高通量筛选瞬时受体势V3通道调节剂细胞模型的建立

为发现TRPV3调节剂,通过荧光成像分析系统检测钙浓度,建立高通量筛选瞬时受体势V3通道(Transient receptor potential V3,TRPV3)调节剂的细胞模型.将TRPV3表达载体转染人胚肾(HEK-293)细胞,抗生素筛选稳定表达TRPV3的细胞系,选取TRPV3特异性调节剂作用于细胞模型,应用荧光成像分析系统测钙实验检测TRPV3高表达细胞系药理学特征,同时优化实验条件,考察模型的稳定性,并评估应用于96孔板及384孔板进行高通量筛选的可靠性及准确性.获得了高表达TRPV3的HEK-293稳定细胞系,通过TRPV3离子通道调节的钙流信号与TRPV3特异性调节剂成剂量依赖关系,优化得到了最适筛选条件,该模型稳定,灵敏,通过Z因子及Spiking检测,完全符合高通量筛选需求.利用此细胞模型通过检测钙信号可筛选TRPV3调节剂.     

组蛋白去乙酰化酶6(HDAC6)抑制剂分子水平高通量筛选模型的建立

旨在建立分子水平HDAC6小分子抑制剂的高通量筛选模型,用于新型HDAC6特异性小分子抑制剂的发现。建立HDAC6的昆虫表达系统,分离纯化HDAC6蛋白,利用底物Boc-Lys(Ac)-AMC对纯化的HDAC6进行测活,并对测活体系进行优化,以SAHA为阳性抑制剂,确定适合高通量筛选的酶及底物浓度,反应时间等。首先构建HDAC6昆虫真核细胞表达载体,转入昆虫细胞中表达,并利用GST亲和柱纯化获得较高纯度的GST-HDAC6融合蛋白;建立体外HDAC6分子测活方法,表明昆虫表达的GST-HDAC6融合蛋白具有去乙酰化酶活性,并通过对多种参数优化使得Z’因子达到0.60,表明分子水平的HDAC6小分子抑制剂高通量筛选体系成功建立。     

异源表达多巴脱羧酶促进大肠杆菌从头合成多巴胺

多巴胺是多种天然抗氧化药物生物合成的前体物质,在人体内作为神经递质调控中枢神经系统的多种生理功能,常用于多种类型休克的临床治疗.目前,通过微生物合成技术已经实现了多巴胺的从头合成,但是合成效率很低.针对该问题,在左旋多巴(L-DOPA)大肠杆菌工程菌基础上,利用不同拷贝数质粒表达野猪Sus scrofa来源的多巴脱羧酶...     

鸟氨酸脱羧酶高活性微生物菌株的筛选

根据鸟氨酸脱羧酶(ODC)催化底物L-鸟氨酸脱羧生成腐胺,从而引起培养基中pH升高的特点,设计了一种高效、经济的筛选方法,并以此作为初筛手段从土壤中快速分离可产生较高ODC活力微生物菌种。研究发现培养后pH的变化与微生物产酶能力存在着明显的正相关性,R2=0.914 2。对从土壤和活性污泥样品中分离得到的343株细菌进行有效初筛以及经过摇瓶发酵测定酶活的复筛试验后,筛选得到6株高酶活的菌株,其中菌株CJW07和CGW27的ODC活性分别达到了121.32 U/mL和109.25 U/mL。     

蛋白酪氨酸磷酸酶1B抑制剂高通量筛选模型建立和应用

本文以蛋白酪氨酸磷酸酶1B（protein tyrosine phosphatase,PTP1B）作为靶点,采用分子克隆GST融合蛋白的方法,重组表达获得PTP1B,结合自动化操作技术和比色分析,建立了一种PTP1B抑制剂的高通量筛选模型.经在96孔板优化各种反应条件并对17940个样品的筛选结果表明,靶向PTP1B建立的高通量筛选模型具有微量、快速、特异、灵敏等特点,平均日筛选量可达15000样次以上,为寻找新的抗糖尿病药物和先导化合物提供了一种先进的手段.     

