Name: a kinetic fluorescence-based ca2+ mobilization assay to identify g protein-coupled receptor agonists, antagonists, and allosteric modulators
Uploaded: 2018-02-20T13:00:00.000+00:00
Duration: 7 min 41 s
Description: Watch this Scientific Journal Video about A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators at JoVE.com

作者感谢埃里克芳廷承袭和 Geert Schoofs 的出色技术援助。这项工作得到了库鲁汶 (格兰特) 的支持。PF/10/018), 全宗与客厅里 Wetenschappelijk Onderzoek (FWO, 授予 no。G. 485.08) 和基金会 Dormeur 瓦杜兹。

注: 1 和2节所述的所有步骤均在层流柜的无菌条件下进行。
1. 维护 U87。CD4. hCXCR4 细胞
<ol><li>在37°c 和 5% CO2的 T75 培养瓶中生长细胞在一个湿润的孵化器。 注意: 本协议中使用的体外细胞线是 U87 胶质瘤细胞系, 它能稳定地表达分化 4 (CD4) 和人类 CXCR4 的簇, 此前曾被描述为17。流式细胞仪连续监测 CD4 和 CXCR4 的细胞表面表达, 并且随着时间的推移, 表达水平保持恒定 (~ 100% CD4+和 ~ 100% CXCR4+细胞)。对所用细胞系的生成和流式细胞术过程的详细描述, 以调查受体表达水平不在本议定书范围之内。<ol>
<li>亚文化细胞在80-85% 融合。允许所有试剂在细胞培养前达到室温 (RT)。</li>
		<li>将条件培养基从细胞中移除, 用5毫升磷酸盐缓冲生理盐水 (PBS) 冲洗细胞单层。</li>
		<li>添加3毫升0.25% 胰蛋白酶-EDTA, 并均匀分布在细胞单层。然后去除超过胰蛋白酶-EDTA 和孵化高达5分钟, 在37摄氏度, 直到细胞开始分离。</li>
		<li>添加10毫升新鲜完整的生长培养基 (Dulbecco 的改良鹰的培养基 (DMEM) + 10% 胎牛血清 (血清) + 0.01 米 HEPES + 适当的选择剂)。并用重悬的细胞通过轻轻吹打上下。将细胞悬浮液转移至无菌50毫升管。</li>
		<li>使用选择方法计算可行单元格的数目。在 U87 胶质瘤细胞的情况下, 生存能力一般达到100%。 注意: 细胞数可以通过多种方式确定。我们经常使用基于台盼蓝染色的自动细胞分析系统 (参见材料表), 根据其标准处理程序, 但其他礼仪也应同样良好。</li>
		<li>将 2 x 106或 3 x 106 (可行) 单元格添加到 T75 培养瓶中的最终体积为25毫升的新鲜生长培养基, 并在37°c 和 5% CO2处孵化。 注: 如果使用 3 x 106单元格, 则在2天后单元格将达到80-90% 融合。如果使用 2 x 106单元格, 则在3天后它们将达到相同的融合。如果使用其他细胞系, 细胞的生长速率应该首先进行经验主义的测定。</li>
	</ol>
</li>
</ol>2. 为 Ca2 +动员化验的细胞播种
<ol><li>在细胞传代天 (0 天), 外套黑壁聚苯乙烯96井板以清楚的底部 (参见材料表) 与0.1% 明胶溶液促进细胞附件。 注: 如果使用的是商用 (参见材料表) 的预涂板, 可以省略96井板与明胶的涂层。建议在继续执行《议定书》之前, 对所调查的每一条细胞线使用预涂钢板而不是手动涂敷进行评估。<ol>
<li>将1克明胶加入100毫升 PBS, 制备明胶溶液, 以获得1% 溶液。在进一步使用前, 用 PBS 稀释10x。为了提高明胶的溶解度, 将溶液加热到37摄氏度。</li>
		<li>添加100µL 0.1% 明胶溶液每井的96井板使用多通道吸管。在 RT 中孵化2小时。</li>
	</ol>
</li>
	<li>同时, 准备在包覆的96井板中播种的细胞。<ol>
<li>将细胞从培养瓶中分离出来, 并用重悬在新鲜生长培养基中, 用台盼蓝染色法进行计数。</li>
		<li>在50毫升管中的细胞旋转5分钟, 在 400 x g 的 RT。</li>
		<li>并用重悬新鲜生长培养基中的细胞 (DMEM + 10%, 1% HEPES, 无抗生素) 获得 0.1 x10 6 (可行) 细胞/mL 细胞密度。</li>
