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.     

急性Q热并横纹肌溶解1例

 Q热是由贝纳柯克斯体感染引起的人畜共患疾病,临床表现可引起肺炎、肝炎、心肌炎、心内膜炎等,但合并横纹肌溶解病例相对少见。贝纳柯克斯体感染诊断相对困难,而高通量测序,包括纳米孔三代测序(ONT)在内,可作为诊断Q热较为敏感的检测方法,早期诊断和及时治疗,一般Q热预后良好。本文报告1例经ONT检测确诊为急性Q热合并横纹肌溶解的病例,该病例经治疗后病情好转出院。     

基于肠道菌群探讨甘草泻心汤对伊立替康致小鼠腹泻的影响

目的 研究甘草泻心汤对化疗相关腹泻小鼠肠道菌群的影响。方法 将小鼠随机分为对照组、模型组、洛哌丁胺（阳性药,0.4 mg·kg^-1）组和甘草泻心汤高、低剂量（16.4、4.1 g·kg^-1）组;采用连续4 d ip 55 mg·kg^-1伊立替康诱导小鼠化疗相关腹泻模型;记录小鼠首次排便时间、稀便级、稀便率和腹泻指数;采用试剂盒测定小鼠肠道消化酶活力及炎症因子水平;采用高通量测序技术分析结肠内容物肠道菌群变化。结果 与对照组比较,模型组小鼠首次排便时间显著缩短（P<0.01）,稀便级、稀便率和腹泻指数显著升高（P<0.01）;消化酶乳酸脱氢酶（LDH）、淀粉酶（AMS）和脂肪酶（LPS）活力显著降低（P<0.01）;炎症因子白细胞介素1β （IL-1β）、环氧化酶2 （COX-2）、细胞间黏附分子1 （ICAM-1）和肿瘤坏死因子α （TNF-α）水平显著升高（P<0.01）;肠道菌群α多样性Simpson指数显著升高（P<0.05）;肠道菌群β多样性差异较大;变形菌门、梭杆菌门、变形菌纲、梭杆菌纲、肠杆菌目、梭杆菌目、拟杆菌科、丹毒丝菌科、埃格特菌科和拟杆菌属丰度显著升高（P<0.01）,Muribaculaceae和norank_f__Muribaculaceae丰度显著降低（P<0.01）。与模型组比较,甘草泻心汤高、低剂量组小鼠首次排便时间显著延长（P<0.05、0.01）、稀便率和腹泻指数显著降低（P<0.05、0.01）,高剂量组稀便级显著降低（P<0.01）;高、低剂量组各消化酶活力显著回升（P<0.05、0.01）;高、低剂量组各炎症因子水平显著降低（P<0.05、0.01）;高剂量组Simpson指数显著降低（P<0.05）,β多样性更趋近对照组,显著回调各优势物种丰度的显著性变化（P<0.05、0.01）。结论 甘草泻心汤对于伊立替康诱导的腹泻小鼠具有明显的治疗作用,可能是其通过调整腹泻小鼠肠道菌群实现。     

Heteromers of μ-δ opioid receptors: new pharmacology and novel therapeutic possibilities

Several studies suggest that heteromerization between μ (MOP) and δ (DOP) opioid receptors modulates the signalling properties of the individual receptors. For example, whereas activation of MOP receptors by an agonist induces G protein-mediated signalling, the same agonist induces β-arrestin-mediated signalling in the context of the MOP-DOP receptor heteromer. Moreover, heteromer-mediated signalling is allosterically modulated by a combination of MOP and DOP receptor ligands. This has implications in analgesia given that morphine-induced antinociception can be potentiated by DOP receptor ligands. Recently reagents selectively targeting the MOP-DOP receptor heteromer such as bivalent ligands, antibodies or membrane permeable peptides have been generated; these reagents are enabling studies to elucidate the contribution of endogenously expressed heteromers to analgesia as well as to the development of side-effects associated with chronic opioid use. Recent advances in drug screening technology have led to the identification of a MOP-DOP receptor heteromer-biased agonist that activates both G protein-mediated and β-arrestin-mediated signalling. Moreover, this heteromer-biased agonist exhibits potent antinociceptive activity but with reduced side-effects, suggesting that ligands targeting the MOP-DOP receptor heteromer form a basis for the development of novel therapeutics for the treatment of pain. In this review, we summarize findings regarding the biological and functional characteristics of the MOP-DOP receptor heteromer and the in vitro and in vivo properties of heteromer-selective ligands.

LINKED ARTICLES

This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2     

基于报告基因检测的PXR、FXR和LXRα激动剂高通量筛选模型的建立

目的建立基于报告基因法的高通量筛选细胞模型,用来发现PXR、FXR和LXRα受体激动剂。方法利用Real-time定量PCR方法比较HEK293、Hep G2和LS174T细胞中内源性核受体PXR、FXR和LXRα的表达量,将p SG5-h PXR和p GL3-XREM-CYP3A4、p EGFP-N3-h FXR和EcRETK-Luc、p CMX-FLAG-h LXRα和p GL3-XREM-CYP3A4等质粒分别共转染到工具细胞中,优化共转染比例,并考察阳性药与萤光素酶报告基因表达强度的量效关系、模型特异性和稳定性。结果 1根据Real-time定量PCR结果,模型选用低表达PXR、FXR和LXRα的HEK293细胞作为工具细胞;2根据不同共转染比例对报告基因活性的结果,PXR、FXR和LXRα报告基因药物筛选模型的报告基因和过表达质粒比例,最终分别选择1∶1、2∶1和2∶1;3模型中,报告基因活性均与相应阳性药物(PXR/Rif、FXR/CDCA和LXRα/T0901317)呈剂量依赖性增长;4仅PXR激动剂Rif、FXR激动剂CDCA和LXRα激动剂T0901317可分别明显增加相应筛选模型的报告基因活性,分别重复5次试验后,计算得Z'值分别为0.58、0.66和0.63。结论该研究建立的PXR、FXR和LXRα激动剂高通量筛选模型,具有良好的特异性和稳定性,适用于对PXR、FXR和LXRα受体激动剂的筛选,进而开发以核受体作为药物靶点的药物。     

