Redox active or thiol reactive? Optimization of rapid screens to identify less evident nuisance compounds

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity are routinely encountered in assays at various stages of drug discovery. We observed that assays for the investigation of thiol-reactive and redox-active compounds have not been collected in a comprehensive review. Here, we review these assays and subject them to experimental optimization to improve their reliability. We demonstrate the usefulness of our assay cascade by assaying a library of bioactive compounds, chemical probes, and a set of approved drugs. These high-throughput assays should complement the array of wet-lab and in silico assays during the initial stages of hit discovery campaigns to pursue only hit compounds with tractable mechanisms of action.     

G6PD缺陷对 1,4-苯醌致K562细胞DNA甲基化的影响及其机制

目的：研究G6PD缺陷对1，4-苯醌（1，4-BQ）致K562细胞DNA甲基化的影响及其机制。方法：分别采用10和20 μmol/L的1，4-BQ溶液对G6PD缺陷K562细胞和正常表达细胞进行染毒，以未染毒组为对照，各组均设置12、24、48 h共3个染毒时间，另在20 μmol/L 1，4-BQ染毒组细胞中加入1.5 mmol/L谷胱甘肽（GSH）进行干预。采用比色法检测全基因组DNA甲基化相对水平，qPCR法检测甲基转移酶DNMT1、DNMT3a、DNMT3b mRNA表达水平，Western blot法检测DNMT1、DNMT3a蛋白表达水平。结果：10和20 μmol/L 1，4-BQ染毒后的K562细胞全基因组DNA甲基化水平升高（P<0.05），除20 μmol/L 1，4-BQ染毒48 h组外，其他各染毒组的G6PD缺陷K562细胞全基因组DNA甲基化水平均高于正常细胞（P<0.05）。在1，4-BQ染毒后，G6PD缺陷细胞的DNMT1、DNMT3a、DNMT3b mRNA水平以及DNMT1、DNMT3a蛋白表达水平均高于正常细胞（P<0.05）。加入GSH后，G6PD缺陷细胞和正常细胞的全基因组DNA甲基化相对水平、DNMTs mRNA及蛋白表达水平的差异的无统计学意义（P均>0.05）。结论：G6PD缺陷可能以抑制K562细胞GSH合成的方式增加氧化应激水平，最终导致细胞DNMTs生成的增多以及全基因组DNA甲基化程度的升高。     

基于血热证小鼠模型紫草素清热凉血与血液氧化还原稳态调控的研究

目的 探究紫草素（SK）对活性酵母诱导的血热证小鼠模型的清热凉血作用与血液氧化还原稳态调控的关系。方法 小鼠随机分为对照组、模型组、SK（10 mg/kg）组、阿司匹林（Asp，200 mg/kg）组、L-丁硫氨酸亚砜亚胺（BSO，600 mg/kg）组、N-乙酰-L-半胱氨酸（NAC，400 mg/kg）组、BSO联合SK组、NAC联合SK组。小鼠颈背部sc 0.2 g/mL活性酵母提取物（10 mL/kg），造模0.5 h后ip SK，造模8 h后ig Asp，于实验开始前ip BSO、NAC，2次/d，连续1周。考察小鼠肛温（t_R）、基本活动、代谢、凝血、血细胞计数等指标，采用OPA荧光法测定小鼠血清中还原型谷胱甘肽（GSH）、氧化型谷胱甘肽（GSSG）水平。结果 与对照组相比，模型组小鼠造模4 h后t_R显著升高（P<0.05），10 h达到（38.07±0.11）℃；小鼠出现烦躁、口唇色红、喜饮、碳末推进率降低、粪便含水量降低、凝血时间延长现象（P<0.05）；肺组织红细胞、炎性细胞浸润；血清LPO、MDA水平显著升高、SOD活性降低、GSH/GSSG比率减小（P<0.05）。与模型组相比，SK组小鼠t_R显著降低，10 h达到（37.51±0.12）℃；各指标均得到显著改善；血清LPO、MDA水平降低、SOD活性升高、GSH/GSSG比率增大（P<0.05）。BSO组可降低血清GSH/GSSG比率（P<0.05），减弱SK对血热证小鼠的治疗作用；NAC组可增加血清GSH水平，增加GSH/GSSG比率（P<0.05），增强SK对血热证小鼠的治疗效果。结论 SK通过调控血液氧化还原稳态，缓解血热证小鼠高热、出血瘀血、血液氧化损伤，从而发挥清热凉血作用。     

