一种基于流式细胞术的高通量技术筛选整合素抑制药物
1Department of Immunology, School of Medicine,UConn Health, 2Center for Molecular Discovery,University of New Mexico Health Sciences Center, 3Comprehensive Cancer Center,University of New Mexico Health Sciences Center, 4Department of Pathology,University of New Mexico Health Sciences Center, 5Autophagy, Inflammation, & Metabolism (AIM) Center,University of New Mexico
Summary
该协议描述了一种基于流式细胞术的高通量筛选方法，用于鉴定抑制人中性粒细胞上β2整合素活化的小分子药物。
Abstract
该方案旨在建立一种利用构象变化报告抗体和高通量流式细胞术鉴定β2整合素活化的小分子拮抗剂的方法。该方法还可以作为其他基于抗体的高通量筛选方法的指南。β2整合素是白细胞特异性粘附分子，在免疫反应中至关重要。中性粒细胞依靠整合素活化排出血液，不仅可以抵抗感染，还可以参与多种炎症性疾病。控制β2整合素活化是治疗中性粒细胞相关炎症性疾病的可行方法。在该方案中，单克隆抗体 mAb24 与 β2 整合素的高亲和力头片特异性结合，用于定量分离的原代人中性粒细胞上的 β2 整合素活化。N-甲酰甲硫酰-亮氨酰-苯丙氨酸 （fMLP） 用作激活中性粒细胞 β2 整合素的刺激物。本研究使用了能够自动运行 384 孔板样品的高通量流式细胞仪。在3小时内评估320种化学物质对β2整合素抑制的影响。通过这种方法可以识别直接靶向β2整合素的分子或G蛋白偶联受体启动的整合素由内而外的激活信号通路中的靶分子。
Introduction
许多炎症性疾病的特征在于中性粒细胞浸润在肿胀或损伤部位1。为了浸润这些组织，中性粒细胞必须完成中性粒细胞募集级联反应，包括阻滞到内皮细胞，外渗穿过血管壁，然后募集到组织中2。循环中性粒细胞需要β2整合素激活才能完成这一级联反应，特别是在停滞阶段。因此，减少中性粒细胞粘附、外渗和募集的整合素抑制药物可有效治疗炎症性疾病 3,4。
β2整合素以前曾被靶向用于炎症性疾病。依法珠单抗是一种直接靶向整合素αLβ2的单克隆抗体，用于治疗银屑病5。然而，依法珠单抗因其致命的副作用而被撤回 – JC病毒再激活导致的进行性多灶性白质脑病6,7。基于整合素的新型抗炎疗法应考虑维持白细胞的抗感染功能，以尽量减少副作用。依法珠单抗的副作用可能是由于单克隆抗体在血液中的循环时间延长，从长远来看可能会抑制免疫功能8.最近的一项研究表明，依法利珠单抗介导 αLβ2 交联和 α4 整合素的不需要的内化，为副作用提供了另一种解释9.因此，短寿命的小分子拮抗剂可能会避免这个问题。
本文介绍了一种使用人中性粒细胞筛选小分子β2整合素拮抗剂的高通量方法。β2 整合素激活需要整合素胞外结构域的构象变化才能进入并增加其与其配体的结合亲和力。在规范的弹簧刀模型中，弯曲闭合的整合素胞外结构域首先延伸到扩展-闭合构象，然后打开其头件以完全激活的扩展-开放构象10,11,12,13。还有另一种途径，从弯曲闭合到弯曲打开和扩展打开，最终是14、15、16、17、18、19。构象特异性抗体 mAb24 与人 β2-I 样结构域中的表位结合，当胞外结构域的头饰打开时20,21,22,23。
在这里，mAb24-APC 用于确定 β2 整合素是否被激活。为了激活中性粒细胞和整合素，N-甲酰甲硫酰-亮氨酰-苯丙氨酸 （fMLP），一种细菌衍生的短趋化肽，可以激活中性粒细胞 β2 整合素24，在该方案中用作刺激物。当 fMLP 与中性粒细胞上的 Fpr1 结合时，涉及 G 蛋白、磷脂酶 Cβ 和磷酸肌醇 3-激酶γ的下游信号级联被激活。这些信号转导事件最终通过由内而外的信号通路18,25 导致整合素激活。除了直接与β2整合素结合并阻止整合素活化构象变化的小分子拮抗剂26外，该方法还可以检测出可以抑制β2整合素由内而外的激活信号通路中成分的化合物。自动流式细胞仪可实现高通量筛选。识别新的拮抗剂不仅可以加深我们对整合素生理学的理解，还可以为基于整合素的抗炎治疗提供转化见解。
Protocol
根据《赫尔辛基宣言》的原则，在获得知情同意后，从去识别化的健康人类供体处获得肝素化全血样本，经康涅狄格大学健康机构审查委员会批准。已获得所有捐赠者的知情同意。本研究的纳入/排除标准经过精心制定，以确保受试者的适用性并将潜在风险降至最低。符合条件的参与者年龄在 18 至 65 岁之间，不分种族，英语流利，能够提供知情同意。被排除在外的受试者包括那些无法为自己提供?…
Representative Results
