仓鼠糜酶2抑制剂高通量筛选模型的建立及其应用

本研究拟建立重组仓鼠糜酶2 (chymase 2) 抑制剂的体外高通量筛选模型, 寻找新型的糜酶2抑制 剂。首先, 利用大肠杆菌表达、制备成熟的重组仓鼠糜酶2蛋白, 以384孔微板为媒介, 建立基于荧光方法测定酶活性的抑制剂筛选模型, 结果表明所建立的糜酶2抑制剂高通量筛选模型具有灵敏、稳定、重复性好的特点 (Z' = 0.84)。利用建立的模型对40 080种样品 (包括28 060种化合物和12 020种天然产物提取物) 进行抑制活性筛选, 取抑制率大于90%的613种样品进行复筛。最终确定化合物J16647和J16648具有较强的抑制活性, 其IC₅₀值分别为0.823和0.690 μmol·L⁻¹。     

聚腺苷二磷酸核糖聚合酶-1抑制剂高通量筛选模型

目的建立体外聚腺苷二磷酸核糖聚合酶-1[Poly(ADP-ribose)polymerase-1,PARP-1]抑制剂的高通量筛选模型,筛选潜在的PARP-1抑制剂。方法将PARP-1、裸DNA与底物NAD+反应,再将剩余底物NAD+转化为荧光物质,通过测定其荧光强度来决定PARP-1的活性,并以此筛选PARP-1的抑制剂。建立384孔板的高通量筛选模型,对9280个化合物(包括合成化合物、天然产物、微生物发酵提取物)组成的随机库进行体外筛选。结果筛选出148个活性化合物对PARP-1的抑制作用大于70%,最终确定3个化合物具有较高的抑制活性。结论建立的PARP-1抑制剂高通量筛选模型具有灵敏度高、快速、微量、准确的特点。     

P-gp蛋白功能抑制剂高通量筛选模型的建立与应用

目的建立高通量筛选模型,寻找疗效好、毒副作用小的肿瘤多药耐药逆转剂。方法以罗丹明123荧光染料胞内积累实验为基础,建立基于细胞水平的高通量筛选模型,对480个中药样品进行了高通量筛选,并采用剂量—活性依赖实验和抗性增敏实验验证阳性样品。结果模型Z′因子为0.72,筛选得到2个中药样品。结论建立的P-gp蛋白功能抑制剂高通量筛选模型稳定性好,灵敏度高,筛选得到的2种中药样品具有较好的P-gp蛋白功能抑制活性。     

糖原磷酸化酶抑制剂高通量筛选模型的建立*

目的:建立糖原磷酸化酶(GPa)抑制剂的体外高通量筛选模型。方法:用梯度离心方法提取大鼠肝脏GPa,以葡萄糖-1-磷酸作为底物,通过测定反应中磷酸根的释放量,间接反映肝脏GPa的活性。通过对反应体系的优化,调整反应条件,建立96孔微孔板的高通量筛选模型,用咖啡因对此模型进行验证,并评价高通量技术参数[Z′-因子(Z-′factor),信号本底比(signal to background,S/B),信噪比(signal to noise,S/N)]。用此模型对5 000个样品(包括合成化合物、天然提取物)组成的随机库进行体外筛选,考察这些样品对GPa的抑制作用。结果:确定反应体系条件是:反应温度25℃,反应时间30 m in,底物浓度0.5 mmol.L-1,大鼠肝脏GPa的用量为250 ng。用此条件测定咖啡因对GPa的抑制曲线,计算其半数抑制浓度(IC50)为(285.3±39.6)μmol.L-1,与文献报道(IC50=240μmol.L-1)基本一致;本模型技术参数Z′-因子=0.79,S/B=0.45,S/N=11.32,表明此模型可以用于高通量筛选。应用此模型筛选得到有活性的化合物9个,其IC50在3.56~45.75μmol.L-1。结论:建立的体外GPa抑制剂高通量筛选模型具有快速、微量、准确的特点,可以作为研究降糖药的工具。     

一氧化氮合酶抑制剂和增强剂的高通量筛选

目的建立一氧化氮合酶(NOS)活性的高通量检测方法，筛选调节NOS活性的药物。方法通过NADPH荧光值的变化，间接反映NOS活性。通过对反应体系的优化，调整各反应底物(NADPH，L-Arg，NOS)及抑制剂(L-NNA)的浓度，建立高通量筛选模型并对5 600个样品进行筛选。结果方法简便、容易操作、灵敏度高、结果稳定，发现了一批对NOS具有抑制或增强作用的化合物。结论此法适用于高通量药物筛选。     

