荧光检测技术在蛋白酪氨酸激酶抑制剂高通量筛选中的应用

介绍荧光共振能量转移技术、荧光偏振免疫分析技术、荧光素酶-激酶检测技术和毛细管电泳-激光诱导荧光技术在酪氨酸激酶抑制剂高通量筛选模型中的应用情况。蛋白酪氨酸激酶与多种疾病的发生和发展密切相关,通过高通量筛选寻找其抑制剂已成为新药研发的热点之一。荧光检测技术因具有灵敏、易于检测、适于微量化和安全性高等优势,在药物的高通量筛选中被广泛使用。     

Detection of IgG Aggregation by a High Throughput Method Based on Extrinsic Fluorescence

The utility of extrinsic fluorescence as a tool for high throughput detection of monoclonal antibody aggregates was explored. Several IgG molecules were thermally stressed and the high molecular weight species were fractionated using size-exclusion chromatography (SEC). The isolated aggregates and monomers were studied by following the fluorescence of an extrinsic probe, SYPRO Orange. The dye displayed high sensitivity to structurally altered, aggregated IgG structures compared to the native form, which resulted in very low fluorescence in the presence of the dye. An example of the application is presented here to demonstrate the properties of this detection method. The fluorescence assay was shown to correlate with the SEC method in quantifying IgG aggregates. The fluorescent probe method appears to have potential to detect protein particles that could not be analyzed by SEC. This method may become a powerful high throughput tool to detect IgG aggregates in pharmaceutical solutions and to study other protein properties involving aggregation. It can also be used to study the kinetics of antibody particle formation, and perhaps allow identification of the species, which are the early building blocks of protein particles. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:2598–2608, 2010     

Application of a High Throughput and Automated Workflow to Therapeutic Protein Formulation Development

Rapid and efficient formulation development is critical to successfully bringing therapeutic protein drug products into a competitive market under increasingly aggressive timelines. Conventional application of high throughput techniques for formulation development have been limited to lower protein concentrations, which are not applicable to late stage development of high concentration therapeutics. In this work, we present a high throughput (HT) formulation workflow that enables screening at representative concentrations via integration of a micro-buffer exchange system with automated analytical instruments. The operational recommendations associated with the use of such HT systems as well as the efficiencies gained (reduction in hands-on time and run time by over 70% and 30%, respectively), which enable practical characterization of an expanded formulation design space, are discussed. To demonstrate that the workflow is fit for purpose, the formulation properties and stability profiles (SEC and CEX) from samples generated by the HT workflow were compared to those processed by ultrafiltration/diafiltration, and the results were shown to be in good agreement. This approach was further applied to two case studies, one focused on a formulation screen that studied the effects of pH and excipient on viscosity and stability, and the other focused on selection of an appropriate viscosity mimic solution for a protein product.     

啤酒酵母等表达的G蛋白偶联受体在药物高通量筛选中的应用

G蛋白偶联受体具有重要的生理功能和病理意义,已成为一类重要的药物靶点。针对靶标G蛋白偶联受体从大量化合物中筛选出理想的药物,就需要开发特异且灵敏的高通量筛选体系。真核酵母细胞具有易操作、培养条件简单稳定、基因组学研究透彻、外源蛋白表达工具丰富多样、检测分析方法成熟等优点,使它成为一种理想的药物筛选工具。文章对酵母表达G蛋白偶联受体用于构建药物高通量筛选模型的研究现状和发展前景做综述。     

A Strategy for Primary High Throughput Cytotoxicity Screening in Pharmaceutical Toxicology