	</ol>
</li>
	<li>通过翻转盘子并将其干燥到组织中, 从透明底部的黑色壁板上取出明胶溶液。添加200µL/井 PBS, 去除多余的明胶, 再翻转盘子。</li>
	<li>分配200µL 的细胞悬浮 (对应于 0.2 x 105细胞) 每井在明胶涂覆的96井板 (从现在一板将进一步被称为 "测量板")。</li>
	<li>在37摄氏度和 5% CO2上隔夜孵化盘。</li>
</ol>3. 使用荧光 Ca2 +敏感染料加载单元格
<ol><li>在1天, 执行实际的 Ca2 +动员化验, 从加载的种子细胞与荧光 Ca2 +结合染料 fluo-2 AM 开始。<ol>
<li>通过加入40毫升 HEPES (1 米) 到200毫升的平衡盐溶液 (HBSS, 10x, 无酚红, 不含碳酸氢钠) 制备化验缓冲器。加入超纯水, 获得2升的最终容积, 并添加4克牛血清白蛋白 (BSA)。通过磁搅拌溶解 BSA。将 pH 值调整为 7.4 (用氢氧化钠) 并过滤溶液。 注意: 在所有后续步骤中, 使用相同的缓冲区 (称为 "检测缓冲区")。</li>
		<li>在荧光 Ca2 +敏感染料 fluo-2, 准备一个股票溶液 (二甲基亚砜) (4 毫米)。避免过度暴露于光线下。溶解1毫克 fluo-2 AM (分子量: 1061 克/摩尔) 在235.6 µL 亚砜。</li>
		<li>准备 fluo-2 的工作解决方案。对于一个96井的检测板混合12.5 µL fluo-2 的股票溶液 (4 毫米) 和12.5 µL 非离子表面活性剂多元醇 (例如, pluronic f-127) 溶液 (20% 重量/体积在亚砜, 见材料表)。将此混合物的22µL 添加到15毫升管中的11毫升检测缓冲器中。fluo-2 的最后浓度是4µM。 注: 非离子表面活性剂用作分散剂, 以改善 fluo-2 的水溶性, 从而提高细胞的负荷。在亚砜中溶解表面活性剂, 将样品加热37摄氏度, 并定期混合以获得均匀溶液。</li>
		<li>通过翻转盘子并将其干燥在纸巾上, 从原来的96井测量板中的种子细胞中去除生长培养基。</li>
		<li>在黑暗中, 使用多通道吸管和孵育45分钟, 在µL 中添加100个负载染料溶液。</li>
	</ol>
</li>
</ol>4. 制备含有趋化因子配体 CXCL12 或受调查化合物的96井聚丙烯板
<ol><li>获得圆底聚丙烯 (PP) 96 井板 (请参阅材料表)。<ol>
<li>允许 CXCL12 (1 毫克/毫升) 的库存溶液和被调查的化合物在 RT 平衡的库存溶液。 注: CXCL12 的库存解决方案是在超纯水中辅以 0.01% Tween20, 并以单一用途整除数储存在-20 摄氏度。</li>
		<li>在检测缓冲器 (250 CXCL12/毫升, 等于 31.25 nM) 和5x 浓缩稀释每个化合物 (在化验缓冲器中) 制备一个5x 浓缩溶液。</li>
		<li>根据预定义的板材布局, 将库存解决方案放入适当的96井板中。在含有 CXCL12 ("趋化物板") 的板块中分配75µL/井, 在含有化合物的板块中分配50µL/井 ("复合板")。 注: 两种化合物和 CXCL12 最终将被稀释5x 后, 分配在测量板。同时, 在复合板和趋化因子板中包含只有检测缓冲器的负控井也很重要。还需要包括正控井 (即, 在趋化因子板中有 CXCL12 的井, 但在复合板的相应井中测定缓冲液)。</li>
	</ol>
</li>
</ol>5. 荧光微板块读取器的协议设置
注意: 此协议中使用的荧光微板块读取器在材料表中引用。
<ol><li>首先打开荧光板读取器的冷却单元, 然后打开荧光读取器。让我们启动几分钟, 打开系统的软件。</li>
	<li>使用包含以下步骤的拖放菜单创建检测协议:<ol>
<li>在 "设置" 框中, 选择 "Read_Mode" 与激发波长: 470-495 毫微米;发射波长: 515-575 nM。 注意: 所选波长适合与 Ca2 +敏感染料 fluo2 一起使用。</li>
		<li>包括 "混合 TF" (转移液) 盒, 以自动混合的化合物在复合板, 将放在源2位置的设备 (见步骤 6.4)。选择15µL 的溶液在每个井将自动吸气和混合三倍。调整吸管尖端的高度在20µL 在液体表面之下。设定50µL 的抽吸速度和分配。 注: 吸管尖的高度指的是在吸管尖端下方留下的体积。例如, 如果复合板的井包含50µL, 高度30µL 对应于20µL 在液体表面之下。使用该软件可以调整混合溶液的体积、混合速度、吸管尖端在混合过程中的位置以及混合循环的数量。</li>
