自动声胶肽受体激动剂的效价测定测定的连续稀释
Summary
Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.
Abstract
与小分子药物的发现，筛选肽激动剂要求的肽，以产生浓度 – 响应曲线的连续稀释。筛选的肽，得到复杂的附加层作为常规的基于针尖 – 样品处理方法暴露肽塑料制品的一个大的表面面积，提供用于通过吸附肽损失增加的机会。防止过度暴露于塑料制品通过加入塑料降低肽损失，因而在效价的预测最小误差，我们先前已经描述了非接触式的声学分配用于肽激动剂1的体外高通量筛选的好处。这里我们讨论了在利用在小鼠胰高血糖素样肽1受体（GLP-1R）筛选肽激动剂的例子中微滴定板的肽系列稀释的非接触声学制备一个完全集成的自动化解决方案。我们的方法允许高-throughput细胞为基础的分析来筛选激动剂，很容易扩展到支持增加的样品通量，或允许检测板拷贝数量增加（ 例如 ，对于更多的靶细胞系面板）。
Introduction
针对GLP-1R是2型糖尿病2的治疗已建立的药物靶标。对于该受体的天然肽激动剂，GLP-1，具有2-3分钟3 的体内半衰期。 GLP-1的其G蛋白偶联靶受体的结果在下游生产通过天然G蛋白偶联的第二信使cAMP对腺苷酸环化酶的活化的结合。累积的cAMP测量提供了可靠的测定法来监测受体激活和筛选活性GLP-1类似物与优选的物理化学性质。这样的测定需要测试样品的连续稀释来构建浓度 – 响应曲线，而这移交肽样品时，是特别复杂。从基于尖连续稀释准备的潜在错误先前已经1,4,5描述。肽将吸附到塑料制品，造成不可靠的效力的估计。肽损失，可以通过T为最小化他纳入缓冲器牛血清白蛋白（BSA）和使用硅化塑料制品的，但蛋白质结合的仍无法预测。特别是，在实验容器GLP-1的结合的变体已经描述6。有在实验室塑料制品中使用可从提示和微量滴定板成水测定缓冲液浸出，并与蛋白质功能7,8干扰该稳定剂进一步复杂化。因此，方法，以减少暴露于塑料制品是必要的，以增加测量的准确度。
声学液体分配器集中的高频声信号到流体样品的表面上，从而导致在精确纳升液滴喷射到相邻测定板9。利用声学弹射的是制药行业的准备和大量的合成化合物库的筛选标准，而且技术已经很好的验证了小moleculES 10。据我们所知，我们是来形容声配药重组和合成肽的制备第一组和我们先前已经报道了更高的精度比传统的基于技巧的方法1。
本文介绍了在完全自动化的处理板的机器人系统由非接触式声学传递肽序列和直接稀释配制的整合。许多围绕样本声传输方法先前已被描述11。我们利用两步法来制备中间库存浓度和以串行稀释肽类似物为全剂量反应曲线的产生。制备的肽温育的细胞表达靶小鼠GLP-1R，并且我们使用市售均相时间分辨荧光（HTRF）测定法来测量这些细胞中的cAMP积累作为肽激动剂ACTIV的读出性。该测定是健壮和适合于高通量384孔格式和常规应用到分析开发和药物筛选项目12。
Protocol
Representative Results
Discussion
这个协议描述自动声学分配的成功应用到在3×10 6个要求小于1微升样品的浓度范围连续稀释的肽样品。这种方法的主要优点是通过经由还原样品暴露于所通常所需的试剂转移和混合试验容器和塑料制品（如枪头）最小化的肽吸附到塑料制品，以提高数据质量。虽然声学分配不能完全排除使用塑料制品的，它确实显著酌减。此外，消除基于尖部-移液还消除了样品的交叉污染，和错误不会在?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
None.
Materials
| Hanks’ Balanced Salt solution | Sigma-Aldrich | H8264 | |
| HEPES | Sigma-Aldrich | H3375 | |
| Bovine Serum Albumin | Sigma-Aldrich | A9418 | |
| 3-Isobutyl-1-methylxanthine | Sigma-Aldrich | I7018 | Prepared as a 0.5 M stock in DMSO |
| GLP-1 (7-36) amide | Bachem | H-6795 | Prepared as a 1 mg/ml stock in PBS, referred to as '100X reference control' |
| Test peptides | Produced in-house at MedImmune | Supplied at various concentrations in DMSO or PBS as appropriate | |
| 100X peptide stock | Produced in-house at MedImmune | Test peptide diluted into assay buffer to 100X final required concentration | |
| Trypan Blue Solution, 0.4% | Thermo Fisher Scientific | 15250-061 | |
| Cedex XS Cell Analyzer | Innovatis | ||
| Corning 384 well plates, low volume | Sigma-Aldrich | 4514 | |
| Echo Qualified 384-Well Polypropylene Microplate | Labcyte Inc. | P-05525 | |
| Echo Qualified Reservoir | Labcyte Inc. | ER-0055 | |
| Echo 550 Liquid Handler | Labcyte Inc. | Droplet transfer volumes in increments of 2.5 nl | |
| Echo 525 Liquid Handler | Labcyte Inc. | Droplet transfer volumes in increments of 25 nl | |
| ACell Benchtop Automation | HighRes Biosolutions | MC522 | |
| Cellario Lab Automation Scheduling software for Life Science Robotics | HighRes Biosolutions | ||
| MultidropCombi Reagent Dispenser | ThermFisher Scientific | 5840300 | Referred to as 'bulk reagent dispenser' |
| HTRF cAMP Dynamic 2 kit | Cisbio Bioassays | 62AM4PEJ | |
| EnVision Multilabel Reader | PerkinElmer |
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