Purpose. Recent advances in combinatorial chemistry and high throughput screens for pharmacologic activity have created an increasing demand for in vitrohigh throughput screens for toxicological evaluation in the early phases of drug discovery. Methods. To develop a strategy for such a screen, we have conducted a data mining study of the National Cancer Institute's Developmental Therapeutics Program (DTP) cytotoxicity database. Results. Using hierarchical cluster analysis, we confirmed that the different tissues of origin and individual cell lines showed differential sensitivity to compounds in the DTP Standard Agents database. Surprisingly, however, approaching the data globally, linear regression analysis showed that the differences were relatively minor. Comparison with the literature on acute toxicity in mice showed that the predictive power of growth inhibition was marginally superior to that of cell death. Conclusions. This datamining study suggests that in designing a strategy for high throughput cytotoxicity screening: a single cell line, the choice of which may not be critical, can be used as a primary screen; a single end point may be an adequate measure and a cut off value for 50% growth inhibition between 10^–6 and 10^–8 M may be a reasonable starting point for accepting a cytotoxic compound for scale up and further study.     

A High Throughput Protein Formulation Platform: Case Study of Salmon Calcitonin

Purpose  The feasibility of using high throughput spectroscopy for characterization and selection of physically stable protein formulations was studied. Materials and Methods  A hundred aqueous formulations of salmon calcitonin (sCT) were prepared using 20 buffer compositions. The solutions had pH values between 2.5 and 10.5. The stability of the sCT formulations was analyzed over 1 week by the following assays: (1) protein concentration, (2) volume control by measuring pathlength, (3) turbidity (absorbance at 350 nm), (4) intrinsic tyrosine fluorescence, (5) 1-anilino-naphthalene-8-sulfonate (ANS) fluorescence, (6) Nile Red fluorescence. Addition of the dyes (Nile Red and ANS) was used to study protein conformational changes. Results  After 1 day, 27 out of the 100 formulations of salmon calcitonin were stable. After 7 days, 12 stable sCT formulations remained. The best salmon calcitonin formulation was in 10 mM sodium acetate buffer with pH values between 3.5 and 5.5. Conclusions  The findings are in accordance with the sCT formulations that were patented and used commercially. This can be considered as a proof of concept for the high throughput protein formulation platform. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

模糊综合评价法在处方筛选中的应用研究

目的:探讨模糊综合评价法在处方筛选中的应用。方法:以复方氨酚那敏颗粒为模型药物,在单因素试验基础上,以阿司帕坦、山楂粉末香精、鲜奶精粉末香精、蔗糖加入量为因素,咖啡因溶出度为指标,进行4因素3水平正交试验设计筛选处方;应用模糊综合评价法对正交试验9个处方所制颗粒从滋味、香气、色泽、溶化性方面进行感官评价。对优选处方进行验证试验并与原处方进行比较。结果:正交试验与模糊综合评价的最优处方均为处方5,结果一致;与原处方的感官综合得分和咖啡因溶出度(63.12分、91.3%)比较,优选处方3次验证试验中2个指标的平均值分别为84.00分、99.07%(RSD<2.0%,n=3)。结论:模糊综合评价法可用于制剂中感官评价指标的处方筛选,其方法科学、结果准确。     

High Throughput Differential Scanning Fluorimetry (DSF) Formulation Screening with Complementary Dyes to Assess Protein Unfolding and Aggregation in Presence of Surfactants

Purpose

The purpose was to evaluate DSF for high throughput screening of protein thermal stability (unfolding/ aggregation) across a wide range of formulations. Particular focus was exploring PROTEOSTAT® – a commercially available fluorescent rotor dye – for detection of aggregation in surfactant containing formulations. Commonly used hydrophobic dyes (e.g. SYPRO? Orange) interact with surfactants, complicating DSF measurements.

Methods

CRM197 formulations were prepared and analyzed in standard 96-well plate rT-PCR system, using SYPRO? Orange and PROTEOSTAT® dyes. Orthogonal techniques (DLS and IPF) are employed to confirm unfolding/aggregation in selected formulations. Selected formulations are subjected to non-thermal stresses (stirring and shaking) in plate based format to characterize aggregation with PROTEOSTAT®.

