用于重组G蛋白偶联受体高通量筛选的“双加法”钙荧光测定
Summary
在这项工作中，描述了一种用于384孔板的高通量细胞内钙荧光测定，以筛选重组G蛋白偶联受体（GPCR）上的小分子文库。靶标是来自牛瘟蜱的激肽受体， Rhipicephalus microplus， 在CHO-K1细胞中表达。该测定法在一个“双重添加”测定中使用相同的细胞识别激动剂和拮抗剂。
Abstract
G蛋白偶联受体（GPCR）是受体的最大超家族，是许多人类药物的靶标。针对GPCR的随机小分子文库的高通量筛选（HTS）被制药行业用于靶标特异性药物发现。在这项研究中，使用HTS鉴定无脊椎动物特异性神经肽GPCRs的新型小分子配体，作为致命人类和兽医病原体载体生理研究的探针。
选择无脊椎动物特异性激肽受体作为靶标，因为它调节无脊椎动物的许多重要生理过程，包括利尿、摄食和消化。此外，许多无脊椎动物GPCR的药理学特征不佳或根本没有表征;因此，这些受体组相对于其他后生动物，特别是人类中的相关GPCR的差异药理学，为GPCR作为一个超家族的结构 – 活性关系增加了知识。为384孔板中的细胞开发了一种HTS测定法，用于从牛热蜱或南方牛蜱， Rhipicephalus microplus中发现激肽受体的配体。蜱激肽受体在CHO-K1细胞中稳定表达。
激肽受体被内源性激肽神经肽或其他小分子激动剂激活时，触发钙储存的Ca2+ 释放到细胞质中。这种钙荧光测定与“双重添加”方法相结合，可以检测同一测定板中的功能激动剂和拮抗剂“命中”分子。每次测定都是使用携带320个随机小分子阵列的药物板进行的。获得可靠的 Z’ 因子为 0.7，当 HTS 处于 2 μM 终浓度时，鉴定出 3 个激动剂和 2 个拮抗剂命中分子。此处报告的钙荧光测定可以适用于筛选激活Ca2+ 信号级联的其他GPCR。
Introduction
G蛋白偶联受体（GPCRs）存在于酵母到人类，是许多生物体中最大的受体超家族1。它们在调节动物几乎所有生物过程中起着关键作用。节肢动物的基因组中有50-200个GPCR，这意味着它们代表了最大的膜受体超家族2。它们根据序列相似性和功能分为六大类，A-F3。GPCR转导各种细胞外信号，例如激素，神经肽，生物胺，谷氨酸，质子，脂糖蛋白和光子的信号4。GPCR与异源三聚体G蛋白（Gα，Gβ和Gγ）偶联以传递下游信号。与Gαs 或Gαi / o 蛋白偶联的GPCR通过激活或抑制腺苷环化酶分别增加或降低细胞内3’，5′-环磷酸腺苷（cAMP）水平。与Gαq/11 偶联的GPCR通过激活磷脂酶C（PLC）-肌醇-1，4，5-三磷酸（IP3）途径，诱导钙从内质网钙储存中释放。与Gα12/13 偶联的GPCR激活RhoGTP酶核苷酸交换因子5，6。GPCR是超过50%的人类药物和杀螨剂amitraz4的靶标。由于GPCR转导了如此多样化的信号，它们是开发破坏无脊椎动物特异性生理功能的新型杀虫剂的有希望的目标。
HTS的目标是识别可以调节受体功能的命中分子。HTS 涉及检测开发、小型化和自动化7。节肢动物神经肽GPCR参与大多数生理功能，如发育、蜕皮和蜕皮、排泄、能量动员和繁殖4。节肢动物和后生动物的大多数神经肽GPCR通过钙信号级联反应2，6，8，9，10发出信号，例如在黑腿蜱肩突硬蜱的肌抑制肽和SIFamide受体中;它们的配体在后肠运动测定中具有拮抗作用，SIF引起收缩，MIP抑制收缩11，12。黄热病蚊子的NPY样受体埃及伊蚊调节雌性宿主寻找13。与其他替代钙动员测定（例如水杉钙生物发光测定14）相比，钙荧光测定易于执行，不需要转染其他重组钙检测蛋白，并且具有成本效益。与在水杉钙生物发光测定中获得的快速动力学信号相比，钙荧光测定产生更长的信号14，15.