双氢青蒿素诱导肿瘤细胞铁死亡及其机制研究

目的阐明双氢青蒿素(DHA)诱导肿瘤细胞铁死亡的作用及其机制。方法利用3,3′,5,5′-四甲基联苯胺(TMB)检测DHA与FeSO_4体外芬顿样(Fenton)反应生成氧自由基(·OH)的能力;MTT法检测DHA对人肝癌HepG2细胞的毒性(包括FeSO_4与去铁胺预处理组)。MTT法考察谷胱甘肽(GSH)与铁死亡抑制剂(Fer-1)对DHA细胞毒性的影响;采用DCFH-DA染料考察DHA(包括FeSO_4预处理组)诱导的细胞内活性氧的生成能力;采用C11-BODIPY581/591与DiO分别考察DHA(包括FeSO_4预处理组)对细胞内脂质过氧化物生成能力以及细胞膜结构的影响;利用谷胱甘肽过氧化物酶4(GPX-4)试剂盒测定DHA(包括FeSO_4预处理组)对HepG2细胞内GPX-4活性的影响。结果 Fe~(2+)能够催化DHA发生芬顿样反应并生成·OH;DHA的半数抑制浓度(IC_(50))为(39.96±8.78)μmol/L,FeSO_4与去铁胺分别能够增加或者降低DHA的细胞毒性;DHA处理后细胞内活性氧含量与脂质过氧化物含量升高,细胞形态变大,细胞膜呈散点状分布并呈现解离状态。FeSO_4预处理组与DHA组相比较进一步增加细胞内活性氧含量与脂质过氧化物含量,并且细胞膜形态完全破坏。FeSO_4能够增强DHA对GPX-4活性的抑制作用。结论 DHA通过芬顿样反应升高细胞内活性氧而最终诱导肿瘤细胞铁死亡。另外,外源性铁可以加速DHA发生芬顿样反应进而加速肿瘤细胞铁死亡的发生与发展。     

宫颈鳞状细胞癌对DDP和DDP+5-FU的体外药敏检测及与耐药蛋白表达的关系研究

目的通过对宫颈鳞状细胞癌进行顺铂(DDP)和DDP+5-氟尿嘧啶(5-FU)体外药敏检测,比较两种化疗方案的体外有效率。同时检测其耐药蛋白P糖蛋白(P-gp)、谷胱甘肽S-转移酶-π(GST-π)、DNA拓扑异构酶Ⅱ(TopoⅡ)、胸苷酸合成酶(TS)的表达,探讨DDP和DDP+5-FU体外药敏与耐药蛋白表达的关系。方法收集35例宫颈鳞状细胞癌患者的新鲜肿瘤组织,采用三磷酸腺苷生物荧光法(ATP-TCA)对DDP和DDP+5-FU进行体外药敏检测,同时采用EnVision二步法检测宫颈鳞状细胞癌组织中耐药蛋白P-gp、GST-π、TopoⅡ、TS的表达,并分析DDP和DDP+5-FU的体外药敏与耐药蛋白P-gp、GST-π、TopoⅡ、TS表达的关系。结果 35例标本均进行药敏试验,DDP的体外有效率为37.14%,与DDP+5-FU的51.43%比较,差异无统计学意义(P> 0.05)。经多因素分析发现,患者年龄、临床分期、分化程度均不为DDP、DDP+5-FU药物敏感性的影响因素(P> 0.05)。P-gp、GST-π、TopoⅡ、TS蛋白阳性表达率分别为57.14%(20/35)、51.43%(18/35)、71.43%(25/35)、57.14%(20/35)。GST-π蛋白阳性表达是宫颈鳞状细胞癌对DDP耐药的影响因素(P=0.002);TS蛋白阳性表达是宫颈鳞状细胞癌对DDP+5-FU耐药的影响因素(P=0.001)。结论 宫颈鳞状细胞癌ATP-TCA法检测DDP+5-FU与单药DDP相比,体外有效率无显著差异。GST-π、TS蛋白阳性表达可用于临床宫颈癌患者对DDP、5-FU化疗耐药的预测指标。     