		<li>在 "转移液" 框中, 定义20µL 从复合板将被转移到测量板。将吸管尖放在液体表面下面的20µL 上, 在µL 的吸入过程中, 在高60µL, 在测量板上配药时, 在高度30的位置. 设定50µL 的吸速速度和25µL/秒的分配速度。 注: 复合板包含50µL 的解决方案 (见步骤 4.1.3), 因此高度为30µL 对应于位置20µL 在液体表面之下。测量板将包含每井80µL 的化验缓冲器 (参见步骤 6.3), 因而高度60µL 对应于提示的一个相似的位置。</li>
		<li>选择 "使用 TF 读取" 按钮。在第一个间隔期间, 选择60读取 (荧光测量), 读取间隔时间为1秒. 定义在将化合物分配到测量盘之前记录10读取, 然后50读取。使用三十年代和18读取的读间隔时间定义第二个间隔。 注意: 在这个步骤中荧光将被测量动力学 (在定义的时间间隔) 为 ~ 10 分钟总计。</li>
		<li>在三次协议中包括 "洗涤提示" 按钮。在每个洗涤步骤中, 选择流体类型: 液体 A (超纯水) 或流体 B (70% 乙醇), 洗涤周期的数量 (1), 泵速度 (快速), 和次数 (5) 在每个洗涤步骤。 注: 在第一和最后洗涤步骤液体 A 使用, 在第二洗涤阶段流体 B 使用。</li>
		<li>包括 "暂停 Pipettor" 功能。将 pipettor 设置为300s 暂停。 注: 通过在协议中包括此步骤, pipettor 头 (集成在荧光板读取器设备中) 将在继续执行该协议之前暂停5分钟。</li>
		<li>包括 "与 TF 混合" 框, 以允许自动混合在趋化板中的 CXCL12 溶液, 将定位在设备的源3位置 (见步骤 6.4)。选择15µL 的溶液在每个井将自动吸气和混合三倍。在吸入和混合过程中, 将吸管尖端的位置放在液体表面下面20µL。吸入和配药的速度设定在50µL/秒。 注: 类似于步骤 5.2.2, 这些参数可以调整。</li>
		<li>在随后的 "转移流体" 框中, 定义25µL 从趋化因子板的每一个井将被转移入测量板材。位置提示20µL 下面的液体表面即, 在高度55µL 在吸入的趋化因子板块, 其中包含75µL/井 (见步骤 4.1.3), 并在高度80µL 在分配的测量板。设定50µL 的吸速速度和25µL/秒的分配速度。</li>
		<li>包括 "读取 TF" 按钮。在第一个间隔中, 选择145读取, 读取间隔时间为1秒. 定义在将 CXCL12 分配到测量盘之前记录5读取, 然后140读取。定义第二个间隔, 读取间隔时间为6秒, 然后选择20读取。 注: 在整个协议243读取 (荧光测量) 被记录在一起:78 步5.2.4 和165在步骤5.2.9。</li>
		<li>类似于步骤 5.2.5, 在协议中包含 "洗涤提示" 按钮三次。 注: 在协议末尾包括这一步骤, 可以使提示重新用于执行另一种检测, 而无需更改提示。</li>
	</ol>
</li>
	<li>通过单击 "设定温度" 按钮并选择37°c, 将设备的温度设置为37摄氏度。</li>
</ol>6. 荧光检测的运行
<ol><li>在测量板与加载缓冲器45分钟的孵化后 (见步骤 3.1.5), 通过翻转板并将其干燥到组织上, 取出缓冲器。</li>
	<li>通过添加150µL/井的化验缓冲液和孵育2分钟来冲洗种子细胞。</li>
	<li>通过翻转板再次删除缓冲区。添加80µL/井的检测缓冲区与多通道吸管。</li>
	<li>把所有的盘子放到设备的适当位置: 在源2位置的复合板, 在源3位置的趋化板, 和测量板在读取位置。在源1提示位置放置一盒黑色提示。在继续执行协议之前, 关闭设备的门并孵化5分钟。</li>
	<li>选择 "协议信号测试" 按钮, 以确定背景相对光单位 (RLUs) 和荧光方差在板上。将弹出一个新窗口。选择 "测试信号"。 注意: 在这一步中, 背景 RLUs 是由 iccd/cop 相机决定的, 该摄像机集成在荧光读取器器件的光学隔间中。这些 RLUs 起因于 Ca2 +敏感染料 (fluo-2) 通过发光二极管 (led 输出波长 470-495 nm) 的激发。800万 RLUs 的值非常适合此应用程序。如有必要, RLUs 可以通过改变激发强度 (选择激动强度) 或相机门 (选择门打开) 来适应。板上的方差最好小于7.5%。</li>