Results

Agreement is observed between SYPRO? Orange (unfolding) and PROTEOSTAT® (aggregation) DSF melt temperatures across wide range of non-surfactant formulations. PROTEOSTAT® can clearly detect temperature induced aggregation in low concentration (0.2 mg/mL) CRM197 formulations containing surfactant. PROTEOSTAT® can be used to explore aggregation due to non-thermal stresses in plate based format amenable to high throughput screening.

Conclusions

DSF measurements with complementary extrinsic dyes (PROTEOSTAT®, SYPRO? Orange) are suitable for high throughput screening of antigen thermal stability, across a wide range of relevant formulation conditions – including surfactants –with standard, plate based rT-PCR instrumentation.

     

HPV疫苗和筛查预防宫颈癌

宫颈癌仅出现在感染特异性、高危型人乳头瘤病毒(human papillomavirus,HPV)的妇女,这一发现促进了非细胞学宫颈癌预防策略的发展.用灵敏的分子手段检测HPV可显著提高对高度恶性宫颈癌前期损害的检测能力.防御HPV 16和18型引起的宫颈癌的预防性HPV疫苗已开发成功.本文讨论怎样更好地将HPV DNA检测和HPV疫苗纳入宫颈癌预防工作,给妇女提供最大裨益并降低全球宫颈癌负荷.     

High Throughput Screening of a Prescription Drug Library for Inhibitors of Organic Cation Transporter 3, OCT3

Pharmaceutical Research - The organic cation transporter 3 (OCT3, SLC22A3) is ubiquitously expressed and interacts with a wide array of compounds including endogenous molecules, environmental...     

Screening for Potential Adjuvants Administered by the Pulmonary Route for Tuberculosis Vaccines

Tuberculosis (TB) infects one third of the world’s population, and new infections occur at a rate of 1/s. Better vaccines are needed than the live mycobacterium Bacille Calmette-Guérin (BCG). Alveolar macrophages (AMΦs) play a central role in pulmonary manifestations of TB. Targeting immunomodulators to AMΦs, the first line of defense against Mycobacterium tuberculosis (Mtb), may initiate a potent cell-mediated immune response. Muramyl dipeptide (MDP) and trehalose dibehenate (TDB) have elicited strong immune response when delivered to the lungs as aerosols. AMΦs show toxicity in response to some immunomodulators. The objective of this work was to screen the immunomodulators MDP and/or TDB encapsulated in microparticles (MPs) and to evaluate certain indicators of toxicity in human AMΦ-like cells. Poly(lactide-co-glycolide) (PLGA) MPs containing MDP and/or TDB were prepared by spray-drying. The morphology, particle size distribution, and immunomodulator encapsulation efficiency of MPs were examined. THP-1 cells were exposed to these MPs for 24 h and characteristics of cell morphology, tumor necrosis factor-alpha (TNF-α) release, lactate dehydrogenase (LDH), N-acetyl-β-d-glucosaminidase (NAG) and alkaline phosphatase (ALP) activity in AMΦ culture supernatants were measured. MTT assay was used to assess the viability of cells. Spray-drying produced low-density MPs having volume median diameters between 4 and 6 μm as measured by laser diffraction and projected area diameter between 3 and 5 μm calculated by microscopy. More TNF-α was produced by THP-1 cells exposed to MPs composed of PLGA-MDP or PLGA alone than PLGA-TDB. LDH release following exposure to MPs of PLGA-MDP and PLGA alone was greater than controls. NAG release was higher following exposure to MPs of PLGA alone or PLGA-MDP 0.1% than PLGA-TDB (0.1% and 1.0%). Cells remained viable after exposure to MPs as per MTT assay. PLGA-MDP MPs demonstrated statistically elevated indicators of biochemical responses in cell culture compared to PLGA-TDB MPs, but the extent of their potential to elicit adverse effects in vivo must be studied independently.     

High Throughput Prediction Approach for Monoclonal Antibody Aggregation at High Concentration

Purpose

Characterization of the monoclonal antibody aggregation process and identification of stability factors that could be used as indicators of aggregation propensity with an emphasis on a large number of samples and low protein material consumption.

