Synthesis and biological evaluation of 4,5-dihydro-1H-pyrazole derivatives as potential nNOS/iNOS selective inhibitors. Part 2: Influence of diverse substituents in both the phenyl moiety and the acyl group

In a preliminary article, we reported a series of 4,5-dihydro-1H-pyrazole derivatives as neuronal nitric oxide synthase (nNOS) inhibitors. Here we present the data about the inhibition of inducible nitric oxide synthase (iNOS) of these compounds. In general, we can confirm that these pyrazoles are nNOS selective inhibitors. In addition, taking these compounds as a reference, we have designed and synthesized a series of new derivatives by modification of the heterocycle in 1-position, and by introduction of electron-donating or electron-withdrawing substituents in the aromatic ring. These derivatives have been evaluated as nNOS and iNOS inhibitors in order to identify new compounds with improved activity and selectivity. Compound 3r, with three methoxy electron-donating groups in the phenyl moiety, is the most potent nNOS inhibitor, showing good selectivity nNOS/iNOS.     

Voltage-dependent anion channel (VDAC) participates in amyloid beta-induced toxicity and interacts with plasma membrane estrogen receptor α in septal and hippocampal neurons

Voltage-dependent anion channel (VDAC) is a porin known by its role in metabolite transport across mitochondria and participation in apoptotic processes. Although traditionally accepted to be located within mitochondrial outer membrane, some data has also reported its presence at the plasma membrane level where it seems to participate in regulation of normal redox homeostasis and apoptosis. Here, exposure of septal SN56 and hippocampal HT22 cells to specific anti-VDAC antibodies prior to amyloid beta (Aβ) peptide was observed to prevent neurotoxicity. In these cell lines, we identified a VDAC form associated with the plasma membrane that seems to be particularly abundant in caveolae. The two membrane-related isoforms of estrogen receptor α (mERα) (80 and 67 kDa), known in SN56 cells to participate in estrogen-induced neuroprotection against Aβ injury, were also observed to be present in caveolae. Interestingly, we demonstrated for the first time that both VDAC and mERα interact at the plasma membrane of these neurons as well as in microsomal fractions of the corresponding murine septal and hippocampal tissues. These proteins were also shown to associate with caveolin-1, thereby corroborating their presence in caveolar microdomains. Taken together, these results suggest that VDAC-mERα association at the plasma membrane level may participate in the modulation of Aβ-induced cell death.     

Functional consequences of retro-inverso isomerization of a miniature protein inhibitor of the p53–MDM2 interaction

Peptide retro-inverso isomerization is thought to be functionally neutral and has been widely used as a tool for designing proteolytically stable d-isomers to recapitulate biological activities of their parent l-peptides. Despite success in a wide range of applications, exceptions amply exist that clearly defy this rule of thumb when parent l-peptides adopt an α-helical conformation in their bound state. The detrimental energetic effect of retro-inverso isomerization of an α-helical l-peptide on its target protein binding has been estimated to be 3.0–3.4 kcal/mol. To better understand how the retro-inverso isomer of a structured protein works at the molecular level, we chemically synthesized and functionally characterized the retro-inverso isomer of a rationally designed miniature protein termed stingin of 18 amino acid residues, which adopts an N-terminal loop and a C-terminal α-helix stabilized by two intra-molecular disulfide bridges. Stingin emulated the transactivation peptide of the p53 tumor suppressor protein and bound with high affinity and via its C-terminal α-helix to MDM2 and MDMX—the two negative regulators of p53. We also prepared the retro isomer and d-enantiomer of stingin for comparative functional studies using fluorescence polarization and surface plasmon resonance techniques. We found that retro-inverso isomerization of l-stingin weakened its MDM2 binding by 720 fold (3.9 kcal/mol); while enantiomerization of l-stingin drastically reduced its binding to MDM2 by three orders of magnitude, sequence reversal completely abolished it. Our findings demonstrate the limitation of peptide retro-inverso isomerization in molecular mimicry and reinforce the notion that the strategy works poorly with biologically active α-helical peptides due to inherent differences at the secondary and tertiary structural levels between an l-peptide and its retro-inverso isomer despite their similar side chain topologies at the primary structural level.¹     

TOPS-MODE model of multiplexing neuroprotective effects of drugs and experimental-theoretic study of new 1,3-rasagiline derivatives potentially useful in neurodegenerative diseases