蛋白酪氨酸磷酸酶1B抑制剂的分子对接及三维定量构效关系研究

目的对文献报道的一系列芳环取代噻唑类蛋白酪氨酸磷酸酶1B(PTP1B)抑制剂进行分子对接及三维定量构效关系(3D-QSAR)研究。方法应用Surflex-Dock进行分子对接结合模式研究,并用比较分子力场分析(CoMFA)和比较分子相似性指数分析(CoMSIA)方法进行三维定量构效关系研究,建立具有良好预测能力的3D-QSAR模型。结果对接结果表明,该类结构可以很好地占据PTP1B的3个关键结合位点,大大提高了抑制剂与酶的亲和力。所建立的CoMFA模型交叉验证系数q~2为0.644,CoMSIA模型交叉验证系数q~2为0.719。结论获得的CoMFA和CoMSIA模型具有可靠的预测能力,可应用于指导该类化合物的设计。     

新型冠状病毒奥密克戎变异株主蛋白酶的制备及其抑制剂的高通量筛选

目的 基于荧光共振能量转移(fluorescence resonance energy transfer,FRET)原理,建立奥密克戎变异株主蛋白酶(Omicron variant main protease,OM-Mpro)抑制剂高通量筛选模型,筛选新型OM-Mpro抑制剂。方法 利用大肠杆菌进行OM-Mpro原核表达,再以HisTrap^TM亲和层析柱进行分离纯化。以FRET法进行OM-Mpro与野生型新型冠状病毒主蛋白酶(wild-type main protease,WT-Mpro)的酶活性测定并评价奈玛特韦的酶抑制活性。利用OM-Mpro抑制剂FRET高通量筛选模型对上市药物库进行高通量筛选。结果 利用大肠杆菌成功进行了OM-Mpro原核表达与分离纯化,其与WTMpro酶活性无显著差异。奈玛特韦对OM-Mpro和WT-Mpro具有等同的酶抑制活性,说明奈玛特韦对奥密克戎变异株依然有效。通过对上市药物库进行高通量筛选,发现西吡氯铵是混合型OM-Mpro抑制剂,其半数抑制浓度值为8.76μmol·L^-1。结论 成功制备了高活性OM-Mpr...     

BACE1抑制剂分子水平高通量筛选体系的建立

目的建立体外分子水平β-分泌酶（BACE1）抑制剂高通量筛选体系。方法采用均相时间分辨荧光法（HTRF）,通过优化反应时间、酶浓度、检测仪器等实验条件,建立BACE1抑制剂高通量筛选体系,根据待测样品对BACE1活性抑制程度筛选其抑制剂。结果采用HTRF法建立了BACE1抑制剂高通量筛选体系,其中反应时间确定为6h、BACE1浓度为670U/L,采用VICTOR3酶标仪测得信噪比为649.6,信背比为44.6,Z′因子为0.91,变异系数小于10%,并筛选到一系列IC50小于10-6mol/L的化合物。结论采用HTRF法建立的BACE1抑制剂筛选体系灵敏度高、特异性和稳定性好,可用于BACE1抑制剂的高通量筛选。     

以FK506结合蛋白52为靶点的高通量药物筛选模型的建立及应用#br#

目的 发现新的FKBP52抑制剂类药物,建立以人FKBP52为靶点的高通量药物筛选模型。方法 通过大肠埃希菌表达系统重组表达FKBP52蛋白,并进行纯化。基于FKBP52的肽基-脯氨酰顺反异构酶(peptidyl-prolyl cis-trans isomerase, PPIase)的活性特性,建立FKBP52高通量药物筛选体系。对本公司化合物库中的31558个放线菌及真菌次级代谢产物粗提物进行筛选。结果 成功构建重组表达菌株BL21(rosetta)/pET-30a/FKBP52。纯化后的重组表达人FKBP52蛋白纯度大于90%,并具有很好的PPIase活性。阳性药FK506能剂量依赖地抑制FKBP52活性,IC50为12.035ng/mL。筛选得到121个抑制率大于80%的样品,其中11株放线菌的次级代谢产物中含FK506,6株放线菌的次级代谢产物中含FK520,7株放线菌的次级代谢产物中含雷帕霉素。对随机抽取的100块96孔板筛选数据进行统计,模型的信号/本底比平均值为2.35±0.26,Z'因子平均值为0.78±0.1。结论 本研究成功建立了人FKBP52抑制剂高通量药物筛选模型,该方法具有快捷、灵敏度高、稳定性好的特点,不仅可以快速进行新FKBP52抑制剂的筛选,还可以实现对FKBP52已知抑制剂如FK506、FK520和雷帕霉素等产生菌的定向筛选。     