Methods

Differential scanning calorimetry, dynamic light scattering and size exclusion chromatography were used as the main methodological approaches. Conformational stability, colloidal stability and aggregation kinetics were assessed for two different IgG monoclonal antibody (mAbs) subclasses. Aggregation was induced by exposing the mAbs to 55°C for 3 weeks. mAb samples were prepared in different formulations and concentrations from 1 mg/mL to 50 mg/mL.

Results

High temperature stress of mAb samples revealed that monoclonal antibodies followed first order aggregation kinetics, which suggests that the rate-limiting step of monomer loss was unimolecular. Conformational stability of mAbs was estimated with denaturation temperature measurements. Colloidal stability was assessed with dynamic interaction parameter k _D . The correlation between aggregation kinetics and colloidal and conformational stability factors was evaluated and the dynamic interaction parameter was found to be a promising predictor of aggregation propensity of monoclonal antibodies. The meaning of using an intermolecular interaction parameter for prediction of what is essentially a unimolecular process is also discussed.

Conclusions

This work estimates the significance of different predictors of aggregation propensity at high concentrations as a part of a high throughput, low resource screening method and is a contribution towards determining protein aggregation phenomena in actual systems used for the development and production of biopharmaceuticals.

     

The Design,Synthesis and Potential Utility of Fluorescence Probes that Target DFG‐out Conformation of p38α for High Throughput Screening Binding Assay

The design, synthesis and utility of fluorescence probes that bind to the DFG‐out conformation of p38α kinase are described. Probes that demonstrate good affinity for p38α, have been identified and one of the probes, PF‐04438255, has been successfully used in an high throughput screening (HTS) assay to identify two novel non‐classical p38α inhibitors. In addition, a cascade activity assay was utilized to validate the selective binding of these non‐classical kinase inhibitors to the unactive form of the enzyme.     

LC—MS／MS高通量快速筛查中成药和保健品中未知非法添加药物

目的建立LC—MS／MS的筛查方法,高通量快速筛查中成药和保健品中的非法添加药物。方法色谱条件采用ZORBAXXDB—C18柱,乙腈：1mM甲酸铵（含0．1％甲酸）梯度洗脱;质谱条件采用电喷雾离子化源正离子化筛查方式（ESI＋）,通过相关信息扫描模式（IDA）,最终在已建立的谱库中自动搜索出中成药和保健品中未知的非法添加物。结果对于怀疑有未知的非法添加物样本,经18min的快速筛查,可以同时筛查到包括降糖、镇静催眠、解热镇痛、非甾体抗炎、抗生素、抗癫痫等多种类的非法添加药物。结论本文建立的LC—MS／MS方法简单、快速、准确、高通量,可用于筛查中成药和保健品中未知的非法添加物。     

氯波必利生物黏附缓释片的处方筛选

目的:优化氯波必利生物黏附缓释片的处方和制备工艺。方法:以累积释放度为指标,采用正交设计方法,对制剂辅料羟丙基甲基纤维素(HPMC)、卡波姆、淀粉及乳糖的用量进行考察,确定氯波必利生物黏附缓释片处方。结果:缓释片规格为每片0.1 g,以HPMC和卡波姆为生物黏附和骨架材料,乳糖为稀释剂,淀粉为填充剂和崩解剂,每100片用量分别为5.5、2.2、1.2、2.0g。与氯波必利普通片比较,缓释片具有显著缓释和黏附性能。结论:本制剂制备工艺简单,符合缓释、黏附要求。     

High Throughput Receptor-Based Virtual Screening Under ZINC Database,Synthesis, and Biological Evaluation of Ketol-Acid Reductoisomerase Inhibitors

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes the second common step in branched-chain amino acid biosynthesis. This enzyme is an important target for drug design. Based on the crystal structure of ketol-acid reductoisomerase/N-hydroxy-N-isopropyloxamate (IpOHA) complex, we have carried out high throughput receptor-based virtual screening of the ZINC/drug like database (2 000 000 compounds) to look for novel inhibitors of KARI for the first time. Some novel compounds were found to inhibit rice KARI in vitro among 15 procured compounds. This method can provide useful information for further design and discovery of KARI inhibitors.     