PPARδ激动剂高通量筛选模型的建立

为建立基于细胞瞬时转染的过氧化物酶体增殖物激活受体δ(peroxisome proliferator-activated receptor delta PPARδ)激动剂高通量筛选模型,用RT-PCR技术从肝的总RNA中扩增PPARδ基因序列,将其连至T克隆载体进行测序。将序列正确的PPARδ片段连接至pTARGET载体上构建表达载体pTARGET-ppARδ;将合成的3个拷贝的PPRE(peroxisome proliferator receptorresponse element)插入pGL3-promoter构成报告质粒pGl3-PPRE×3-luc。用脂质体转染技术将表达载体与报告质粒共转染细胞系,通过检测荧光素酶基因的表达状况评价化合物对PPARδ的激动活性。通过多种条件的优化,得到了最佳的共转染条件。阳性药苯扎贝特明显提高荧光素酶的表达,最大上调倍增数可达10倍,并且在一定浓度下阳性药与相对荧光酶的活性表达有较好的量效关系。该筛选模型灵敏、稳定,为对PPARδ激动剂进行药物研发和PPARδ机理的研究打下基础。     

日本脑炎病毒NS3蛋白酶活性检测及抑制剂高通量筛选方法的建立

日本脑炎病毒(Japanese encephalitis virus,JEV)是单股正链RNA病毒,全基因组仅含有一个开放阅读框,编码一条多聚蛋白前体,病毒编码的NS3蛋白酶在JEV多聚蛋白加工过程中起着重要作用,是重要的药物靶标。通过PCR扩增了NS2BH-NS3蛋白酶的编码区,构建了原核表达质粒并转化到大肠杆菌BL21(DE3),经IPTG诱导得到可溶性的NS3蛋白酶,用镍亲和层析方法进行了纯化。建立了基于荧光共振能量转移的NS3蛋白酶活性检测方法,并确定了最佳的反应条件,对113个化合物进行了筛选,发现其中两个化合物对JEV NS3蛋白酶具有一定的抑制活性。本研究为JEV NS3蛋白酶的活性研究及抑制剂筛选提供了一种操作方便、成本低廉的方法。     

适用于高通量筛选的脂肪酶表达系统

突变库容量和高通量筛选方法是影响酶分子定向进化的两个决定因素,虽然巴斯德毕赤酵母pPIC9K表达系统已被广泛使用[1],但由于外源基因可通过单插入整合入基因组,产生多拷贝突变基因,从而干扰后续重组子的筛选;另一方面,pPIC9K表达系统需要甲醇诱导,需要每日补加甲醇来诱     

抗甲病毒化合物高通量筛选模型的建立与应用

建立抗甲病毒化合物高通量筛选模型可为筛选和分析抗甲病毒药靶提供技术基础。本研究以含有分泌型荧光素酶(Gaussialuciferase,GLUC)报告基因标记的重组辛德毕斯病毒XJ160-GLUC为分子基础,建立了抗甲病毒化合物的高通量筛选模型,并对感染复数、检测时间等参数进行优化。经过优化,筛选模型的Z’因子值可达到0.71,表明我们所建立的筛选模型具有较好的稳定性。应用该筛选模型对8080个五合一样品进行筛选,最终获得19个具有抗甲病毒活性的阳性化合物。本研究所建立的抗甲病毒化合物高通量筛选模型及抗甲病毒阳性化合物的发现为抗甲病毒靶标相关的研究提供了新的思路和信息。     

异色瓢虫多巴脱羧酶基因序列及低温诱导表达分析

多巴脱羧酶(dopa decarboxylase,DDC)是把多巴降解成多巴胺的重要酶类,在昆虫行为和发育中具有重要作用。本研究在转录组获得异色瓢虫Harmonia axyridis DDC基因序列的基础上,从ORF两端设计特异性引物进行扩增并测序验证,获得了DDC基因的c DNA的ORF全长序列,其包含1431 bp,编码476个氨基酸,软件分析预测显示该基因编码蛋白的分子量为53.88 k Da,理论等电点为5.80。本实验分为升温、降温、低温储存以及不同发育阶段(包括预蛹,1-3 d蛹,1-3 d 4龄幼虫以及羽化1-3 d成虫)4个处理组,采用荧光定量PCR技术研究异色瓢虫不同处理组DDC基因的表达水平。结果表明:DDC基因在蛹期第1天的表达量最高,在升温诱导条件下表达无差异,降温诱导下表达量上升;黄色雌成虫低温储存条件下基因表达量先显著上升后显著下降,黑色雌成虫表达量无差异,表明DDC基因可以在低温胁迫下高表达促使异色瓢虫适应环境变化。     

基于受体的药物高通量筛选研究进展

受体是药物筛选的重要靶标,基于受体的药物高通量筛选是药物筛选的主要类型之一。本文根据受体作用原理,按照检测对象的不同,从直接与间接检测的角度,将基于受体的药物高通量筛选进行了分类,总结了基于受体的几种不同的药物筛选模型,并简要介绍了高通量筛选技术在中药研究中的应用,对药物筛选的发展进行了展望。     

Expression, purification, and characterization of human malonyl-CoA decarboxylase

