Syntheses,antiviral activities and induced resistance mechanisms of novel quinazoline derivatives containing a dithioacetal moiety

A series of novel quinazoline derivatives containing a dithioacetal moiety were designed and synthesized, and their structures were characterized by ¹H nuclear magnetic resonance, ¹³C nuclear magnetic resonance, and high-resolution mass spectrometry. Bioassay results indicated that compound 4b exhibited remarkable protective activity against cucumber mosaic virus (CMV, EC₅₀ = 248.6 μg/mL) and curative activity against potato virus Y (EC₅₀ = 350.5 μg/mL), which were better than those of ningnanmycin (357.7 μg/mL and 493.7 μg/mL, respectively). Moreover, compound 4b could increase the chlorophyll content in plants, improve photosynthesis, and effectively induce tobacco anti-CMV activity.     

Role of labile iron in the toxicity of pharmacological ascorbate

Pharmacological ascorbate has been shown to induce toxicity in a wide range of cancer cell lines. Pharmacological ascorbate in animal models has shown promise for use in cancer treatment. At pharmacological concentrations the oxidation of ascorbate produces a high flux of H₂O₂ via the formation of ascorbate radical (Asc^•-). The rate of oxidation of ascorbate is principally a function of the level of catalytically active metals. Iron in cell culture media contributes significantly to the rate of H₂O₂ generation. We hypothesized that increasing intracellular iron would enhance ascorbate-induced cytotoxicity and that iron chelators could modulate the catalytic efficiency with respect to ascorbate oxidation. Treatment of cells with the iron-chelators deferoxamine (DFO) or dipyridyl (DPD) in the presence of 2 mM ascorbate decreased the flux of H₂O₂ generated by pharmacological ascorbate and reversed ascorbate-induced toxicity. Conversely, increasing the level of intracellular iron by preincubating cells with Fe-hydroxyquinoline (HQ) increased ascorbate toxicity and decreased clonogenic survival. These findings indicate that redox metal metals, e.g., Fe³⁺/Fe²⁺, have an important role in ascorbate-induced cytotoxicity. Approaches that increase catalytic iron could potentially enhance the cytotoxicity of pharmacological ascorbate in vivo.     

On the use of fluorescence lifetime imaging and dihydroethidium to detect superoxide in intact animals and ex vivo tissues: A reassessment

Recently, D.J. Hall et al. reported that ethidium (E⁺) is formed as a major product of hydroethidine (HE) or dihydroethidium reaction with superoxide (O₂⁻) in intact animals with low tissue oxygen levels (J. Cereb. Blood Flow Metab. 32:23–32, 2012). The authors concluded that measurement of E⁺ is an indicator of O₂⁻ formation in intact brains of animals. This finding is in stark contrast to previous reports using in vitro systems showing that 2-hydroxyethidium, not ethidium, is formed from the reaction between O₂⁻ and HE. Published in vivo results support the in vitro findings. In this study, we performed additional experiments in which HE oxidation products were monitored under different fluxes of O₂⁻. Results from these experiments further reaffirm our earlier findings (H. Zhao et al., Free Radic. Biol. Med. 34:1359, 2003). We conclude that whether in vitro or in vivo, E⁺ measured by HPLC or by fluorescence lifetime imaging is not a diagnostic marker product for O₂⁻ reaction with HE.     

Norepinephrine stimulation of alpha1D-adrenoceptor promotes proliferation of pulmonary artery smooth muscle cells via ERK-1/2 signaling