在这里的示例中，来自牛瘟蜱的激 肽受体Rhipicephalus microplus在CHO-K1细胞系中重组表达并用于钙荧光测定。在 R. microplus中仅发现一个激肽受体基因;受体通过Gq蛋白依赖性信号通路发出信号，并触发Ca2+ 从钙储存到细胞内空间的外排16。该过程可以通过荧光团进行检测和定量，荧光团在结合钙离子时引发荧光信号（图1）。
激肽受体是无脊椎动物特异性GPCR，属于A类视紫红质样受体。激肽是一种古老的信号神经肽，存在于软体动物、甲壳纲、昆虫和螨科4，17，18 中。鞘翅目动物（甲虫）缺乏激肽信号系统;在蚊子埃及伊蚊中，只有一个激肽受体结合三个伊蚊素，而黑腹果蝇有一个激肽受体，果蓿素作为独特的配体19，20，21。脊椎动物中没有同源激肽或激肽受体。虽然激肽的确切功能在蜱中尚不清楚，但激肽受体RNAi沉默的雌性R. microplus显示出显着降低的生殖适应性22。激肽是昆虫中的多效性肽。在黑腹果蝇中，它们参与中枢和周围神经系统调节系统23，蜕皮前期24，喂养25，代谢26和睡眠活动模式26，27，以及幼虫运动28。激肽调节蚊子埃及伊蚊的后肠收缩、利尿和摄食 29，30，31。激肽具有保守的C端五肽Phe-X1-X2-Trp-Gly-NH2，这是生物活性所需的最小序列32。节肢动物的特异性、内源性配体的小尺寸（使其易于小分子干扰）以及昆虫中的多效性功能使激肽受体成为害虫防治的有希望的靶标4。
“双重加成”测定（图2）允许在同一HTS测定15中鉴定激动剂或拮抗剂。它改编自制药行业中通常用于药物发现的“双重添加”测定33。简而言之，与溶剂对照的应用相比，首次将药物添加到细胞板中允许在检测到更高的荧光信号时鉴定化学库中的潜在激动剂。用这些小分子孵育5分钟后，将已知的激动剂（激肽）施用于所有孔。与在第一次添加中接受溶剂的对照孔相比，那些从药物板随机接受拮抗剂的孔在加入激动剂时显示出较低的荧光信号。然后，该测定允许鉴定具有相同细胞的潜在激动剂和拮抗剂。在标准的HTS项目中，这些命中分子将通过剂量反应测定和额外的生物活性测定进一步验证，此处未显示。
图1：钙荧光测定机理图示。Gq蛋白触发细胞内钙信号通路。激肽受体（G蛋白偶联受体）在CHO-K1细胞中重组表达。当激动剂配体与受体结合时，与激肽受体相关的Gq蛋白激活PLC，从而催化PIP2分子转化为IP3和DAG。然后，IP 3 与内质网表面的 IP3 R 结合，导致 Ca 2+ 释放到细胞质中，其中Ca 2+ 离子与荧光团结合并引发荧光信号。荧光信号可以通过在490nm处激发并在514nm处检测获得。缩写：GPCR = G蛋白偶联受体;PLC = 磷脂酶 C;PIP2 = 磷脂酰肌醇 4，5-二磷酸;IP3 = 肌醇三磷酸;DAG = 二酰基甘油;IP3 R = IP3 受体。用 BioRender.com 创建。请点击此处查看此图的大图。
图 2：在 CHO-K1 细胞中表达的 G 蛋白偶联受体上高通量筛选小分子的工作流程。 （A）使用液体处理系统（25μL/孔）将稳定表达激肽受体的重组CHO-K1细胞添加到384孔板（10，000个细胞/孔）中，并在加湿的CO2培养箱中孵育12-16小时。）使用液体处理系统将含有荧光染料（25μL/孔）的测定缓冲液加入细胞板中。将板在37°C下孵育30分钟30分钟，并在室温下再平衡30分钟。 （C）用读板器测量每个孔中细胞的背景荧光信号。（D） 使用液体处理系统将来自 384 孔文库板和空白溶剂（均为 0.5 μL/孔）的药物溶液添加到细胞测定板中。（E）加入药物溶液后立即用酶标仪测量细胞钙荧光反应;引起高于平均水平荧光信号的化合物被挑选出来作为激动剂命中。在步骤G期间加入肽激动剂后，揭示了阻断GPCR的拮抗剂命中（下图）。（F）在同一测定板中，将细胞与筛选化合物孵育5分钟后，将蜱激肽受体的内源性激动剂肽Rhimi-K-1（QFSPWGamide）加入到每个孔（1μM）中。（G）加入激动剂肽后立即用酶标仪测量细胞荧光反应。选择抑制荧光信号的化合物作为拮抗剂命中。缩写：GPCR = G蛋白偶联受体;RT = 室温;RFU = 相对荧光单位。用 BioRender.com 创建。请点击此处查看此图的大图。
Protocol
Representative Results
Discussion
HTS的目标是通过筛选大量小分子来识别命中分子。因此，本例的结果仅代表常规HTS实验的一小部分。此外，鉴定出的命中分子需要在下游检测中验证，例如在同一重组细胞系和仅携带空载体的 CHO-K1 细胞系上进行剂量依赖性检测，可以同时进行以保存小分子。细胞毒性测定将有助于证明第二次添加时缺乏反应不是由于细胞死亡率。应进行其他生物测定以验证 体外分离组织中的活性，或者通?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国农业部-NIFA-AFRI动物健康和福祉奖（奖项编号2022-67015-36336，PVP [项目总监]）的支持，以及德克萨斯州A&M AgriLife研究昆虫媒介疾病资助计划（FY’22-23）的竞争性资金给P.V.P. TAMU农业与生命科学学院的A.W.E.S.O.M.E.教师小组因帮助编辑手稿而受到认可。 补充表S2 包含来自德克萨斯A&M大学James Sacchettini博士实验室和德克萨斯A&M AgriLife Research的内部随机小分子库的数据。
Materials
| 0.25% trypsin-EDTA | Gibco Invitrogen | 15050-065 | with phenol red |
| 0.4% trypan blue | MilliporeSigma | T8154 | liquid, sterile |
| 1.5 mL microcentrifuge tubes | Thermo Fisher | AM12400 | RNase-free Microfuge Tubes |