The recombinant human malonyl-CoA decarboxylase (hMCD) was overexpressed in Escherichia coli with and without the first 39 N-terminal amino acids via a cleavable MBP-fusion construct. Proteolytic digestion using genenase I to remove the MBP-fusion tag was optimized for both the full length and truncated hMCD. The apo-hMCD enzymes were solubilized and purified to homogeneity. Steady-state kinetic characterization showed similar kinetic parameters for the MBP-fused and apo-hMCD enzymes with an apparent Km value of approximately 330-520 microM and a turnover rate kcat of 13-28s(-1). For the apo-hMCD enzymes, the N-terminal truncated hMCD was well tolerated over a broad pH range (pH 4-10); whereas the full-length hMCD appeared to be stable only at pH >/= 8.5. Our results showed that the N-terminal region of hMCD has no effect on the catalytic activity of the enzyme but plays a role in the folding process and conformation stability of hMCD.     

The establishment of mouse embryonic stem cell cultures on 96-well plates for high-throughput screening

Embryonic stem (ES) cells can be valuable for monitoring differentiation processes and for improving applications in basic developmental biology. The application of ES cells can be a useful tool for drug discovery and toxicology. Therefore, we suggest the high-throughput screening (HTS) system based on ES cells in this study. Firstly, we optimized the feeder-free condition and seeding cell number which can maintained for at least 7 days without over-confluency. We analyzed the system by cell viability, proliferation activity, RT-PCR and morphologic/immunohistochemical evaluations. The optimal cell seeding number was 30/well that was maintained the typical colonial morphology over 9 d with 1,000 U/ml LIF in the limited space. The cell in optimized condition expressed ALP, SSEA-1, Oct 4 and Nanog and the genetic expressions showed similar to protein expressions. The cell lineage marker expressions showed faint or none. The cell viability and proliferation activity were increased in time-dependent manner in our optimized HTS system. In conclusion, the novel HTS system using ES cells can by useful for developing models for drug discovery as well as toxicological screening in the near future.     

新型冠状病毒主蛋白酶抑制剂的筛选方法研究进展

新型冠状病毒肺炎(coronavirus disease 2019,COVID-19)席卷全球,具有较高的传染性和死亡率,但目前尚缺乏安全有效的COVID-19疫苗与治疗药物.新型冠状病毒主蛋白酶(main protease,Mpro)的进化高度保守,在调控新冠病毒RNA复制中具有重要的生物学功能,已成为新型广谱抗冠状...     

In silico high-throughput virtual screening and molecular dynamics simulation study to identify inhibitor for AdeABC efflux pump of Acinetobacter baumannii

Emergence of multi-drug resistant strains of Acinetobacter baumannii has caused significant health problems and is responsible for high morbidity and mortality. Overexpression of AdeABC efflux system is one of the major mechanisms. In this study, we have focused on overcoming the drug resistance by identifying inhibitors that can effectively bind and inhibit integral membrane protein, AdeB of this efflux pump. We performed homology modeling to generate structure of AdeB using MODELLER v9.16 followed by model refinement using 3D-Refine tool and validated using PSVS, ProsaWeb, ERRAT, etc. The energy minimization of modeled protein was done using Protein preparation wizard application included in Schrodinger suite. High-throughput virtual screening of 159,868 medicinal compounds against AdeB was performed using three sequential docking modes (i.e. HTVS, SP and XP). Furthermore, absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis was done using QIKPROP. The selected 123 compounds were further analyzed for binding free energy by molecular mechanics (using prime MM-GBSA). We have also performed enrichment study (ROC curve analysis) to validate our docking results. The selected molecule and its interaction with AdeB were validated by molecular dynamics simulation (MDS) using GROMACS v5.1.4. In silico high-throughput virtual screening and MDS validation identified ZINC01155930 ((4R)-3-(cycloheptoxycarbonyl)-4-(4-etochromen-3-yl)-2-methyl-4,6,7,8-tetrahydroquinolin-5-olate) as a possible inhibitor for AdeB. Hence, it might be a suitable efflux pump inhibitor worthy of further investigation in order to be used for controlling infections caused by Acinetobacter baumannii.     

Discovery of LRRK2 inhibitors by using an ensemble of virtual screening methods

In this paper, we present the results of a ligand- and structure-based virtual screen targeting LRRK2, a kinase that has been implicated in Parkinson’s disease. For the ligand-based virtual screen, the structures of 12 competitor compounds were used as queries for a variety of 2D and 3D searches. The structure-based virtual screen relied on homology models of LRRK2, as no X-ray structure is currently available in the public domain. From the virtual screening, 662 compounds were purchased, of which 35 showed IC₅₀ values below 10 μM in wild-type and/or mutant LRRK2 (a hit rate of 5.3%). Of these 35 hits, four were deemed to have potential for medicinal chemistry follow-up.     