Pulse Proteolysis: An Orthogonal Tool for Protein Formulation Screening

Protein formulation stability is difficult to predict a priori and generally involves long-term stability studies. It is of interest to develop an analytical method that can predict stability trends reliably. Here, pulse proteolysis was evaluated as an analytical tool to predict solution-state stability in different formulations. Four proteins formulated in different buffer and excipient compositions were subjected to urea-induced unfolding and brief enzymatic digestion (“pulse” proteolysis), and relative resistance to proteolysis was measured by microfluidics-based capillary electrophoresis–sodium dodecyl sulfate. Biophysical properties of each formulation were measured using orthogonal biophysical techniques such as differential scanning fluorimetry, differential scanning calorimetry, dynamic light scattering, circular dichroism, and fluorescence spectroscopy. Protein stability in all formulations was monitored by size exclusion chromatography on storage at 5°C and 40°C. For all 4 proteins, formulations with greater proteolytic resistance also showed higher monomer content on thermal stability. In contrast, standard biophysical techniques showed reasonable-to-no correlation with size exclusion chromatography data. The data support the use of pulse proteolysis as an orthogonal, quantitative, and predictive tool to measure protein conformational stability and rank-order formulations.     

Application of Second-Derivative Fluorescence Spectroscopy to Monitor Subtle Changes in a Monoclonal Antibody Structure

In this study, the tertiary structure of a monoclonal antibody was analyzed under thermal and chemical stresses using second‐derivative fluorescence spectroscopy. The effect of polyols, sucrose, and ethylene glycol on the tertiary structure of monoclonal antibody‐U (mAb‐U) (pH 7.0) was studied under thermal stress (25°C–75°C). The tertiary structure of mAb‐U was also analyzed upon chemical denaturation using urea (2.0–8.0 M). The second derivative of mAb‐U showed three bands corresponding to the three spectral classes of tryptophan, class I (330 nm), class II (340 nm), and class III (350 nm). Class II was higher in intensity in the presence of polyols compared with the solution without any polyol. Thermally denatured structure of mAb‐U in sucrose and ethylene glycol was distinctly different than that in buffer. Addition of urea resulted in a decrease in intensity of class I and II, and an increase in intensity of class III implying unfolding. This study showed that second‐derivative fluorescence spectroscopy is an effective tool to monitor subtle alterations in the tertiary structure of proteins. The unfolding of a protein is reflected as an increase in the intensity of the polar class III accompanied with a decrease in the intensity of class I.     

Microfluidic System Based High Throughput Drug Screening System for Curcumin/TRAIL Combinational Chemotherapy in Human Prostate Cancer PC3 Cells

We have developed a fully automated high throughput drug screening (HTDS) system based on the microfluidic cell culture array to perform combinational chemotherapy. This system has 64 individually addressable cell culture chambers where the sequential combinatorial concentrations of two different drugs can be generated by two microfluidic diffusive mixers. Each diffusive mixer has two integrated micropumps connected to the media and the drug reservoirs respectively for generating the desired combination without the need for any extra equipment to perfuse the solution such as syringe pumps. The cell array is periodically exposed to the drug combination with the programmed LabVIEW system during a couple of days without extra handling after seeding the cells into the microfluidic device and also, this device does not require the continuous generation of solutions compared to the previous systems. Therefore, the total amount of drug being consumed per experiment is less than a few hundred micro liters in each reservoir. The utility of this system is demonstrated through investigating the viability of the prostate cancer PC3 cell line with the combinational treatments of curcumin and tumor necrosis factor-alpha related apoptosis inducing ligand (TRAIL). Our results suggest that the system can be used for screening and optimizing drug combination with a small amount of reagent for combinatorial chemotherapy against cancer cells.