来自代表性的 384 孔板筛选的数据（图 4）显示，阴性对照的 mAb24-APC MFI 为 3236 ± 110，而阳性对照的 mAb24-APC MFI 为 7588 ± 858。该板的 Z’ 因子约为 0.33，在可接受的范围内31。然而，Z’需要在二次测定中进一步验证。 为了对数据进行归一化，对所有值进行了缩放，以将最大值 1 分配给正均值，将最小值 0 分配给负均值。Z’因子将在二次测定?…
Discussion
中性粒细胞刺激和染色的开始和终止由添加中性粒细胞和固定剂PFA来确定。因此，确保将中性粒细胞或PFA移液到每根色谱柱中的时间间隔相同至关重要。这确保了每个孔中嗜中性粒细胞的刺激和染色时间保持一致。由于中性粒细胞的寿命较短，从从供体采集血液到完成流式细胞术的整个实验必须在同一天进行。中性粒细胞对温度变化高度敏感，当暴露于温度快速升高时，例如从4°C过渡到室温或从…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢康涅狄格大学健康中心流式细胞术核心的 Evan Jellison 博士和 Li Zhu 女士在流式细胞术方面的帮助，感谢康涅狄格大学健康中心免疫学系的 Lynn Puddington 博士对仪器的支持，感谢康涅狄格大学健康中心临床研究核心的 Slawa Gajewska 女士和 Paul Appleton 博士在获取血液样本方面的帮助。我们感谢康涅狄格大学医学院的 Christopher “Kit” Bonin 博士和 Geneva Hargis 博士在科学写作和编辑本手稿方面提供的帮助。这项研究得到了美国国立卫生研究院、美国国家心脏、肺和血液研究所 （R01HL145454）、美国国家普通医学科学研究所 （P20GM121176）、美国心脏协会职业发展奖 （18CDA34110426） 和康涅狄格大学健康中心的启动基金的支持。 图 1 是使用 BioRender.com 创建的。
Materials
| 16-channel pipettes | Thermo | 4661090N | Instrument |
| 384-well plate | Greiner | 784201 | Materials |
| APC anti-human CD11a/CD18 (LFA-1) Antibody Clone: m24 | BioLegend | 363410 | Reagents |
| Bravo Automated Liquid Handling Platform | Agilent | 16050-102 | 384 multi-channel liquid handler |
| Centrifuge | Eppendorf | Model 5810R | Instrument |
| FlowJo | Becton, Dickinson & Company | NA | Software |
| Human Serum Albumin Solution (25%) | GeminiBio | 800-120 | Reagents |
| Lifitegrast | Thermofisher | 50-208-2121 | Reagents |
| Nexinhib20 | Tocris | 6089 | Reagents |
| N-Formyl-Met-Leu-Phe (fMLP) | Sigma | F3506 | Reagents |
| Paraformaldehyde 16% solution | Electron Microscopy Sciences | 15710 | Reagents |
| Plate buckets | Eppendorf | UL155 | Accessory |
| Plate shaker | Fisher | 88-861-023 | Instrument |
| PolymorphPrep | PROGEN | 1895 (previous 1114683) | Reagents |
| Prestwick Chemical Library Compound Plates (10 mM) | Prestwick Chemical Libraries | Ver19_384 | 1520 small molecules, 98% marketed approved drugs (FDA, EMA, JAN, and other agencies approved) |
| RPMI 1640 Medium, no phenol red | Gibco | 11-835-030 | Reagents |
| Swing-bucket rotor | Eppendorf | A-4-62 | Rotor |
| ZE5 Cell Analyzer | Bio-Rad Laboratories | Model ZE5 | Instrument |
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