促神经细胞分化小分子活性物质的筛选研究

目的　建立便捷有效的高通量筛选方法用以NGF样促神经细胞分化小分子活性物质的初级筛选。方法　采用经NGF诱导的PC12细胞,以NGF为阳性对照,通过对分化细胞百分率的测定和存活细胞的MTT染色,建立模型并对样品进行筛选。结果　所建模型定量反映NGF生物活性;应用该模型对1 600多种化合物进行筛选,发现了多个不同活性类型的样品。其中代号为B的化合物作用持续时间长,量效关系明显,且较少引起细胞死亡。结论　该工作为深入筛选促进中枢神经再生药物和神经细胞肿瘤分化诱导剂提供了模型和样品基础。     

Molecular docking and high-throughput screening for novel inhibitors of protein tyrosine phosphatase-1B

High-throughput screening (HTS) of compound libraries is used to discover novel leads for drug development. When a structure is available for the target, computer-based screening using molecular docking may also be considered. The two techniques have rarely been used together on the same target. The opportunity to do so presented itself in a project to discover novel inhibitors for the enzyme protein tyrosine phosphatase-1B (PTP1B), a tyrosine phosphatase that has been implicated as a key target for type II diabetes. A corporate library of approximately 400 000 compounds was screened using high-throughput experimental techniques for compounds that inhibited PTP1B. Concurrently, molecular docking was used to screen approximately 235 000 commercially available compounds against the X-ray crystallographic structure of PTP1B, and 365 high-scoring molecules were tested as inhibitors of the enzyme. Of approximately 400 000 molecules tested in the high-throughput experimental assay, 85 (0.021%) inhibited the enzyme with IC50 values less than 100 microM; the most active had an IC50 value of 4.2 microM. Of the 365 molecules suggested by molecular docking, 127 (34.8%) inhibited PTP1B with IC50 values less than 100 microM; the most active of these had an IC50 of 1.7 microM. Structure-based docking therefore enriched the hit rate by 1700-fold over random screening. The hits from both the high-throughput and docking screens were dissimilar from phosphotyrosine, the canonical substrate group for PTP1B; the two hit lists were also very different from each other. Surprisingly, the docking hits were judged to be more druglike than the HTS hits. The diversity of both hit lists and their dissimilarity from each other suggest that docking and HTS may be complementary techniques for lead discovery.     

Discovery of a novel competitive inhibitor of PTP1B by high-throughput screening

AIM: The aim of the present study was to discover novel protein tyrosine phosphatase 1B (PTP1B) inhibitors. We expressed and purified the human PTP1B catalytic domain and set up a molecular level high-throughput screening (HTS) assay to screen a set of 48,000 pure compounds. RESULTS: HTS was finished with an averaged Zo factor of 0.63, and LGH00081, a competitive inhibitor of PTP1B with novel structure and relatively good selectivity for receptor-type protein tyrosine phosphatases, was identified. CONCLUSION: We established a molecular level assay which is useful for the screening of PTP1B inhibitors with therapeutic potential. The novel competitive PTP1B inhibitor LGH00081 offers a good start for structure modification and cellular functional activity study.     

High-throughput screening of human soluble epoxide hydrolase inhibitors

To screen potential human soluble epoxide hydrolase (hsEH) inhibitors, a high-throughput screening model in 384-well microplate with total volume of 50 microL was established. Recombinant hsEH was cloned and expressed in E. coli. and its specific substrate PHOME was synthesized. The HTS model was based on fluorescence analysis with enhanced sensitivity and specificity (Z' = 0.65). A total of 47 360 samples (including 25 040 compounds and 22 320 natural products) were screened, of which 950 samples with inhibition greater than 80% were selected for further rescreening. Finally, two compounds with high inhibitory activity were identified, whose IC50 value were 8.56 and 4.31 micromol x L(-1), separately. The results indicated that the method was stable, sensitive, reproducible and also suitable for high-throughput screening.     

A high-throughput model for screening anti-tumor agents capable of promoting polymerization of tubulin in vitro

INTRODUCTION Discovery compounds that exhibit specificfeatures, such as binding, inhibiting or catalyticproperties, is a central task in drug development andmedical applications. The advances in genomics,bioinformatics, combinatorial chemistry, cell-basedassays, and high-throughput screening (HTS) have ledthe Pacific yew tree Taxus brevifolia[3], has undergone Co.extensive clinical development as a result of its effi- Preparation of tubulin The method[12,13…     

基于深孔板培养高通量筛选rakicidin B1高产菌的研究

目的 获得rakicidin B1的高产突变菌株。方法 建立深孔板发酵rakicidin B1的方法,结合常压室温等离子体技术(ARTP)及核糖体工程对rakicidin B1的产生菌进行高通量诱变选育。结果 获得一株高产突变菌株R36-27,其摇瓶发酵产量达到490mg/L左右,较出发菌株提高了237.9%;经遗传稳定性考察验证了该菌株稳定性较好。结论 采用ARTP为诱变源,以庆大霉素抗性为选择压力,结合深孔板高通量筛选,可以快速有效地获得高产菌株。     

