A novel approach for LC-MS/MS-based chiral metabolomics fingerprinting and chiral metabolomics extraction using a pair of enantiomers of chiral derivatization reagents

Chiral metabolites are found in a wide variety of living organisms and some of them are understood to be physiologically active compounds and biomarkers. However, the overall analysis of chiral metabolomics is quite difficult due to the high number of metabolites, the significant diversity in their physicochemical properties, and concentration range from metabolite-to-metabolite. To solve this difficulty, we developed a novel approach for chiral metabolomics fingerprinting and chiral metabolomics extraction, which is based on the labeling of a pair of enantiomers of chiral derivatization reagents (i.e., DMT-(S,R)-Pro-OSu and DMT-3(S,R)-Apy) and precursor ion scan chromatography of the derivatives. The multivariate statistics is also required for this strategy. The proposed procedures were evaluated by the detection of a diagnostic marker (i.e., d-lactic acid) using the saliva of diabetic patients. This method was used for the determination of biomarker candidates of chiral amines and carboxyls in Alzheimer's disease (AD) brain homogenates. As the results, l-phenylalanine (L-Phe) and l-lactic acid (L-LA) were identified as the decreased and increased biomarker candidates in the AD brain, respectively. Therefore, the proposed approach seems to be helpful for the determination of non-target chiral metabolomics possessing amines and carboxyls.     

A reversed‐phase compatible thin‐layer chromatography autography for the detection of acetylcholinesterase inhibitors

A dual readout autographic assay to detect acetylcholinesterase inhibitors present in complex matrices adsorbed on reversed‐phase or normal‐phase thin‐layer chromatography plates is described. Enzyme gel entrapment with an amphiphilic copolymer was used for assay development. The effects of substrate and enzyme concentrations, pH, incubation time, and incubation temperature on the sensitivity and the detection limit of the assay were evaluated. Experimental design and response surface methodology were used to optimize conditions with a minimum number of experiments. The assay allowed the detection of 0.01% w/w of physostigmine in both a spiked Sonchus oleraceus L. extract chromatographed on normal phase and a spiked Pimenta racemosa (Mill.) J.W. Moore leaf essential oil chromatographed on reversed phase. Finally, the reversed‐phase thin‐layer chromatography assay was applied to reveal the presence of an inhibitor in the Cymbopogon citratus (DC.) Stapf essential oil. The developed assay is able to detect acetylcholinesterase inhibitors present in complex matrixes that were chromatographed in normal phase or reversed‐phase thin‐layer chromatography. The detection limit for physostigmine on both normal and reversed phase was of 1×10^?4 μg. The results can be read by a change in color and/or a change in fluorescence.     

A one-pot synthesis of unsymmetrical bis-styrylbenzenes

An efficient and widely applicable one-pot synthesis of unsymmetrical (E,E)-bis-styrylbenzenes using successive Heck and Horner-Wadsworth-Emmons reactions is described.     

人类乙酰胆碱酯酶（ACHE）与抑制剂小分子的对接研究

阿尔茨海默病（Alzheimer’s disease,AD）是一种中枢神经系统致死性神经原发性退行病变,是老年痴呆症中最常见的一种类型．乙酰胆碱酯酶抑制剂（AChEI）是目前治疗AD的主要药物．我们选择他克林、利伐斯的明、石杉碱甲、在研药物TV-3326、多奈哌齐和Anseculin,分别与AChE进行分子对接研究．结果表明：抑制剂与AChE结合能力的大小顺序为：他克林〈利伐斯的明〈石杉碱甲〈TV．3326〈多奈哌齐〈Anseculin,这与实验中测得ACHE1的IC50值反应活性大小一致．上述6类药物分子,Anseculin与AChE的相互作用能力最强．     

Highly sensitive detection of S-nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence

