Drug Development and modern biotechnology
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Overview of drug development. Procedure of drug development and biotech. One case. Frequent problems. A quick quiz.
Fluorescence Polarization (FP) Assay 荧光偏振分析
• Tracer: a small binding partner labeled with a fluorescent tag. When excited, it rotates rapidly, thereby depolarizing the emission Low FP
Seeing is believing. A picture is worth a thousand words!
Assay Validation
Plate layout
Assay Validation
• STABILITY AND PROCESS STUDIES 稳定性和分析过程 • PLATE UNIFORMITY AND SIGNAL VARIABILITY ASSESSMENT 板均一性和信号变异性评估
AlphaScreen
AlphaScreen
•Homogenous •Flexibility: Use in any cell line •Sensitivity: physiologically •Automatable •Ultra HTS compatible •Multiple assay options
A real hit!!
Assay establishment
Fluorescence Polarization (FP) Assay 荧光偏溶液中的荧光物质，后者可吸收并 释放出相应的偏振荧光。如果被激发的荧光物质处于静止状态，该物 质仍将保持原有激发光的偏振性，如果其处于运动状态，该物质发出 的偏振光将区别于原有激发光的偏振特性，也就是所谓荧光去偏振现 象。
Nat. Biotech. (2006) 24:167
Biochemical Society Transaction (2006) 34:313
From a target to a real hit
• Target identification and validation（药靶的发现与验证） • Assay establishment（筛选方法的建立） • Assay validation（筛选方法的验证） • Primary screening（初筛） • Hit identification（活性样品的发现） • Hit confirmation (secondary screening) （活性样品的确定/复筛）
High Content Screening/Analysis (HCS/HCA)
Automated Imaging System - ImageXpress Micro From Molecular Devices (MDC)
What is High Content Screening?
• Cell-based assay • Automated microscopy in 96 or 384-well format • High throughput image acquisition & processing • Visualization of sub-cellular & intracellular events • Multiple colors & parameters are measured/image • Information rich
How is it done?
• Probes/indicators/dyes reveal: – Sub-cellular localization – Cell or organelle morphology – Ion concentration (e.g., Ca2+, pH) – Membrane potential – Pathology markers (e.g., apoptosis) Measuring fluorophores – Intensity – Spatial – Spectral – Temporal 100x oil objective Actin: green; Mitochondria: yellow Nuclei: red. Image from MDC
荧光偏振分析
利用荧光偏振的原理，通过检测荧光素标记的小分子与其它分子 相互作用前后分子量的变化，计算水平方向及垂直方向 的荧光偏振 值作相关分析。如果被检测分子大，激发时运动慢，测得的荧光偏 振光值高。如果分子小，分子旋转或翻转速度快，发射光相对于激 发光平面将去偏振化，测得的偏振光值低，从而计算出样品的偏振 值（偏振值单位MP）。
B2. Reaction Stability Over Projected Assay Time
• Conduct time-course experiments to determine the range of acceptable times for each incubation step in the assay. This information will greatly aid in addressing logistic and timing issues. • The stability studies will require running assays under standard conditions, but with one of the reagents held for various times before addition to the reaction. The results will be useful in generating a convenient protocol and understand the tolerance of the assay to potential delays. • If possible, reagents should be stored in aliquots suitable for daily needs. However, some information pertinent to saving leftover reagents (particularly expensive ones) for future assays should be obtained. • New lots of critical reagents should be validated using the bridging studies
• REPLICATE-EXPERIMENT STUDY 实验可重复性
STABILITY AND PROCESS STUDIES
• Reagent Stability and Storage Requirements • Reaction Stability Over Projected Assay Time • DMSO Compatability • Fermentation Compatability ?
B3. DMSO Compatability
• DMSO concentrations from 0 to 10% are tested. • For cell based assays, it is recommended that the final %DMSO be kept under 1%. • Note that this study should be done relatively early in development of the assay.
• Binder: a larger unlabeled partner
• Binding of two partners: - the molecular volume of tracer increases - overall rotation slows down - emission light is highly polarized
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Plate Reader-based HTS vs. Image-based HCS
• Numerical data • Population average/well
• Image • Numerical data (cellular and population)
HCS addresses needs for assays: • Multi-colors & multi-parameters • Location & quantity of molecules • Morphology
High FP
Bioluminescence Resonance Energy Transfer (BRET) Assay 生物发光共振能转移分析
Fluorescence Resonance Energy Transfer (FRET) Assay 荧光共振能转移分析
Time Resolved-Fluorescence Assay 时间分辨荧光
A
B
Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay
PerkinElmer’s Lance Ultra kinase assay
• Donor: Eu（镧系元素）-labeled antiphospho-substrate antibody • Acceptor: Ulight-labeled substrate • Eu-Ab binds to phosporylated Ulight-substrate → high TR-FRET • Eu-Ab does not bind to Ulight-substrate → low TR-FRET