	<li>如果获得 800万 RLUs 的背景值, 请选择 "更新"。通过单击 "保存" 按钮保存主协议。 注意: 通过这样做, 协议信号测试的设置将被执行到主协议中。</li>
	<li>通过推 "运行" 按钮来运行检测。</li>
</ol>7. 数据分析和质量
<ol><li>检测完毕后, 在系统软件中打开 "分析" 框。<ol>
<li>转到 "配置更正" 并选择 "在基线上响应"。定义基准线 1, 从测量1开始, 在测量5结束;平均荧光将计算在每一个井之间的测量1和5。 注: 从某一井的所有进一步测量被划分由这个明确的基准线1。<ol>
<li>定义基线 2, 从测量78开始, 直到测量 83 (CXCL12 在测量83之后被配发; 再次在测量78和83之间的每个井中计算平均值荧光, 并将此校正因子应用于以下所有测量测量 83)。</li>
		</ol>
</li>
		<li>激活刻度框 "显示为百分比" 和 "减去背景"。</li>
		<li>转到 "配置动能还原"。要评估化合物对 CXCL12-induced Ca2 +响应的抑制作用, 请选择从测量84开始的最大分钟数 (即, 将 CXCL12 的第一个度量值分配给 243) (即, 最终测量)。 注意: 通过选择最大极小值, 报告了测量84和测量243之间基线的最小和最大响应之间的差异。如果从测量11和测量78之间开始选择最大最小值, 则报告相对于基线1的最小和最大响应之间的差异。此值可用于分析化合物的潜在竞赛活动, 请参见讨论)。</li>
		<li>数据将被可视化在系统的软件中。如果需要, 将原始数据导出到其他通用分析软件包中的其他分析和可视化。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

本文所述的 Ca2 +-动员检测以前被证明是一种有价值的工具, 用于识别和描述针对 CXCR417的受体拮抗剂。但是, 预计此方法可以更广泛地应用于其他受体的大组, 在激活后触发胞浆 Ca2 +释放, 如相关的趋化因子受体 CCR5 所示。而在 CCR5 的情况下, 可以应用相同的实验条件, 协议中的几个步骤 (例如, 在电镀时的细胞数, 与荧光染料的细胞的加载) 需要重新优化, 然后才将化验结果转移到其他受体, 以获得良好的信噪比。这里的一个重要问题是选择合适的体外蜂窝主机, 允许 GPCR 的高级别表达式和功能耦合到 Ca2 +通路。在 CXCR4 的情况下, 人类胶质瘤 U87 细胞的选择是因为: (1) 缺乏内源性表达的 CXCR7 (一个相关的趋化因子受体与 CXCR4), (2) 他们的能力, 以 CXCR4 激活 Ca2 +-信号通路, (3) 通过 Ca2 +敏感染料, 在电池加载后获得的荧光响应的有利动态范围, (4) 它们极好地粘附在检测板上, 最大限度地减少了整个过程中的细胞紊乱。所有这些参数可能需要实验性地确定每一个新的 GPCR 的兴趣和每个细胞表达系统。
在为另一 GPCR 建立类似的检测方法时, 可以考虑在该协议中的几种替代步骤或试剂。例如, 其他 Ca2 +敏感显影 (例如、fluo-4、fluo-3) 可能用作此协议中使用的荧光 (fluo-2) 的替代方法。在松散黏附细胞的情况下, 通过引入无洗涤染料来最小化细胞逐出24, 可以省去对细胞的洗涤。此外, 排除该协议的洗涤步骤将进一步增加化验的吞吐量。虽然这种检测也可能与显示 CXCR4 内源表达的细胞一起执行, 例如, 包括几种类型的人类癌细胞系10,25, 应注意的是, 稳定转染的细胞线高 GPCR可以获得在一段时间内保持稳定的表达式级别。这将导致更明显的化验测量, 一个更大的窗口, 以可靠的数据解释, 并一致的检测性能较长的时间。当使用细胞内在表达 CXCR4 时, 要达到这个效果将会困难得多。
该检测的一个主要缺点是它依赖于荧光测量。因此, autofluorescent 化合物可以干扰该检测的测量, 因为不可靠的数据解释而排除了它们的分析。此外, 此 Ca2 +动员分析是理想的执行使用一个细胞筛选系统配备了集成的吹打系统, 允许标准化混合和 dispension 的化合物和受体激动剂进入测量板中的所有井同时。然而, 该化验可以适应与其他, 较便宜的荧光微板块阅读器与动能测量能力。添加化合物和激动剂, 然后需要使用多通道吸管手动执行, 这将增加动手时间和降低化验的吞吐量。此外, 在某一特定时间间隔内获得类似数量的荧光测量, 将很难实现, 因为许多微板块的读者都很好地测量信号, 而高端筛分系统测量所有油井同时.