It has been shown that the sympathetic nervous system is activated in pulmonary arterial hypertension (PAH). Norepinephrine (NE) levels are increased by chemoreflex-dependent sympathetic overactivation and involved in pulmonary vascular remodeling. However, the underlying mechanisms of the remodeling induced by NE are poorly understood. In this study, we found that, in vivo, the expression of tyrosine hydroxylase and the concentration of plasma NE were increased in PAH rats compared with normal rats. Increases in ventricular hypertrophy and medial width of the pulmonary arteries were reversed by prazosin, α₁-adrenoceptor (α₁-AR) antagonists, in PAH rats. Elevated expression of α_1D-AR was detected in PAH rats. In addition, prazosin reduced the increasing expression of PCNA, CyclinA and CyclinE induced by hypoxia. In vitro, MTT assay, flow cytometry, Western blotting and immunofluorescence were performed to investigate the effects of NE on proliferation of pulmonary artery smooth muscle cells (PASMCs). We revealed that NE promoted PASMCs viability, increased the expression of PCNA, CyclinA and CyclinE, made more cells from G₀/G₁ phase to G₂/M + S phase and enhanced the microtubule formation. Above NE-induced changes could be suppressed by BMY 7378, an inhibitor of α_1D-AR. Furthermore, ERK-1/2 pathway was activated by NE. U0126, a specific inhibitor for ERK-1/2, attenuated the NE-induced proliferation of PASMCs under normoxia and hypoxia. Taken together, our results suggest that NE which stimulates α_1D-AR promotes proliferation of PASMCs and the effect is, at least in part, mediated via the ERK-1/2 pathway.     

Preliminary studies on the involvement of glutathione metabolism and redox status against zinc toxicity in radish seedlings by 28-Homobrassinolide

The effect of exogenous application of 28-Homobrassinolide (HBR) on radish (Raphanus sativus L.) seedlings under zinc (Zn²⁺) stress on glutathione (GSH) production, consumption and changes in redox status was investigated. Zinc toxicity resulted in oxidative burst as evidenced by increased accumulation of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) content. These stress indices were significantly decreased by HBR supplementation. Under Zn²⁺ stress, GSH pool was decreased, while the contribution of oxidized glutathione (GSSG) to total GSH increased (GSSH/GSH ratio), this translated into significant reduction of GSH redox homeostasis. In addition, an increase of phytochelatins (PCs) was observed. In radish seedlings under Zn²⁺ stress, the activities of gamma-glutamylcysteine synthetase (γ-ECS), glutathione synthetase (GS), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and cysteine (Cys) levels increased but the activity of glutathione reductase (GR) decreased. However, application of HBR increased the GSH pool and maintained their redox ratio by increasing the enzyme activities of GSH biosynthesis (γ-ECS and GS) and GSH metabolism (GR, GPX and GST). The results of present study are novel in being the first to demonstrate that exogenous application of HBR modulates the GSH synthesis, metabolism and redox homeostasis to confer resistance against Zn²⁺ induced oxidative stress.     

Targeting the upregulation of reactive oxygen species subsequent to hyperglycemia prevents type 1 diabetic cardiomyopathy in mice

Cardiac oxidative stress is an early event associated with diabetic cardiomyopathy, triggered by hyperglycemia. We tested the hypothesis that targeting left-ventricular (LV) reactive oxygen species (ROS) upregulation subsequent to hyperglycemia attenuates type 1 diabetes-induced LV remodeling and dysfunction, accompanied by attenuated proinflammatory markers and cardiomyocyte apoptosis. Male 6-week-old mice received either streptozotocin (55 mg/kg/day for 5 days), to induce type 1 diabetes, or citrate buffer vehicle. After 4 weeks of hyperglycemia, the mice were allocated to coenzyme Q₁₀ supplementation (10 mg/kg/day), treatment with the angiotensin-converting-enzyme inhibitor (ACE-I) ramipril (3 mg/kg/day), treatment with olive oil vehicle, or no treatment for 8 weeks. Type 1 diabetes upregulated LV NADPH oxidase (Nox2, p22^phox, p47^phox and superoxide production), LV uncoupling protein UCP3 expression, and both LV and systemic oxidative stress (LV 3-nitrotyrosine and plasma lipid peroxidation). All of these were significantly attenuated by coenzyme Q₁₀. Coenzyme Q₁₀ substantially limited type 1 diabetes-induced impairments in LV diastolic function (E:A ratio and deceleration time by echocardiography, LV end-diastolic pressure, and LV −dP/dt by micromanometry), LV remodeling (cardiomyocyte hypertrophy, cardiac fibrosis, apoptosis), and LV expression of proinflammatory mediators (tumor necrosis factor-α, with a similar trend for interleukin IL-1β). Coenzyme Q_10's actions were independent of glycemic control, body mass, and blood pressure. Coenzyme Q₁₀ compared favorably to improvements observed with ramipril. In summary, these data suggest that coenzyme Q₁₀ effectively targets LV ROS upregulation to limit type 1 diabetic cardiomyopathy. Coenzyme Q₁₀ supplementation may thus represent an effective alternative to ACE-Is for the treatment of cardiac complications in type 1 diabetic patients.     