A new homogeneous high-throughput screening assay for profiling compound activity on the human ether-a-go-go-related gene channel

Long QT syndrome, either inherited or acquired from drug treatments, can result in ventricular arrhythmia (torsade de pointes) and sudden death. Human ether-a-go-go-related gene (hERG) channel inhibition by drugs is now recognized as a common reason for the acquired form of long QT syndrome. It has been reported that more than 100 known drugs inhibit the activity of the hERG channel. Since 1997, several drugs have been withdrawn from the market due to the long QT syndrome caused by hERG inhibition. Food and Drug Administration regulations now require safety data on hERG channels for investigative new drug (IND) applications. The assessment of compound activity on the hERG channel has now become an important part of the safety evaluation in the process of drug discovery. During the past decade, several in vitro assay methods have been developed and significant resources have been used to characterize hERG channel activities. However, evaluation of compound activities on hERG have not been performed for large compound collections due to technical difficulty, lack of throughput, and/or lack of biological relevance to function. Here we report a modified form of the FluxOR thallium flux assay, capable of measuring hERG activity in a homogeneous 1536-well plate format. To validate the assay, we screened a 7-point dilution series of the LOPAC 1280 library collection and reported rank order potencies of ten common hERG inhibitors. A correlation was also observed for the hERG channel activities of 10 known hERG inhibitors determined in this thallium flux assay and in the patch clamp experiment. Our findings indicate that this thallium flux assay can be used as an alternative method to profile large-volume compound libraries for compound activity on the hERG channel.     

Comparative expression and purification of human glutamic acid decarboxylase from Saccharomyces cerevisiae and Pichia pastoris

The yeast cell factory is a potentially useful source of proteins in general. They include glutamic acid decarboxylase (GAD), which is one of the major autoantigens for Type 1 diabetes. We have created a hybrid form of GAD consisting of amino acids 1–101 of the human GAD67 protein fused to amino acids 96–585 of the human GAD65 protein, and have modified this to include a C-terminal hexa-Histidine (H6) tag sequence. This hybrid GAD67/65-H6 was expressed in two yeast hosts: constitutively under the control of the plasmid phosphoglycerate kinase promoter (PGK1) in Saccharomyces cerevisiae, and inducibly under the control of the chromosomal alcohol oxidase promoter (AOX1) in Pichia pastoris. Enzymatically active hybrid GAD was prepared from yeast lysates by purification either on an affinity column based on the GAD-1 monoclonal antibody, or by metal-affinity chromatography. The purified GAD67/65-H6 was radiolabelled with iodine-125 and tested with Type 1 diabetes sera in a radioimmunoprecipitation assay, and results were compared with those using untagged GAD67/65 and those using porcine brain GAD. The results of enzymatic and immunological assays show hybrid GAD67/65 is isolated at high specific activity and moderate yield, and the addition of the H6 tag sequences or the choice of yeast strain did not appreciably affect enzyme activity, percentage recovery of GAD, protein purification, or the utility in diagnosis of diabetes in terms of specificity and sensitivity to the various sera.     

Development of a high-throughput screening assay for cytoprotective agents in rotenone-induced cell death

Parkinson’s disease (PD) is a neurodegenerative disease featured by selective loss of substantia nigra neurons. Rotenone administration in animals induces neurodegeneration accompanied by α-synuclein-positive Lewy body-like inclusions, recapturing typical histopathological features of PD. In an effort to screen for small-molecule agents to reverse rotenone-induced cytotoxicity, we developed and validated a sensitive and robust assay with neuroblastoma SK-N-SH cells. This assay was amenable to a high-throughput screening format with Z′ factor of 0.56. Robotic screening of a bioactive compound library led to the identification of carnosic acid that can effectively protect cells from rotenone treatment. Using a high-content image-based assay and Western blot analysis, we demonstrated that carnosic acid protects cells from rotenone stress by significant induction of HSP70 expression. Therefore, the assay reported here can be used to identify novel cytoprotective agents for clinical therapeutics of PD.     

Discovery of novel indolinone-based,potent, selective and brain penetrant inhibitors of LRRK2

Mutations in leucine-rich repeat kinase-2 (LRRK2) are the most common genetic cause of Parkinson’s disease (PD). The most frequent kinase-enhancing mutation is the G2019S residing in the kinase activation domain. This opens up a promising therapeutic avenue for drug discovery targeting the kinase activity of LRRK2 in PD. Several LRRK2 inhibitors have been reported to date. Here, we report a selective, brain penetrant LRRK2 inhibitor and demonstrate by a competition pulldown assay in vivo target engagement in mice.