S-nitrosylated proteins are biomarkers of oxidative damage in aging and Alzheimer's disease (AD). Here, we report a new method for detecting and quantifying nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF). Dylight 488 maleimide was used to specifically label thiol group (SH) after switching the S-nitrosothiol (S-NO) to SH in cysteine using the "fluorescence switch" assay. In vitro nitrosylation model-BSA subjected to S-nitrosoglutathione (GSNO) optimized the labeling reactions and characterized the response of the LIF detector. The method proves to be highly sensitive, detecting 1.3 picomolar (pM) concentration of nitrosothiols in nanograms of proteins, which is the lowest limit of detection of nitrosothiols reported to date. We further demonstrated the direct application of this method in monitoring protein nitrosylation damage in MQ mediated human colon adenocarcinoma cells. The nitrosothiol amounts in MQ treated and untreated cells are 14.8±0.2 and 10.4±0.5 pmol/mg of proteins, respectively. We also depicted nitrosylated protein electrophoretic profiles of brain cerebrum of 5-month-old AD transgenic (Tg) mice model. In Tg mice brain, 15.5±0.4 pmol of nitrosothiols/mg of proteins was quantified while wild type contained 11.7±0.3 pmol/mg proteins. The methodology is validated to quantify low levels of S-nitrosylated protein in complex protein mixtures from both physiological and pathological conditions.     

Discovery of potential inhibitor against human acetylcholinesterase: a molecular docking and molecular dynamics investigation

Alzheimer’s disease (AD) is a progressive neurodegenerative disease of central nervous system among elderly people. Human acetylcholinesterase (hAChE), an important enzyme in neuronal signaling, is responsible for the degradation of acetylcholine which in turn prevents the post synaptic signal transmissions. hAChE has been an attractive target of drug discovery for the search of therapeutics against AD. In the recent past hAChE has become hot target for the investigation of new potential therapeutics. We performed virtual screening of entire database against hAChE. Further, the extra precision molecular docking was carried out to refine the docking results and the best complex was passed for molecular dynamics simulations in order of understanding the hAChE dynamics and its behavior in complex with the ligand which corroborate the outcomes of virtual screening. This also provides binding free energy data that establishes the ligands efficiency for inhibiting hAChE. The computational findings discussed in this paper provide initial information of inhibitory effects of ligand, (drugbank entry DB00983), over hAChE.     

Influence of functional moiety in lupane-type triterpenoids in BACE1 inhibition

Lupane-type triterpenoids have shown a potential effect against neurodegenerative disorders. Alzheimer’s disease, one of the common neurodegenerative disease, is evident by the accumulation of amyloid-beta (Aβ) plaque in the extracellular regions of the brain. β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is a key enzyme for the Aβ formation viathe cleavage of amyloid precursor protein (APP). Therefore, to find the potent BACE1 inhibitors and furthermore to explore the role of the functional group responsible for the strong BACE1 inhibitory activity, we synthesized a series of triterpenoids with lupane skeleton starting from the natural compounds calenduladiol and lupeol. Compound 1 revealed a potent competitive BACE1 inhibitory activity (IC₅₀ = 16.77 ± 1.16 μM; K_i = 19.38). Furthermore, the molecular docking simulation revealed the importance of Tyr198 residue along with the other hydrophobic interactions for the strong affinity of 1‒BACE1 complex. To sum up, our results demonstrated the importance of carbonyl moiety at 3 and 16 position of lupane-type triterpenoid over the hydroxyl group at the same position.     