Rationally designed molecules for resurgence of cyanide mitigated cytochrome c oxidase activity

Cytochrome c oxidase (CcOX) containing binuclear heme a₃-Cu B centre (BNC) mechanises the process of electron transfer in the last phase of cellular respiration. The molecular modelling based structural analysis of CcOX – heme a₃-Cu B complex was performed and the disturbance to this complex under cyanide poisoning conditions was investigated. Taking into consideration the results of molecular docking studies, new chemical entities were developed for clipping cyanide from the enzyme and restoring its normal function. It was found that the molecules obtained by combining syringaldehyde, oxindole and chrysin moieties bearing propyl/butyl spacing groups occupy the BNC region and effectively remove cyanide bound to the enzyme. The binding constant of compound 2 with CN^– was 2.3 × 10⁵ M⁻¹ and its ED₅₀ for restoring the cyanide bound CcOX activity in 10 min was 16 µM. The compound interacted with CN^– over the pH range 5–10. The comparison of the loss of enzymatic activity in the presence of CN^– and resumption of enzymatic activity by compound 2 mediated removal of CN^– indicated the efficacy of the compound as antidote of cyanide.     

Investigation of the XCAT phantom as a validation tool in cardiac MRI tracking algorithms

PurposeTo describe our magnetic resonance imaging (MRI) simulated implementation of the 4D digital extended cardio torso (XCAT) phantom to validate our previously developed cardiac tracking techniques. Real-time tracking will play an important role in the non-invasive treatment of atrial fibrillation with MRI-guided radiosurgery. In addition, to show how quantifiable measures of tracking accuracy and patient-specific physiology could influence MRI tracking algorithm design.MethodsTwenty virtual patients were subjected to simulated MRI scans that closely model the proposed real-world scenario to allow verification of the tracking technique’s algorithm. The generated phantoms provide ground-truth motions which were compared to the target motions output from our tracking algorithm. The patient-specific tracking error, e_p, was the 3D difference (vector length) between the ground-truth and algorithm trajectories. The tracking errors of two combinations of new tracking algorithm functions that were anticipated to improve tracking accuracy were studied. Additionally, the correlation of key physiological parameters with tracking accuracy was investigated.ResultsOur original cardiac tracking algorithm resulted in a mean tracking error of 3.7 ± 0.6 mm over all virtual patients. The two combinations of tracking functions demonstrated comparable mean tracking errors however indicating that the optimal tracking algorithm may be patient-specific.ConclusionsCurrent and future MRI tracking strategies are likely to benefit from this virtual validation method since no time-resolved 4D ground-truth signal can currently be derived from purely image-based studies.     

Translational studies on regulation of brain docosahexaenoic acid (DHA) metabolism in vivo

One goal in the field of brain polyunsaturated fatty acid (PUFA) metabolism is to translate the many studies that have been conducted in vitro and in animal models to the clinical setting. Doing so should elucidate the role of PUFAs in the human brain, and effects of diet, drugs, disease and genetics on this role. This review discusses new in vivo radiotracer kinetic and neuroimaging techniques that allow us to do this, with a focus on docosahexaenoic acid (DHA). We illustrate how brain PUFA metabolism is influenced by graded reductions in dietary n-3 PUFA content in unanesthetized rats. We also show how kinetic tracer techniques in rodents have helped to identify mechanisms of action of mood stabilizers used in bipolar disorder, how DHA participates in neurotransmission, and how brain DHA metabolism is regulated by calcium-independent iPLA₂β. In humans, regional rates of brain DHA metabolism can be quantitatively imaged with positron emission tomography following intravenous injection of [1-¹¹C]DHA.     