The interest on computational techniques for the discovery of neuroprotective drugs has increased due to recent fail of important clinical trials. In fact, there is a huge amount of data accumulated in public databases like CHEMBL with respect to structurally heterogeneous series of drugs, multiple assays, drug targets, and model organisms. However, there are no reports of multi-target or multiplexing Quantitative Structure–Property Relationships (mt-QSAR/mx-QSAR) models of these multiplexing assay outcomes reported in CHEMBL for neurotoxicity/neuroprotective effects of drugs. Accordingly, in this paper we develop the first mx-QSAR model for multiplexing assays of neurotoxicity/neuroprotective effects of drugs. We used the method TOPS-MODE to calculate the structural parameters of drugs. The best model found correctly classified 4393 out of 4915 total cases in both training and validation. This is representative of overall train and validation Accuracy, Sensitivity, and Specificity values near to 90%, 98%, and 80%, respectively. This dataset includes multiplexing assay endpoints of 2217 compounds. Every one compound was assayed in at least one out of 338 assays, which involved 148 molecular or cellular targets and 35 standard type measures in 11 model organisms (including human). The second aim of this work is the exemplification of the use of the new mx-QSAR model with a practical case of study. To this end, we obtained again by organic synthesis and reported, by the first time, experimental assays of the new 1,3-rasagiline derivatives 3 different tests: assay (1) in absence of neurotoxic agents, (2) in the presence of glutamate, and (3) in the presence of H₂O₂. The higher neuroprotective effects found for each one of these assays were for the stereoisomers of compound 7: compound 7b with protection = 23.4% in assay (1) and protection = 15.2% in assay (2); and for compound 7a with protection = 46.2% in assay (3). Interestingly, almost all compounds show protection values >10% in assay (3) but not in the other 2 assays. After that, we used the mx-QSAR model to predict the more probable response of the new compounds in 559 unique pharmacological tests not carried out experimentally. The results obtained are very significant because they complement the pharmacological studies of these promising rasagiline derivatives. This work paves the way for further developments in the multi-target/multiplexing screening of large libraries of compounds potentially useful in the treatment of neurodegenerative diseases.     

New structure–activity relationship studies in a series of N,N-bis(cyclohexanol)amine aryl esters as potent reversers of P-glycoprotein-mediated multidrug resistance (MDR)

As a continuation of previous research on a new series of potent and efficacious P-gp-dependent multidrug resistant (MDR) reversers with a N,N-bis(cyclohexanol)amine scaffold, we have designed and synthesized several analogs by modulation of the two aromatic moieties linked through ester functions to the N,N-bis(cyclohexanol)amine, aiming to optimize activity and to extend structure–activity relationships (SAR) within the series. This scaffold, when esterified with two different aromatic carboxylic acids, gives origin to four geometric isomers (cis/trans, trans/trans, cis/cis and trans/cis).The new compounds were tested on doxorubicin-resistant erythroleukemia K562 cells (K562/DOX) in the pirarubicin uptake assay. Most of them resulted in being potent modulators of the extrusion pump P-gp, showing potency values ([I]_0.5) in the submicromolar and nanomolar range. Of these, compounds 2b, 2c, 3d, 5a–d and 6d, showed excellent efficacy with a α_max close to 1. Selected compounds (2d, 3a, 3b, 5a–d) were further studied to evaluate their doxorubicin cytotoxicity potentiation (RF) on doxorubicin-resistant erythroleukemia K562 cells and were found able to enhance significantly doxorubicin cytotoxicity on K562/DOX cells.The results of both pirarubicin uptake and the cytotoxicity assay, indicate that the new compounds of the series are potent P-gp-mediated MDR reversers. They present a structure with a mix of flexible and rigid moieties, a property that seems critical to allow the molecules to choose the most productive of the several binding modes possible in the transporter recognition site.In particular, compounds 5c and 5d, similar to the already reported analogous isomers 1c and 1d,²⁹ are potent and efficacious modulators of P-gp-dependent MDR and may be promising leads for the development of MDR-reversal drugs.     

The Cardiac Ryanodine Receptor N-Terminal Region Contains an Anion Binding Site that Is Targeted by Disease Mutations

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Genome-wide interval mapping using SNPs identifies new QTL for growth,body composition and several physiological variables in an F2 intercross between fat and lean chicken lines

Background

For decades, genetic improvement based on measuring growth and body composition traits has been successfully applied in the production of meat-type chickens. However, this conventional approach is hindered by antagonistic genetic correlations between some traits and the high cost of measuring body composition traits. Marker-assisted selection should overcome these problems by selecting loci that have effects on either one trait only or on more than one trait but with a favorable genetic correlation. In the present study, identification of such loci was done by genotyping an F₂ intercross between fat and lean lines divergently selected for abdominal fatness genotyped with a medium-density genetic map (120 microsatellites and 1302 single nucleotide polymorphisms). Genome scan linkage analyses were performed for growth (body weight at 1, 3, 5, and 7 weeks, and shank length and diameter at 9 weeks), body composition at 9 weeks (abdominal fat weight and percentage, breast muscle weight and percentage, and thigh weight and percentage), and for several physiological measurements at 7 weeks in the fasting state, i.e. body temperature and plasma levels of IGF-I, NEFA and glucose. Interval mapping analyses were performed with the QTLMap software, including single-trait analyses with single and multiple QTL on the same chromosome.