Discovery of a Highly Selective Glycogen Synthase Kinase‐3 Inhibitor (PF‐04802367) That Modulates Tau Phosphorylation in the Brain: Translation for PET Neuroimaging

Glycogen synthase kinase‐3 (GSK‐3) regulates multiple cellular processes in diabetes, oncology, and neurology. N‐(3‐(1H‐1,2,4‐triazol‐1‐yl)propyl)‐5‐(3‐chloro‐4‐methoxyphenyl)oxazole‐4‐carboxamide (PF‐04802367 or PF‐367) has been identified as a highly potent inhibitor, which is among the most selective antagonists of GSK‐3 to date. Its efficacy was demonstrated in modulation of tau phosphorylation in vitro and in vivo. Whereas the kinetics of PF‐367 binding in brain tissues are too fast for an effective therapeutic agent, the pharmacokinetic profile of PF‐367 is ideal for discovery of radiopharmaceuticals for GSK‐3 in the central nervous system. A ¹¹C‐isotopologue of PF‐367 was synthesized and preliminary PET imaging studies in non‐human primates confirmed that we have overcome the two major obstacles for imaging GSK‐3, namely, reasonable brain permeability and displaceable binding.     

在线固相萃取-液相色谱-串联质谱法检测蘑菇中毒患者尿液中痕量α -鹅膏毒肽

建立了在线固相萃取-液相色谱-串联质谱（online SPE-LC-MS/MS）测定蘑菇中毒患者尿液中痕量α -鹅膏毒肽的分析方法。样品经甲酸酸化的乙腈-甲醇（5：1,v/v）沉淀蛋白质,反相液液微萃取去除样品提取液中的有机溶剂,毒素经ODS微柱（5 mm×2.1 mm,5 μm）在线SPE净化,XBridge^TM BEH C₁₈ 色谱柱（150 mm×3.0 mm,2.5 μm）分离,MS/MS测定。采用基于定量环的快速阀切换技术作为在线SPE和LC-MS/MS模块的接口,使得两个分离模块互相独立,无论是流动相还是压力,都不会互相干扰,保证了系统的稳定性;在线系统的精准净化,有效消除了后续质谱检测的基质效应,确保了尿液中痕量水平α -鹅膏毒肽的定性定量检测。尿液中α -鹅膏毒肽在0.1~50 μg/L范围内线性关系良好,相关系数（r ² ）为0.9983;检出限（LOD）为0.03 μg/L;α -鹅膏毒肽的加标（0.1、2.0和20 μg/L）平均回收率为84.3%~91.7%,相对标准偏差（RSD）为3.8%~7.2%。体内鹅膏毒肽代谢迅速,生物基质中痕量水平毒素的检测是其中毒实验室鉴定的主要难题,通过实际样品检测,证明该法操作简单,准确、灵敏;溶剂沉淀蛋白质和反相液液微萃取去除有机相和脂溶性基质的简单操作,可以作为水溶性毒素在线SPE-LC-MS/MS检测时快速且有效的配套前处理方法;基于在线SPE精准净化技术,可以实现尿液中α -鹅膏毒肽的高灵敏度测定（LOD为0.03 μg/L）,解决了中毒时患者体内痕量水平α -鹅膏毒肽定性确证的难题,部分患者α -鹅膏毒肽中毒实验室鉴定的时间可以扩展到90 h以上;同时,痕量水平的定量检测技术,可以为中毒后迅速代谢的α -鹅膏毒肽在体内的剂量反应关系研究提供可靠的技术支撑。     

Network pharmacology-based screening of the active ingredients and potential targets of the genus of Pithecellobium marthae (Britton & Killip) Niezgoda & Nevl for application to Alzheimer’s disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder associated with synaptic dysfunction, pathological accumulation of β-amyloid (Aβ), and neuronal loss. Given the prevalence of AD and the lack of effective long-term therapies, there is a pressing need to discover viable leads that can be developed into clinically approved drugs with disease-modifying effects. The analysis of current reported literatures confirms the importance of the plants of Pithecellobium genus as candidate against AD. Hence, it is necessary to identify selective anti-dementia agents from this genus. To explore potential compounds with marked effect on AD in Pithecellobium genus, a compound database based on the methods of network pharmacology prediction was established in this paper by constructing the compound-disease target network. The result showed that the most effective compound in the plants of this genus might be (7′R,8′R)-7′-methoxyl strebluslignanol, and the most potential target might be Macrophage colony-stimulating factor 1 receptor.