受体拮抗剂, 防止绑定到受体和随后激活的内源性激动药 (CXCL12) 目前形成的主要类别的化合物专门针对 CXCR411,12。AMD3100 是用来说明 Ca2 +动员试验性能的小分子, 是 CXCR4 拮抗剂26的最突出的例子之一。除了受体拮抗剂外, 调节 GPCR 信号的分子也会引起一般的兴趣。这些分子包括反激动剂, 以及 GPCR PAMs 和 NAMs19,20,27。这篇手稿中描述的一个有趣的特点是, 它不仅能识别受体拮抗剂, 而且还能构调节器。然而, 必须考虑到对化验和限制的一些细微的适应。
PAMs 和 NAMs 绑定到受体部位地形不同于 orthosteric 结合部位所占据的受体的内源性配体。而 PAMs 增强受体内源配体的效力和/或功效, NAMs 抑制内源配体的效力和/或功效 (s)19,20。PAMs 没有内在的激动剂活动。它们只是活跃在内源性激动剂的存在, 不像所谓的构激动剂。因为构 GPCR 调制器在临床设置中应用时会引起比传统受体拮抗剂更少的副作用28,29, 它们是宝贵的药理工具。在我们的化验中, 构激动剂会在添加到 CXCR4+单元格后立即引发荧光信号的瞬时增加。该信号的受体特异性, 然后应通过测试相同的化合物相同的化验, 但在缺乏 CXCR4 的细胞。在具体筛选 PAMs 时, 应使用较低浓度的内源性激动剂来诱导荧光 Ca2 +反应。虽然这种较低量的激动剂将产生一个较小的荧光信号, 它留下一个较大的窗口, 以检测额外的受体刺激。通常, 与 ec10-欧共体30的受体刺激值对应的激动剂浓度被选择30,31。在化验的第一部分添加 PAM 不会导致荧光测量的增加。然而, 荧光 Ca2 +信号后, 随后刺激受体与其内源性激动剂将增加。同样, 在激动剂添加前, 不结盟运动不会改变荧光测量, 但在加入激动剂后抑制受体的反应。因此, 受体拮抗剂和不结盟运动的活动剖面看起来也类似。他们都将导致抑制的荧光反应后激动剂添加。因此, 需要进一步的实验研究来区分拮抗剂和不结盟运动。在这里, 受体结合研究, 即未标记化合物竞争与固定数量的标记 CXCL12 绑定在 CXCR417,32可能是一个宝贵的战略, 以区分拮抗剂, 这将防止标记激动剂从绑定到受体, 和不结盟运动, 不会因为它不会共享相同的受体结合网站。
受体的配体独立 (或基底或本构) 活性已观察到许多受体33 , 但当受体表达 recombinantly27时, 基本活动的水平通常相当低。基本的 GPCR 活动可以通过自然发生的突变33和一个点突变导致增加的基础 CXCR4 活动被描述了早先34。通过将此组成性活性 CXCR4 突变体与酵母中的信息素反应通路和 [35] GTPγS 结合实验相耦合, 证明 T140 是一种以肽为基础的 CXCR4 拮抗剂, 具有有效的抗 HIV 活性 17, 35显著减少了这一基本活动, 因此被证明表现为反向激动剂34。值得注意的是, 在基于 Fura-2 荧光的 ca2 +动员实验中, 同一作者观察到, 在 ca2 +相关荧光中添加 T140 到表达突变体 CXCR4 的细胞中的小瞬时减少, 表明基底受体活动34。然而, 当分析一个由 T140 设计的循环五肽 CXCR4 反激动剂的附加面板时, 用 Ca2 + 动员试验检测减少的基底活动是不太直接的 36.当用本手稿中描述的 Ca2 +动员试验分析 T140 对表达野生型 CXCR4 细胞的影响时, 未观察到基底荧光信号的变化17。此外, 荧光的波动是快速和瞬变的, 并增加基 Ca2 +是, 例如, 没有观察到表达组成性活性 G αq 耦合受体4的细胞.两者相结合, 反激动剂的作用将是非常困难的, 如果不是不可能的识别和评估可靠的我们的化验。

受体是细胞表面蛋白的重要超家族, 激活信号转导级联后细胞外配体结合。它们可以由多种刺激激活, 包括肽, 蛋白质荷尔蒙, 生物胺和脂质, 这导致各种细胞内信号通路的启动和最终的生物反应1,2.此外, 受体参与了许多 (如果不是所有的) 发育和生理过程, 许多人类疾病与功能失调的 GPCR 信号或受体过度表达有关。因此, 受体是医学中最有效的药理靶点之一1,3。
通常情况下, GPCR 药物发现工作流程开始于细胞筛选化验, 从而能够识别小分子、单克隆抗体和能调节特定 GPCR 活动的肽等化合物。在 GPCR 药物发现, 有许多不同类型的化验, 以寻找这种化合物, 其中大部分是兼容中-高通量筛选运动。最常用的化验方法包括受体结合实验、荧光或发光测定法检测所谓二次信使水平的波动 (例如、Ca2 +、循环腺苷磷酸 (AMP))、表型筛选化验和β-arrestin 招聘化验4。对某一特定类型的化验的选择可能取决于多种因素, 但也由先验知识决定给定 GPCR 的信令性质。激动剂结合到一个 GPCR 诱导构象变化催化磷酸胍 (GDP) 的交换磷酸胍 (GTP) 在α亚基的 heterotrimeric G 蛋白。随后, gα-GTP 亚基离解从 gβγ亚基和两个亚基将启动进一步的信号通路。水解的 GTP 分子和随后重新联合的 gα-GDP 和 Gβγ亚基将恢复 G 蛋白进入其非活动构象5,6。基于 gα亚基的序列相似性定义了不同类型的 g 蛋白 (gs, gi, Gq, g12/13)7。通过 gα亚基发出信号会产生几个典型的响应, 例如增加 (通过 gs) 或减少 (通过 gi) 循环放大器生产和胞内 Ca2 +动员 (通过 Gq)5 ,7。Gβγ子单元也能够诱导细胞内效应通路。例如, 在激活 gi耦合的受体后, gβγ可以直接刺激磷脂酶 C (PLC β) 产生三磷酸肌醇 (IP3), 触发 Ca2 +从胞内存储7 的释放.继受体活化后, 受体通过 GPCR 激酶 (GRKs) 磷酸化, 促进与β arrestins 的相互作用。这一过程终止 G 蛋白信号, 导致受体脱敏和最终内化。β-arrestins 也能够形成多分子复合物, 可以触发其他信号通路独立的 G 蛋白信号8。