Results

Sixty-seven QTL were detected, most of which had never been described before. Of these 67 QTL, 47 were detected by single-QTL analyses and 20 by multiple-QTL analyses, which underlines the importance of using different statistical models. Close analysis of the genes located in the defined intervals identified several relevant functional candidates, such as ACACA for abdominal fatness, GHSR and GAS1 for breast muscle weight, DCRX and ASPSCR1 for plasma glucose content, and ChEBP for shank diameter.

Conclusions

The medium-density genetic map enabled us to genotype new regions of the chicken genome (including micro-chromosomes) that influenced the traits investigated. With this marker density, confidence intervals were sufficiently small (14 cM on average) to search for candidate genes. Altogether, this new information provides a valuable starting point for the identification of causative genes responsible for important QTL controlling growth, body composition and metabolic traits in the broiler chicken.     

基于SSU序列的串珠藻目植物系统发育分析

通过18S rDNA基因(SSU)序列,构建了串珠藻目植物的系统发育关系.结果显示:SSU基因序列片段长度为1 871 bp,核苷酸变异位点有709个,占序列长度的38％;其中简约信息位点有169个,占序列长度的9％.用最大似然法、邻接法和贝叶斯法构建的系统树拓扑结构基本一致,都显示红索藻目的2个属独立于串珠藻目成单独分支,支持红索藻目的建立;胶串珠藻独立于其他串珠藻组植物,支持将其单独分组;数据同时支持将扭曲组和杂生组合并,建立Kumanoa属;但多芒组、绿色组、沼生组等因分子序列数据涉及的种类较少,其系统关系的确定还需要更多的证据.     

表没食子儿茶素没食子酸酯对高脂饮食大鼠脂肪组织TNF-α表达的影响及意义

目的 探讨表没食子儿茶素没食子酸酯（EGCG）对高脂饮食大鼠脂肪组织中肿瘤坏死因子-α（TNF-α）表达的影响及其与胰岛素敏感性的相关性.方法 将30只SPF级雄性SD大鼠随机分为正常饮食组（ND组,n=10）和高脂饮食组（HFD组,n=20）.喂养16w,当两组大鼠体质量出现显著差异后（P〈0.05）,将HFD组按随机区组原则分为单纯高脂组（HFD组,n=10）和EGCG干预组（HFD＋0.32%EGCG,EGCG组,n=10）.干预16w.留取血清及附睾周脂肪组织.检测每组大鼠空腹血糖（FBG）、胰岛素水平（FINS）及游离脂肪酸（FFAs）,并计算胰岛素抵抗指数（HOMA-IR）;应用Real-time PCR及Western blot方法检测附睾周脂肪组织中TNF-α表达水平.结果 （1）与HFD组相比,EGCG组FINS水平显著下降[分别为（13.83±0.79）mIU/l vs.（31.71±3.61）mIU/l,P〈0.05];HOMA-IR值下降[分别为（3.36±0.31） vs.（7.59±0.99）,P〈0.05];FFAs值亦明显下降[分别为（0.38±0.08）mmol/l vs.（0.81±0.11）mmol/l,P〈0.05];三组大鼠FBG水平无明显统计学差异（P〉0.05）.（2）与ND组相比,HFD组脂肪组织中TNF-αmRNA水平显著升高[分别为（0.0033±0.00070）vs.（0.0010±0.00008）,P〈0.01];而EGCG组较HFD组则明显下降[分别为（0.0018±0.00037）vs.（0.0033±0.00070）,P〈0.05];同时,EGCG组TNF-α蛋白表达量低于HFD组[分别为（0.42±0.09）vs.（0.67±0.09）,P〈0.05];（3）EGCG组与ND组无明显差异（P〉0.05）.结论 EGCG改善高脂饮食大鼠胰岛素敏感性可能与减轻脂肪组织TNF-α介导的炎症状态相关.     

Identification of Fungal Species Associated with Cladode Spot of Prickly Pear and Their Sensitivity to Chitosan

México is the most important producer of prickly pear (Opuntia ficus‐indica) in the world. There are several fungal diseases that can have a negative impact on their yields. In this study, there was a widespread fungal richness on cladodes spot of prickly pears from México. A total of 41 fungi isolates were obtained from cladodes spot; 11 of them were morphologically different. According to the pathogenicity test, seven isolates caused lesions on cladodes. The morphological and molecular identification evidenced the isolation of Colletotrichum gloeosporioides, Alternaria alternata, Fusarium lunatum, Curvularia lunata. All these species caused similar symptoms of circular cladodes spot. However, it is noticeable that some lesions showed perforation and detachment of affected tissues by Fusarium lunatum. To our knowledge, this is the first report of the Fusarium lunatum as phytopathogenic fungus of cladodes of prickly pear. The chitosan inhibited the mycelium growth in the seven isolates of phytopathogenic fungi. Chitosan applications diminished the disease incidence caused by C. gloeosporioies and F. lunatum in 40 and 100%, respectively. Likewise, the lesion severity index in cladodes decreased. There are no previous reports about the application of chitosan on cladodes of prickly pears for the control of phytopathogenic fungi. Therefore, this research could contribute to improve the strategies for the management of diseases in prickly pear.