Development of pyrazole and spiropyrazoline analogs as multifunctional agents for treatment of Alzheimer’s disease

Cholinergic hypothesis of Alzheimer’s disease has been advocated as an essential tool in the last couple of decades for the drug development. Here in, we report de novo fragment growing strategy for the design of novel 3,5-diarylpyrazoles and hit optimization of spiropyrazoline derivatives as acetyl cholinesterase inhibitors. Both type of scaffolds numbering forty compounds were synthesized and evaluated for their potencies against AChE, BuChE and PAMPA. Introduction of lipophilic cyclohexane ring in 3,5-diarylpyrazole analogs led to spiropyrazoline derivatives, which facilitated and improved the potencies. Compound 44 (AChE = 1.937 ± 0.066 µM; BuChE = 1.166 ± 0.088 µM; hAChE = 1.758 ± 0.095 µM; P_e = 9.491 ± 0.34 × 10⁻⁶ cm s¹) showed positive results, which on further optimization led to the development of compound 67 (AChE = 0.464 ± 0.166 µM; BuChE = 0.754 ± 0.121 µM; hAChE = 0.472 ± 0.042 µM; P_e = 13.92 ± 0.022 × 10⁻⁶ cm s¹). Compounds 44 and 67 produced significant displacement of propidium iodide from the peripheral anionic site (PAS) of AChE. They were found to be safer to MC65 cells and decreased metal induced Aβ_1-42 aggregation. Further, in-vivo behavioral studies, on scopolamine induced amnesia model, the compounds resulted in better percentage spontaneous alternation scores and were safe, had no influence on locomotion in tested animal groups at dose of 3 mg/kg. Early pharmacokinetic assessment of optimized hit molecules was supportive for further drug development.     

Inhibition of thrombin,an unexplored function of retinoic acid

Retinoic acid, a derivative of vitamin A, is known to possess in vivo anti-inflammatory, anti-platelet and fibrinolytic activities. We have investigated the in vitro thrombin and platelet aggregation inhibitory activities of vitamin A (retinol) and its derivatives, retinoic acid and retinaldehyde. The thrombin enzymatic assay was performed fluorimetrically to assess the inhibition of thrombin (Sigma and plasma). Retinoic acid, retinaldehyde and retinol exhibited potent inhibition of thrombin, with IC₅₀ values of 67μg/ml, 74μg/ml and 152μg/ml, respectively for the inhibition of thrombin (Sigma); and 49μg/ml, 74μg/ml and 178μg/ml, respectively for the inhibition of thrombin (plasma). Amongst vitamin A and its derivatives, retinoic acid showed the highest inhibition of both the forms of thrombin. Vitamin A and its derivatives also displayed remarkable inhibition of platelet aggregation. This is the first report of vitamin A and its derivatives showing inhibition of thrombin and platelet aggregation in vitro.     

Lambertellin from Pycnoporus sanguineus MUCL 51321 and its anti-inflammatory effect via modulation of MAPK and NF-κB signaling pathways

Lambertellin (1) and ergosta-5,7,22-trien-3-ol (2) were isolated from the solid rice fermentation of the plant pathogenic fungus Pycnoporus sanguineus MUCL 51321. Their structures were elucidated using comprehensive spectroscopic methods. The isolated compounds were tested on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Lambertellin (1) exhibited promising inhibitory activity against nitric oxide (NO) production with IC₅₀ value of 3.19 µM, and it significantly inhibited the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2). Lambertellin (1) also decreased the expression of pro-inflammatory cytokines IL-6 and IL-1β. The study of the mechanistic pathways revealed that lambertellin (1) exerts its anti-inflammatory effect in LPS-stimulated RAW 264.7 macrophage cells by modulating the activation of the mitogen activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways. Therefore, lambertellin (1) could be a promising lead compound for the development of new anti-inflammatory drugs.     

Mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach,Diploptera punctata

The FGLamide allatostatins (FGL/ASTs) are a family of neuropeptides with pleiotropic functions, including the inhibition of juvenile hormone (JH) biosynthesis, vitellogenesis and muscle contraction. In the cockroach, Diploptera punctata, thirteen FGLa/ASTs and one allatostatin receptor (AstR) have been identified. However, the mode of action of ASTs in regulation of JH biosynthesis remains unclear. Here, we determined the tissue distribution of Dippu-AstR. And we expressed Dippu-AstR in vertebrate cell lines, and activated the receptor with the Dippu-ASTs. Our results show that all thirteen ASTs activated Dippu-AstR in a dose dependent manner, albeit with different potencies. Functional analysis of AstR in multiple cell lines demonstrated that activation of the AstR receptor resulted in elevated levels of Ca²⁺ and cAMP, which suggests that Dippu-AstR can act through the Gα_q and Gα_s protein pathways. The study on the target of AST action reveals that FGL/AST affects JH biosynthesis prior to the entry of acetyl-CoA into the JH biosynthetic pathway.     

Magnolol inhibits colonic motility through down-regulation of voltage-sensitive L-type Ca2+ channels of colonic smooth muscle cells in rats

This study aimed to investigate the effect of magnolol (5,5′-diallyl-2,2′-biphenyldiol) on contraction in distal colonic segments of rats and the underlying mechanisms. Colonic segments were mounted in organ baths for isometric force measurement. Whole-cell voltage-sensitive L-type Ca²⁺ currents were recorded on isolated single colonic smooth muscle cells using patch-clamp technique. The spontaneous contractions and acetylcholine (ACh)- and Bay K 8644-induced contractions were inhibited by magnolol (3–100 μM). In the presence of Bay K8644 (100 nM), magnolol (10–100 μM) inhibited the contraction induced by 10 μM ACh. By contrast, tetrodotoxin (100 nM) and N_ώ-nitro-l-arginine methyl ester (l-NAME 100 μM) did not change the inhibitory effect of magnolol (10 μM). In addition, magnolol (3–100 μM) inhibited the L-type Ca²⁺ currents. The present results suggest that magnolol inhibits colonic smooth muscle contraction through downregulating L-type Ca²⁺ channel activity.     

The development of 2-acetylphenol-donepezil hybrids as multifunctional agents for the treatment of Alzheimer’s disease

A series of 2-acetylphenol-donepezil hybrids was designed and synthesized based on multi-target-directed ligands strategy. The biological activities were evaluated by AChE/BChE inhibition and MAO-A/MAO-B inhibition. The results revealed that the tertiary amines and methylene chain length significantly affected the eeAChE inhibitory potency, in particular, compound TM-14 showed the best eeAChE inhibitory activity with IC₅₀ value of 2.9 μM, in addition, both kinetic analysis of AChE inhibition and docking study displayed that TM-14 could simultaneously bind to the catalytic active site and peripheral anionic site of AChE. Moreover, compound TM-14 was a selective metal chelator and could form 1:1 TM-14-Cu²⁺ complex. The structure-active-relationship also indicated that the O-alkylamine fragment remarkably decreased hMAO-B inhibitory activity, compound TM-2 exhibited potent hMAO-B inhibitory activity (IC₅₀ = 6.8 μM), which was supported by the molecular docking study. More interestingly, compounds TM-14 and TM-2 could cross the blood-brain barrier in vitro. Therefore, the structure-active-relationship of 2-acetylphenol-donepezil hybrids could encourage the development of multifunction agents with selective AChE inhibition or selective MAO-B inhibition for the treatment of Alzheimer’s disease.     

