of 9.3 and 11.1% for isolates JMS14 and HLJ4, respectively. However, dimethachlone and prochloraz mixed at their respective stimulatory concentrations had the maximum percent stimulations of 48.1 and 9.3% for isolates JMS14 and HLJ4, respectively. After the mycelia with increased and inhibited growth on fungicide-amended PDA were subcultured on PDA without fungicide, mycelial growth for the second generation increased compared with the nontreated control. After the mycelia grown on fungicide-amended PDA were inoculated on rapeseed leaves, the amplitude of virulence stimulation was much greater than that of mycelial growth on PDA amended with fungicide, and the inhibited mycelia also showed substantially increased virulence on leaves. The mixture of dimethachlone at 100 µg/ml and prochloraz at 0.03 µg/ml in PDA inhibited mycelial growth of isolate JMS14 by 59.4%; however, after the inhibited mycelia were inoculated on rapeseed leaves, virulence was stimulated by 69.0%. Spraying sublethal doses of dimethachlone and prochloraz on rapeseed leaves also exhibited significant stimulatory effects on virulence. For isolate JMS14, the stimulatory concentration ranges for dimethachlone and prochloraz were around 1 to 600 µg/ml and 0.0003 to 0.18 µg/ml, respectively. The fitted curve of virulence stimulation for the mixture of dimethachlone and prochloraz shifted to the left on the x axis, denoting dose-additive interactions between the two fungicides with regard to virulence stimulation. Spraying dimethachlone alone at 10 to 50 µg/ml had significant stimulations on virulence, whereas prochloraz alone at 10 to 50 µg/ml had significant inhibitory effects on virulence, and the mixture of dimethachlone and prochloraz at the concentration ratio of 1:1 had greater inhibitory effects than prochloraz alone, indicating dose-additive interactions for the inhibitory effects. Dimethachlone and prochloraz and their mixtures increased tolerance of mycelia to hydrogen peroxide. Dimethachlone significantly increased, whereas prochloraz reduced mycelial cell membrane permeability, and the mixture of the two fungicides had effect-additive interactions with respect to effects on cell membrane permeability. These studies will advance our understanding of hormesis of fungicide mixtures.

Neuroprotective Effects of MAGL (Monoacylglycerol Lipase) Inhibitors in Experimental Ischemic Stroke.

BACKGROUND AND PURPOSE: MAGL (monoacylglycerol lipase) is an enzyme that hydrolyzes the endocannabinoid 2-arachidonoylglycerol and regulates the production of arachidonic acid and prostaglandins-substances that mediate tissue inflammatory response. Here, we have studied the effects of the selective MAGL inhibitors JZL184 and MJN110 and their underlying molecular mechanisms on 3 different experimental models of focal cerebral ischemia. METHODS: SHR (spontaneously hypertensive rats) and normotensive WKY (Wistar Kyoto) rats were subject to an intracortical injection of the potent vasoconstrictor endothelin-1, permanent occlusion of a distal segment of the middle cerebral artery via craniectomy, or transient occlusion of the middle cerebral artery by the intraluminal suture method. JZL184 or MJN110 was administered 60 minutes after focal cerebral ischemia. Infarct volumes, hemispheric swelling, and functional outcomes were assessed between days 1 to 28 by magnetic resonance imaging, histology, and behavioral tests. RESULTS: Pharmacological inhibition of MAGL significantly attenuated infarct volume and hemispheric swelling. MAGL inhibition also ameliorated sensorimotor deficits, suppressed inflammatory response, and decreased the number of degenerating neurons. These beneficial effects of MAGL inhibition were not fully abrogated by selective antagonists of cannabinoid receptors, indicating that the anti-inflammatory effects are caused by inhibition of eicosanoid production rather than by activation of cannabinoid receptors. CONCLUSIONS: Our results suggest that MAGL may contribute to the pathophysiology of focal cerebral ischemia and is thus a promising therapeutic target for the treatment of ischemic stroke.

Palmitate aggravates proteinuria-induced cell death and inflammation via CD36-inflammasome axis in the proximal tubular cells of obese mice.