Nitric oxide donors offer protection to RBC from storage lesion

BackgroundRed blood cell (RBC), which is the most commonly transfused blood component, due to its ability to save a life in absence of any other blood components, can be stored up to maximum 6 weeks by following standard preservation procedure. During storage, RBC undergoes various biophysical and biochemical changes (commonly known as storage lesion) for which blood transfusion with “old RBC” shows a lot of clinical problems especially relevant to critically ill patients. Recent research on S-nitrosylation of haemoglobin to improve oxygen delivery of banked blood revealed the important role of nitric oxide (NO) in protecting storage lesion.Materials and methodsIn the present study, we used various “NO donating” chemicals with different NO release dynamics and chemistries in RBC storage cocktails to test the effects of NO on storage lesion. Changes in different storage markers were evaluated after 7 days storage of pre-treated RBC.ResultsAll the NO donors have shown protection against hemolysis. However, S-nitroso glutathione (GSNO) ranks first in shielding RBCs from storage lesion and additionally, it helps in elevating the value of 2, 3-di phosphoglycerate (2, 3-DPG), improving the RBC membrane fluidity and decreasing the adhesion towards endothelial monolayer.DiscussionPresent study reveals that NO released from NO donors confers protection against storage lesions of the RBC. Further, the study confirms that pre-treatment with GSNO, a NO donor and a nitrosylating agent, ensures the best protection to RBC during low temperature storage, when compared to other NO donor treatments.     

Oxidative stress markers in seminal plasma of idiopathic infertile men may be associated with glutathione S-transferase M1 and T1 null genotypes

This study aimed to investigate the association between glutathione S-transferase (GST) M1 and T1 null genotypes and thiobarbituric acid reactive substances (TBARS), total antioxidant capacity (TAC) and nitric oxide (NO) levels in male infertility. For this purpose, semen samples were collected from fertile and infertile subjects, and then they were genotyped for GSTT1 and GSTM1 genes using multiplex-PCR. The TBARS, TAC and NO levels in seminal plasma were then measured via the ferric-reducing ability of plasma (FRAP). A significant association was observed between GSTT1 null genotype and oligozoospermia, asthenozoospermia and teratozoospermia. But, the GSTM1 null genotype was merely associated with teratozoospermia. Moreover, the GSTT1−/GSTM1+ combined genotype was associated with all subgroups of male infertility. Besides, an association was observed between GSTT1−/GSTM1− genotype and asthenozoospermia and teratozoospermia. Further analysis showed that the GSTT1 null genotype was associated with increased NO in asthenozoospermia. Also, the GSTT1 null genotype was associated with increased TBARS in oligozoospermia and asthenozoospermia. As well, GSTM1 null genotype was associated with decreased TAC and increased NO in asthenozoospermia respectively. As a preliminary conclusion, the GSTM1 and GSTT1 null genotypes could be considered as genetic risk factors for male infertility, interfering with some oxidative stress markers in infertile men.     

Thiol‐water proton exchange of glutathione,cysteine, and N‐acetylcysteine: Implications for CEST MRI

Amide‐, amine‐, and hydroxyl‐water proton exchange can generate MRI contrast through chemical exchange saturation transfer (CEST). In this study, we show that thiol‐water proton exchange can also generate quantifiable CEST effects under near‐physiological conditions (pH = 7.2 and 37°C) through the characterization of the pH dependence of thiol proton exchange in phosphate‐buffered solutions of glutathione, cysteine, and N‐acetylcysteine. The spontaneous, base‐catalyzed, and buffer‐catalyzed exchange contributions to the thiol exchange were analyzed. The thiol‐water proton exchange of glutathione and cysteine was found to be too fast to generate a CEST effect around neutral pH due to significant base catalysis. The thiol‐water proton exchange of N‐acetylcysteine was found to be much slower, yet still in the fast‐exchange regime with significant base and buffer catalysis, resulting in a 9.5% attenuation of the water signal at pH 7.2 in a slice‐selective CEST NMR experiment. Furthermore, the N‐acetylcysteine thiol CEST was also detectable in human serum albumin and agarose phantoms.