Quantification of cellular viability by automated microscopy and flow cytometry

Cellular viability is usually determined by measuring the capacity of cells to exclude vital dyes such as 4′,6-diamidino-2-phenylindole (DAPI), or by assessing nuclear morphology with chromatinophilic plasma membrane-permeant dyes, such as Hoechst 33342. However, a fraction of cells that exclude DAPI or exhibit normal nuclear morphology have already lost mitochondrial functions and/or manifest massive activation of apoptotic caspases, and hence are irremediably committed to death. Here, we developed a protocol for the simultaneous detection of plasma membrane integrity (based on DAPI) or nuclear morphology (based on Hoechst 33342), mitochondrial functions (based on the mitochondrial transmembrane potential probe DiOC₆(3)) and caspase activation (based on YO-PRO^®-3, which can enter cells exclusively upon the caspase-mediated activation of pannexin 1 channels). This method, which allows for the precise quantification of dead, dying and healthy cells, can be implemented on epifluorescence microscopy or flow cytometry platforms and is compatible with a robotized, high-throughput workflow.     

Dual inhibition of HDAC and EGFR signaling with CUDC-101 induces potent suppression of tumor growth and metastasis in anaplastic thyroid cancer

Anaplastic thyroid cancer (ATC) is one of the most lethal human malignancies that currently has no effective therapy. We performed quantitative high-throughput screening (qHTS) in three ATC cell lines using 3,282 clinically approved drugs and drug candidates, and identified 100 active agents. Enrichment analysis of active compounds showed that inhibitors of EGFR and histone deacetylase (HDAC) were most active. Of these, the first-in-class dual inhibitor of EGFR, HER2 and HDACs, CUDC-101, had the highest efficacy and lower IC₅₀ than established drugs. We validated that CUDC-101 inhibited cellular proliferation and resulted in cell death by inducing cell cycle arrest and caspase-dependent apoptosis. CUDC-101 also inhibited cellular migration in vitro. Mechanistically, CUDC-101 inhibited MAPK signaling and histone deacetylation in ATC cell lines with multiple driver mutations present in human ATC. The anticancer effect of CUDC-101 was associated with increased expression of p21 and E-cadherin, and reduced expression of survivin, XIAP, β-catenin, N-cadherin, and Vimentin. In an in vivo mouse model of metastatic ATC, CUDC-101 inhibited tumor growth and metastases, and significantly prolonged survival. Response to CUDC-101 treatment in vivo was associated with increased histone 3 acetylation and reduced survivin expression. Our findings provide a preclinical basis to evaluate CUDC-101 therapy in ATC.     

急性和慢性髓性白血病细胞中可变剪接事件的识别

目的：为揭示异常的可变剪接在肿瘤中发挥的作用,着重研究急性髓性白血病（ AML）与慢性髓性白血病（CML）内显著差异的可变剪接事件以及剪接异构体。方法基于CML（K562）和AML（THP1、HL－60）细胞系的全转录组测序数据,在全基因组范围内采用TopHat、MATS和Cufflinks计算识别可变剪接事件、重构剪接异构体和分析其差异表达。结果在AML和CML细胞间识别的130个基因在CML细胞系中特异性高表达,80个基因在AML两株细胞系内均表达上调。在CML与AML之间识别了337个差异表达的剪接异构体以及35个差异可变剪接事件。结论 AML与CML细胞系间存在显著差异的可变剪接事件和剪接异构体。白血病相关的可变剪接事件可作为其潜在的标志物或药物靶标。     

Neutralising effects of small molecule toxin inhibitors on nanofractionated coagulopathic Crotalinae snake venoms

Repurposing small molecule drugs and drug candidates is considered as a promising approach to revolutionise the treatment of snakebite envenoming. In this study, we investigated the inhibiting effects of the small molecules varespladib (nonspecific phospholipase A₂ inhibitor), marimastat (broad spectrum matrix metalloprotease inhibitor) and dimercaprol (metal ion chelator) against coagulopathic toxins found in Crotalinae (pit vipers) snake venoms. Venoms from Bothrops asper, Bothrops jararaca, Calloselasma rhodostoma and Deinagkistrodon acutus were separated by liquid chromatography, followed by nanofractionation and mass spectrometry identification undertaken in parallel. Nanofractions of the venom toxins were then subjected to a high-throughput coagulation assay in the presence of different concentrations of the small molecules under study. Anticoagulant venom toxins were mostly identified as phospholipases A₂, while procoagulant venom activities were mainly associated with snake venom metalloproteinases and snake venom serine proteases. Varespladib was found to effectively inhibit most anticoagulant venom effects, and also showed some inhibition against procoagulant toxins. Contrastingly, marimastat and dimercaprol were both effective inhibitors of procoagulant venom activities but showed little inhibitory capability against anticoagulant toxins. The information obtained from this study aids our understanding of the mechanisms of action of toxin inhibitor drug candidates, and highlights their potential as future snakebite treatments.