Nodules from Fynbos legume Virgilia divaricata have high functional plasticity under variable P supply levels

Legumes have the unique ability to fix atmospheric nitrogen (N₂) via symbiotic bacteria in their nodules but depend heavily on phosphorus (P), which affects nodulation, and the carbon costs and energy costs of N₂ fixation. Consequently, legumes growing in nutrient-poor ecosystems (e.g., sandstone-derived soils) have to enhance P recycling and/or acquisition in order to maintain N₂ fixation. In this study, we investigated the flexibility of P recycling and distribution within the nodules and their effect on N nutrition in Virgilia divaricata Adamson, Fabaceae, an indigenous legume in the Cape Floristic Region of South Africa. Specifically, we assessed tissue elemental localization using micro-particle-induced X-ray emission (PIXE), measured N fixation using nutrient concentrations derived from inductively coupled mass-spectrometry (ICP-MS), calculated nutrient costs, and determined P recycling from enzyme activity assays. Morphological and physiological features characteristic of adaptation to P deprivation were observed for V. divaricata. Decreased plant growth and nodule production with parallel increased root:shoot ratios are some of the plastic features exhibited in response to P deficiency. Plants resupplied with P resembled those supplied with optimal P levels in terms of growth and nutrient acquisition. Under low P conditions, plants maintained an increase in N₂-fixing efficiency despite lower levels of orthophosphate (Pi) in the nodules. This can be attributed to two factors: (i) an increase in Fe concentration under low P, and (ii) greater APase activity in both the roots and nodules under low P. These findings suggest that V. divaricata is well adapted to acquire N under P deficiency, owing to the plasticity of its nodule physiology     

The adenosine A2A antagonistic properties of selected C8-substituted xanthines

The adenosine A_2A receptor is considered to be an important target for the development of new therapies for Parkinson’s disease. Several antagonists of the A_2A receptor have entered clinical trials for this purpose and many research groups have initiated programs to develop A_2A receptor antagonists. Most A_2A receptor antagonists belong to two different chemical classes, the xanthine derivatives and the amino-substituted heterocyclic compounds. In an attempt to discover high affinity A_2A receptor antagonists and to further explore the structure–activity relationships (SARs) of A_2A antagonism by the xanthine class of compounds, this study examines the A_2A antagonistic properties of series of (E)-8-styrylxanthines, 8-(phenoxymethyl)xanthines and 8-(3-phenylpropyl)xanthines. The results document that among these series, the (E)-8-styrylxanthines have the highest binding affinities with the most potent homologue, (E)-1,3-diethyl-7-methyl-8-[(3-trifluoromethyl)styryl]xanthine, exhibiting a K_i value of 11.9 nM. This compound was also effective in reversing haloperidol-induced catalepsy in rats, providing evidence that it is in fact an A_2A receptor antagonist. The importance of substitution at C8 with the styryl moiety was demonstrated by the finding that none of the 8-(phenoxymethyl)xanthines and 8-(3-phenylpropyl)xanthines exhibited high binding affinities for the A_2A receptor.     

Single particle 3D reconstruction for 2D crystal images of membrane proteins

In cases where ultra-flat cryo-preparations of well-ordered two-dimensional (2D) crystals are available, electron crystallography is a powerful method for the determination of the high-resolution structures of membrane and soluble proteins. However, crystal unbending and Fourier-filtering methods in electron crystallography three-dimensional (3D) image processing are generally limited in their performance for 2D crystals that are badly ordered or non-flat. Here we present a single particle image processing approach, which is implemented as an extension of the 2D crystallographic pipeline realized in the 2dx software package, for the determination of high-resolution 3D structures of membrane proteins. The algorithm presented, addresses the low single-to-noise ratio (SNR) of 2D crystal images by exploiting neighborhood correlation between adjacent proteins in the 2D crystal. Compared with conventional single particle processing for randomly oriented particles, the computational costs are greatly reduced due to the crystal-induced limited search space, which allows a much finer search space compared to classical single particle processing. To reduce the considerable computational costs, our software features a hybrid parallelization scheme for multi-CPU clusters and computer with high-end graphic processing units (GPUs). We successfully apply the new refinement method to the structure of the potassium channel MloK1. The calculated 3D reconstruction shows more structural details and contains less noise than the map obtained by conventional Fourier-filtering based processing of the same 2D crystal images.