High levels of serum free fatty acids (FFAs) and proteinuria have been implicated in the pathogenesis of obesity-related nephropathy. CD36, a class B scavenger receptor, is highly expressed in the renal proximal tubules and mediates FFA uptake. It is not clear whether FFA- and proteinuria-mediated CD36 activation coordinates NLRP3 inflammasomes to induce renal tubular injury and inflammation. In this study, we investigated the roles of CD36 and NLRP3 inflammasomes in FFA-induced renal injury in high-fat diet (HFD)-induced obesity. HFD-fed C57BL/6 mice and palmitate-treated HK2 renal tubular cells were used as in vivo and in vitro models. Immunohistochemical staining showed that CD36, IL-1ß, and IL-18 levels increased progressively in the kidneys of HFD-fed mice. Sulfo- N-succinimidyl oleate (SSO), a CD36 inhibitor, attenuated the HFD-induced upregulation of NLRP3, IL-1ß, and IL-18 and suppressed the colocalization of NLRP3 and ASC in renal tubular cells. In vitro, SSO abolished the palmitate-induced activation of IL-1ß, IL-18, and caspase-1 in HK2 proximal tubular cells. Furthermore, treatment with SSO and the knockdown of caspase-1 expression by siRNA both inhibited palmitate-induced cell death and apoptosis in HK2 cells. Collectively, palmitate causes renal tubular inflammation, cell death, and apoptosis via the CD36/NLRP3/caspase-1 axis, which may explain, at least in part, the mechanism underlying FFA-related renal tubular injury. The blockade of CD36-induced cellular processes is therefore a promising strategy for treating obesity-related nephropathy.

FAAH, but not MAGL, inhibition modulates acute TLR3-induced neuroimmune signaling in the rat, independent of sex.

Toll-like receptor (TLR)3 is a key component of the innate immune response to viral infection. The present study firstly examined whether sex differences exist in TLR3-induced inflammatory, endocrine, and sickness responses. The data revealed that TLR3-induced expression of interferon- or NFkB-inducible genes (IFN-α/ß, IP-10, or TNF-α), either peripherally (spleen) or centrally (hypothalamus), did not differ between male and female rats, with the exception of TLR3-induced IFN-α expression in the spleen of female, but not male, rats 8 hr post TLR3 activation. Furthermore, TLR3 activation increased plasma corticosterone levels, induced fever, and reduced locomotor activity and body weight - effects independent of sex. Thus, the acute-phase inflammatory, endocrine, and sickness responses to TLR3 activation exhibit minimal sex-related differences. A further aim of this study was to examine whether enhancing endocannabinoid tone - namely, 2-arachidonylglycerol (2-AG) or N-arachidonoylethanolamine (AEA), exhibited similar effects on TLR3-induced inflammatory responses in male versus female rats. Systemic administration of the monoacylglycerol lipase (MAGL) inhibitor MJN110 and subsequent increases in 2-AG levels did not alter the TLR3-induced increase in IP-10, IRF7, or TNF-α expression in the spleen or the hypothalamus of male or female rats. In contrast, the fatty acid amide hydrolase (FAAH) inhibitor URB597 increased levels of AEA and related N-acylethanolamines, an effect associated with the attenuation of TLR3-induced inflammatory responses in the hypothalamus, but not the spleen, of male and female rats. These data support a role for FAAH, but not MAGL, substrates in the modulation of TLR3-induced neuroinflammatory responses, effects independent of sex.

Monoacylglycerol Lipase Inhibitors Reverse Paclitaxel-Induced Nociceptive Behavior and Proinflammatory Markers in a Mouse Model of Chemotherapy-Induced Neuropathy.