在趋化因子受体的亚家族中, Gi耦合 CXCR4 是一种 GPCR, 它引起了许多人的兴趣, 作为药物发现的一个有希望的目标。鉴于其作为人体免疫机能丧失病毒 1 (HIV-1) 病毒进入和感染的主要共同受体的作用, 最初将 CXCR4 作为抗艾滋病毒药物候选者, 被开发成了9。最近, 越来越多的证据表明, CXCR4 在肿瘤发生和肿瘤转移中的重要作用, 使其成为肿瘤治疗的有效靶点, 也是10。CXCR4 在二十多个类型的人类癌症中得到高度表达, 并控制肿瘤细胞的存活、增殖和迁移, 以及肿瘤相关的血管生成10。不同化学类的 CXCR4 拮抗剂以前被描述为11、12, 但目前只有小分子 AMD3100 被批准用于临床中作为淋巴瘤治疗中使用的干细胞动员剂。和骨髓瘤患者13,14。目前正在进行临床试验, 以评估其他几种 CXCR4 拮抗剂在不同的人类疾病中的安全性和有效性, 但重点是肿瘤学12。鉴于许多潜在的应用 CXCR4 拮抗剂, 寻找新的化合物具有改进的药代动力学性质, 改善生物利用度, 或潜在较少的副作用是必要的。
本文介绍了一种基于动态荧光的细胞检测方法, 主要用于筛查能抑制 CXCR4 的化合物。这种方法的荧光测量是基于胞内 Ca2 +浓度在 CXCR4 活化作用下的瞬态增加, 即趋化因子配体 CXCL12 (以前称为基质细胞衍生因子 1α (SDF1-α)), 并可能抑制此 CXCL12-induced Ca2 +响应的特定化合物。本试验采用 U87 人胶质瘤细胞稳定表达人 CXCR4 受体的方法。同时, 这些细胞缺乏内源性表达的 CXCR7, 一个相关的趋化因子受体, 也绑定 CXCL1215,16,17。CXCR7 以前也被证明能够与 CXCR4 形成 heterodimers, 从而调节后一种受体18的信令特性。通过将 CXCR4+单元格与 fluo-2 乙酰甲基 (AM) 酯 (CXCR4)、细胞渗透性的高亲和性荧光 Ca2+ 结合染料一起加载, 监视由由由由被测者介导的胞内 Ca2 + 级的波动。Fluo-2 AM 是一种单波长荧光分子, 可在 490 nm 激发, 而其发射荧光量为 520 nm。此发射荧光在 ca2 +绑定上增加, 在 ca2 +绑定和未绑定状态之间具有较大的动态范围。荧光信号的增加是瞬态的, 在几分钟的时间间隔内发生, 之后会衰减。荧光发射的峰值高度进一步与受体活化水平有关。该检测本身是使用一个荧光微板块读取器, 配备了增强 CCD (iccd/cop) 相机, 拥有一个集成的吹打系统, 允许标准化的吹打步骤在化验 (见材料表).此外, 在微板块的所有井中同时测量荧光信号也是使用荧光读取器的另一个关键优点。在分析的第一部分中, 被调查的化合物 (例如, 在固定浓度或稀释系列中的小分子小组) 被添加到 fluo-2 加载的 CXCR4+ U87 细胞中, 随后是10分钟的孵化期。在此过程中, 化合物的潜在竞赛效应不断地实时测量。然后, 将内源性激动剂 (即, CXCL12) 添加到细胞中, 以唤起胞内 Ca2 +的 CXCR4-mediated 瞬时增加。在这部分的分析中, 可以评估测试化合物的潜在拮抗活性。在图 1中介绍了该检测的一般工作流的示意图。
尽管此 Ca2 +动员分析主要用于确定和确定竞争性 CXCR4 拮抗剂 (即) 的抑菌效力, 但它也可以识别受体激动剂, 此外, 通过结合在构站点 (即, 地形不同于内源性激动剂所占的 orthosteric 结合部位) 发挥作用的化合物。后一类化合物的例子包括构激动剂和 PAMs 和 NAMs19,20。虽然受体特定的拮抗剂以及 NAMs 会抑制 CXCL12-induced Ca2 +响应, 但 PAMs 会增强此响应 (请参见讨论部分)。虽然此处描述的检测具体针对 CXCR4, 但预计此方法可应用于其他受体, 且最小的优化工作, 至少如果它们通过释放胞内 Ca2 +发出信号。

<table><tbody><tr><td>Fluo-2 AM</td><td>Abcam</td><td>ab142775</td><td>fluorescent Ca2+ sensitive dye</td></tr><tr><td>Pluronic F-127</td><td>Sigma</td><td>P2443-250G</td><td>pluronic acid</td></tr><tr><td>Gelatin</td><td>Sigma</td><td>G9391</td><td></td></tr><tr><td>AMD3100</td><td>Sigma</td><td>A5602-5 mg</td><td>specific CXCR4 antagonist</td></tr><tr><td>Maraviroc</td><td>kind gift of AnorMed</td><td></td><td>antiretroviral drug, CCR5 antagonist</td></tr><tr><td>Chemokine ligand