| 5 mL serological pipette | Corning | 29443-045 | Corning Costar Stripette individually wrapped |
| 10 mL serological pipette | Corning | 29443-047 | Corning Costar Stripette individually wrapped |
| 15 mL conical tubes | Falcon | 352196 | sterile |
| 20 µL filter tips | USA Scientifc Inc. | P1121 | sterile, barrier |
| 25 mL serological pipette | Corning | 29443-049 | Corning Costar Stripette individually wrapped |
| 50 mL conical tubes | Corning | 430828 | graduated, sterile |
| 150 mL auto-friendly reservior | Integra Bioscience | 6317 | sterile, individually wrapped for cell seeding in day 1 |
| 150 mL auto-friendly reservior | Integra Bioscience | 6318 | sterile, stacked, for loading dye in day 2 |
| 384/ 12.5 µL low retention tips | Integra Bioscience | 6405 | long, sterile filter |
| 384/ 12.5 µL tips | Integra Bioscience | 6404 | long, sterile filter |
| 384-well plate | Greiner | 781091 | CELLSTAR, clear polystyrene, µClear, Black/Flat |
| Aluminum plate seals | Axygen Scientific | PCR-AS-200 | polyester-based |
| Aluminum foil wrap | Walmart | ||
| Biosafty cabinet II | NuAire | NU-540-300 | |
| Cell counter | Nexcelom | AutoT4 | |
| cell counting slides | Nexcelom | SD-100 | 20 µL chamber |
| CO2 humidified incubator | Thermo Fisher | Forma Series II | |
| Desk Lamp | SunvaleeyTEK | RS1000B | |
| Dimethyl sulfoxide | MilliporeSigma | 276855 | anhydous, >99.9% |
| Drug plate | Corning | 3680 | |
| Dulbecco's phosphate-buffered saline | Corning | 21-031-CV | DPBS, 1x without calcium amd magnesium |
| Ethanol | Koptec | 2000 | |
| F-12K Nutrient Mixture | Corning | 45000-354 | (Kaighn's Mod.) with L-glutamine |
| Fetal bovine serum | Equitech-Bio | SFBU30 | |
| Fluorescent calcium assay kit | ENZO Lifescience | ENZ-51017 | 10×96 tests |
| G418 sulfate | Gibco Invitrogen | 10131-027 | Geneticin selective antibiotic 50 mg/mL |
| Hank's buffer | MilliporeSigma | 55037C | HBSS modified, with calcium, with magnesium, without phenol read |
| HEPES buffer | Gibco Invitrogen | 15630-080 | 1 Molar |
| HTS data storage plateform | CDD vault | https://www.collaborativedrug.com/ | |
| Liquid handling system | Integra Bioscience | Viaflo | 384/12.5 µL |
| Plate centrifuge | Thermo Fisher | Sorvall ST8 | |
| Plate reader | BMG technology | Clariostar | |
| Poly-D-lysine | MilliporeSigma | P6407 | |
| Rhimi-K-1 agonist peptide | Genscript | custom order | QFSPWGamide |
| T-75 flask | Falcon | 353136 |
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