Although paclitaxel effectively treats various cancers, its debilitating peripheral neuropathic pain side effects often persist long after treatment has ended. Therefore, a compelling need exists for the identification of novel pharmacologic strategies to mitigate this condition. As inhibitors of monoacylglycerol lipase (MAGL), the primary hydrolytic enzyme of the endogenous cannabinoid, 2-arachidonyolglycerol, produces antinociceptive effects in numerous rodent models of pain, we investigated whether inhibitors of this enzyme (i.e., JZL184 and MJN110) would reverse paclitaxel-induced mechanical allodynia in mice. These drugs dose dependently reversed allodynia with respective ED50 values (95% confidence limit) of 8.4 (5.2-13.6) and 1.8 (1.0-3.3) mg/kg. Complementary genetic and pharmacologic approaches revealed that the antiallodynic effects of each drug require both cannabinoid receptors, CB1 and CB2 MJN110 reduced paclitaxel-mediated increased expression of monocyte chemoattractant protein-1 (MCP-1, CCL2) and phospho-p38 MAPK in dorsal root ganglia as well as MCP-1 in spinal dorsal horn. Whereas the antinociceptive effects of high dose JZL184 (40 mg/kg) underwent tolerance following 6 days of repeated dosing, repeated administration of a threshold dose (i.e., 4 mg/kg) completely reversed paclitaxel-induced allodynia. In addition, we found that the administration of MJN110 to control mice lacked intrinsic rewarding effects in the conditioned place preference (CPP) paradigm. However, it produced a CPP in paclitaxel-treated animals, suggesting a reduced paclitaxel-induced aversive state. Importantly, JZL184 did not alter the antiproliferative and apoptotic effects of paclitaxel in A549 and H460 non-small cell lung cancer cells. Taken together, these data indicate that MAGL inhibitors reverse paclitaxel-induced neuropathic pain without interfering with chemotherapeutic efficacy.

A new leptin-mediated mechanism for stimulating fatty acid oxidation: a pivotal role for sarcolemmal FAT/CD36.

Leptin stimulates fatty acid oxidation in muscle and heart; but, the mechanism by which these tissues provide additional intracellular fatty acids for their oxidation remains unknown. We examined, in isolated muscle and cardiac myocytes, whether leptin, via AMP-activated protein kinase (AMPK) activation, stimulated fatty acid translocase (FAT/CD36)-mediated fatty acid uptake to enhance fatty acid oxidation. In both mouse skeletal muscle and rat cardiomyocytes, leptin increased fatty acid oxidation, an effect that was blocked when AMPK phosphorylation was inhibited by adenine 9-ß-d-arabinofuranoside or Compound C. In wild-type mice, leptin induced the translocation of FAT/CD36 to the plasma membrane and increased fatty acid uptake into giant sarcolemmal vesicles and into cardiomyocytes. In muscles of FAT/CD36-KO mice, and in cardiomyocytes in which cell surface FAT/CD36 action was blocked by sulfo-N-succinimidyl oleate, the leptin-stimulated influx of fatty acids was inhibited; concomitantly, the normal leptin-stimulated increase in fatty acid oxidation was also prevented, despite the normal leptin-induced increase in AMPK phosphorylation. Conversely, in muscle of AMPK kinase-dead mice, leptin failed to induce the translocation of FAT/CD36, along with a failure to stimulate fatty acid uptake and oxidation. Similarly, when siRNA was used to reduce AMPK in HL-1 cardiomyocytes, leptin failed to induce the translocation of FAT/CD36. Our studies have revealed a novel mechanism of leptin-induced fatty acid oxidation in muscle tissue; namely, this process is dependent on the activation of AMPK to induce the translocation of FAT/CD36 to the plasma membrane, thereby stimulating fatty acid uptake. Without increasing this leptin-stimulated, FAT/CD36-dependent fatty acid uptake process, leptin-stimulated AMPK phosphorylation does not enhance fatty acid oxidation.

Two New Succinimide Derivatives Cladosporitins A and B from the Mangrove-derived Fungus Cladosporium sp. HNWSW-1.

Two new succinimide-containing derivatives, cladosporitins A (1) and B (2), were isolated from the fermentation cultures of the mangrove-derived fungus Cladosporium sp. HNWSW-1, along with a new pyrone, clapone (3), as well as the previously reported talaroconvolutin A (4) and anthraquinone (5). The structures of the isolated compounds were elucidated by 1D, 2D NMR, and HRMS spectral analysis. Compound 2 showed cytotoxicity against BEL-7042, K562 and SGC-7901 cell lines with IC50 values of 29.4 ± 0.35 µM, 25.6 ± 0.47 µM, and 41.7 ± 0.71 µM, respectively, whereas compound 4 exhibited cytotoxicity against Hela and BEL-7042 cell lines with IC50 values of 14.9 ± 0.21 µM and 26.7 ± 1.1 µM, respectively. In addition, compounds 4 and 5 displayed inhibitory activity against α-glycosidase, with IC50 values of 78.2 ± 2.1 µM and 49.3 ± 10.6 µM, respectively.