CXCL12</td><td>PeproTech</td><td>300-28A</td><td></td></tr><tr><td>Chemokine ligand CCL5</td><td>PeproTech</td><td>300-06</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco (Life Technologies)</td><td>10270-106</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A1933-25G</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Gibco (Life Technologies)</td><td>41965-039</td><td></td></tr><tr><td>HBSS (10 x), calcium, magnesium, no phenol red</td><td>Gibco (Life Technologies)</td><td>14065-049</td><td></td></tr><tr><td>HEPES (1 M)</td><td>Gibco (Life Technologies)</td><td>15630-056</td><td></td></tr><tr><td>Trypsin-EDTA (0,25 %), phenol red</td><td>Gibco (Life Technologies)</td><td>25200-056</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (DPBS)</td><td>Gibco (Life Technologies)</td><td>14190-094</td><td></td></tr><tr><td>Falcon tubes, 50 mL</td><td>Greiner Bio-One</td><td>227 261</td><td></td></tr><tr><td>Tissue culture flask (T75)</td><td>Corning</td><td>353024</td><td></td></tr><tr><td>Black plate, 96-well, clear bottom, with lid</td><td>Costar/Fisher Scientific</td><td>10530753</td><td>assay plate (96-well), for cell seeding</td></tr><tr><td>Polypropylene (PP) plates</td><td>Thermo Scientific (VWR)</td><td>732-2661</td><td>plates used to prepare the compound plates and chemokine plates, round bottom</td></tr><tr><td>FLIPR Tetra high throughput cellular screening system</td><td>Molecular Devices</td><td></td><td>Fluorescent plate reader with integrated pipettor head and ICCD camera</td></tr><tr><td>FLIPR Tetra LED Module 470 - 495 nm</td><td>Molecular Devices</td><td>0200-6128</td><td>Light emitting diodes for excitation of the fluorescent Ca2+ sensitive dye</td></tr><tr><td>FLIPR Tetra Emission Filter 515 - 575 nm</td><td>Molecular Devices</td><td>0200-6203</td><td>emission filter compatible with the fluorescent dye</td></tr><tr><td>FLIPR Tetra 96 Head</td><td>Molecular Devices</td><td>0310-4536</td><td>96-well pipettor head, integrated within the fluorescent plate reader</td></tr><tr><td>ScreenWorks</td><td>Molecular Devices</td><td></td><td>software package used for data analysis and visualization on the FLIPR Tetra</td></tr><tr><td>Vi-CELL</td><td>Beckman Coulter</td><td></td><td>cell viability analyzer</td></tr><tr><td>Corning CellBIND 96 Well Flat Clear Bottom Black Polystyrene Microplates, with Lid, Sterile</td><td>Corning</td><td>3340</td><td>Pre-coated 96-well assay plates that may represent an alternative for manual coating of the assay plate.</td></tr></tbody></table>