Identification of ZINC02765569: a potent inhibitor of PTP1B by vHTS

The present study describes identification of a novel lead molecule ZINC02765569 for inhibition of protein tyrosine phosphatase 1B (PTP1B) enzyme by a high-throughput virtual screening of Zinc database against catalytic domain of PTP1B employing docking algorithm Glide. The identified hit molecule ZINC02765569 was synthesized and evaluated for in vitro PTP1B enzyme inhibition, in vitro cellular glucose uptake assay, and animal models of hyperglycemia. ZINC02765569 shows promising inhibition of PTP1B enzyme at 10 μm assay, positively up-regulate the cellular glucose uptake in skeletal cell muscle myotubes and SLM/STZ hyperglycemic animal experiments. The novel hit reported here should provide a platform for the further development of its analogs as potential PTP1B enzyme inhibitors.     

High-throughput fluorescence polarization method for identifying ligands of LOX-1

AIM: To develop and optimize a competitive fluorescence polarization (FP) method, and use it as a high-throughput screening (HTS) assay for drug discovery. METHODS: Human lectin-like oxidized low-density lipoprotein receptor-1 (hLOX-1) and oxidized low-density lipoprotein (oxLDL) were used to establish a high-throughput fluorescence polarization assay to screen ligands of human LOX-1. A 96-well plate assay was performed with a fast plate reader. Three fluorescein isothiocyanate-labeled hLOX-1 concentrations (100, 200, and 400 nmol/L) were selected to be titrated by oxLDL (from 0.05 nmol/L to 100 micromol/L) in order to obtain optimal reactive concentrations. The concentration of Me2SO used (0%, 1%, 3%, 5%) and incubation time (15 min, 30 min, 1 h, 2 h) were optimized. The Z' factor was calculated to estimate the quality of FP-based HTS. RESULTS: Concentrations of 200 nmol/L for human LOX-1 and 50 micromol/L for oxLDL were used in the actual assay. Concentrations of 0% to 5% Me2SO and different reaction times did not affect the FP-based HTS. The Z' value was 0.66. By using this detection and screening system, 12 700 compounds were screened and 3 ligands with an IC50 of less than 4.5 micromol/L were found. CONCLUSION: The established competitive FP-based assay is sensitive, stable, highly reproducible and robust, and suitable for HTS for ligands of the hLOX-1 receptor.     

HTS Technologies--IBC Informa Conference

In 1997, approximately US $6 billion were spent on worldwide screening technologies. The estimate for the total global market for high-throughput screening (HTS) products and services was about US $1.5 billion (source: Medical and Healthcare Marketplace Guide, 15th Edition, 1999-2000). Genomics and combinatorial chemistry demand a technological shift away from present-day laboratory to fully automated HTS and ultra HTS (uHTS), with over 100,000 compounds screened per day. The potential of new technology in miniaturization and automation will ensure faster and cheaper solutions to discovery. The conference explored the rapidly advancing field of HTS and examined numerous areas of HTS application. Identification and prioritization of viable targets, and the development of 'information rich' screens without reducing screening capacity, are important topics. Furthermore, the essential integration of all these screening, analytical and parallel synthesis systems in one coherent process is considered to be of significant interest. Focused sessions addressed the rapidly advancing areas of ADME (absorption, distribution, metabolism, elimination), toxicity and formulation screening, including virtual 'in silico' screening.     

A robust homogeneous binding assay for α4β2 nicotinic acetylcholine receptor

AIM: To develop a homogeneous high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel alpha4beta2 nicotinic acetylcholine receptor (nAChR) modulators. METHODS: Membrane preparation of HEK293 cells expressing alpha4beta2 nAChR, [(3)H]cytisine and wheat germ agglutinin (WGA)-coupled microbeads were used to develop an HTS assay based on SPA technology. This method was validated against a conventional filter binding approach and applied to large-scale screening of a library containing 32 000 synthetic compounds. Intracellular calcium measurement was carried out to verify the bioactivities of the hits found by the SPA assay. RESULTS: IC(50) values of 2 reference compounds (epibatidine and RJR 2403) determined by SPA and filter binding methods were comparable and consistent with those reported elsewhere. A total of 54 compounds, showing more than 60% competitive inhibition on [(3)H]cytisine binding to alpha4beta2 nAChR, were identified initially following an HTS campaign. Secondary screening confirmed that 17 compounds with novel chemical structures possessed relatively high binding affinity to alpha4beta2 nAChR (K(i)<2 micromol/L). Eight compounds displayed antagonistic effects with >50% inhibition on ABT-594-induced calcium mobilization while none showed any agonist activity. CONCLUSIONS: This homogeneous binding assay is a highly efficient, amenable to automation and robust tool to screen potential alpha4beta2 nAChR modulators in an HTS setting. Its application may be expanded to other membrane receptors and ion channels.