a kinetic fluorescence-based ca2+ mobilization assay to identify g protein-coupled receptor agonists, antagonists, and allosteric modulators

G 蛋白偶联受体 (受体) 对于制药业来说是非常重要的, 因为它们涉及许多人类疾病, 包括良好的治疗干预目标。发现铅化合物, 包括小的合成分子, 特别抑制受体的功能, 是药物开发的一个重要的初步步骤, 依赖于敏感的, 特定的, 和强健的细胞检测方法。在这里, 我们描述了一个荧光读数的动态细胞分析, 主要用于识别受体特异拮抗剂, 抑制细胞内 Ca2 +释放诱发后, 激活的 CXC 趋化因子受体 4 (CXCR4) 由其内源配体, CXC 趋化因子配体 12 (CXCL12)。该方法的一个主要优点是, 它还能筛选出具有内在竞赛特性的化合物 (即, 在没有 CXCL12 的情况下, 化合物诱导胞内 Ca2 +浓度增加) 或化合物通过与构绑定站点 (即、正负构调制器 (PAMs 和 NAMs) 的交互来调节受体的功能。在下降的一侧, autofluorescent 化合物可能干扰检测的读数, 从而妨碍可靠的数据解释。最有可能的是, 这种分析可以实施, 以最小的适应, 作为一个通用的药物发现化验的许多其他受体, 其中激活导致释放胞内 Ca2 +。

CXCL12 刺激对 U87 中胞内 Ca2 +动员的影响。CD4. CXCR4+和 U87。用 Ca2 +动员试验评估 CD4 细胞。在协议的第一个吹打步骤 (图 1) 中通常会添加20µL 测试复合, 而不是将检测缓冲区添加到 fluo-2 加载的 U87 中。CD4. CXCR4 在测量板中的+单元格。在第二个配药步骤, 不同浓度的 CXCL12 (0.2-102.4 nM, 最终浓度) 被免除在测量板。通过剂量依赖性增加的荧光, 与剂量相关的增加释放 Ca2 +, 演示 (图 2A)。在这里, 负控制样本代表的是那些在两个增加仅检测缓冲区添加到测量板 (即, 0 nM CXCL12) 的油井, 导致没有响应 (图 2A)。随着时间的推移, CXCL12 的增加会导致荧光量的增加 (图 2A)。从这些数据中, 根据测量 84 (即、CXCL12 添加后的第一个测量) 和测量 243 (i. e) 之间的 "最大最小响应基线" 生成剂量响应曲线, 并在协议的最后测量中).使用非线性回归, EC50值 (即, 唤起半最大响应所需的浓度) 被确定, 对应于 1.14 nM (图 2B)。没有荧光 Ca2 +相关的反应是由 CXCL12, 即使在高浓度 (102.4 nM), 当 U87。CD4 细胞缺乏功能 CXCR4 表达的使用。这说明了 CXCL12 (图 2C) 诱发的测量的受体特异性。
这种基于细胞的检测的一个关键应用是识别专门针对 CXCR4 的小分子, 并能够抑制 CXCL12-induced Ca2 +的动员。如果发现活性化合物 ("命中"), 则可以通过测试化合物的串联稀释和更详细地分析抑制效力来进一步表征。举例来说, bicyclam AMD3100, 一个建立良好的小分子 CXCR4-specific 受体拮抗剂与有效的 anti-HIV-1 活动21,22的效果, 在图 3中说明。AMD3100 的浓度系列 (n = 4, 3 µM 到 1.37 nM 最终浓度在1/3 稀释系列) 被免除 U87。在协议的第一步期间, CD4. CXCR4+单元格。然后, 该化合物被允许孵化的细胞为〜10分钟, 其中荧光信号连续记录。然后将 6.4 nM 的 CXCL12 (50 ng/毫升, 最终浓度) 添加到细胞板块的所有井中同时诱导 CXCR4-mediated Ca2 +动员。AMD3100 的不同稀释对此响应的剂量依赖性抑制在图 3A中显示。在这种情况下, 只显示 CXCL12 添加后的图形部分。作为阴性控制 (蓝线) 仅缓冲被应用了在试验 (没有预先孵化与 AMD3100, 没有 CXCL12 增加到井), 导致期望缺乏反应。正则控制 (红线) 对应于在没有预先孵化 AMD3100 (图 3A) 的情况下, 将 6.4 nM CXCL12 添加到水井中的样本。基于这些定义的负和正控制, 此检测中的 Z 值通常介于0.5 和1之间。为了确定 AMD3100 的抑制效力, 通过非线性回归产生了剂量响应曲线, 结果成了计算出的 IC50值 770.8 nM (图 3B)。为了进一步说明非活性化合物的行为, 本实验包括 maraviroc。Maraviroc 是一个小分子, 专门抑制 CC 趋化因子受体 5 (CCR5) 从而阻断 (CCR5)-热带 HIV-1 感染23 。即使在高浓度 (10 µM 最终浓度), 在 CXCR4-mediated Ca2 +响应 (图 3A) 上未观察到此化合物的抑制效果。
最后, 为了证明此 Ca2 +动员分析也可以应用于研究其他受体, 在表达 CCR5 的细胞中加入了 CC 趋化因子配体 5 (CCL5) 的序列稀释, CCR5 的内源配体。CD4. CCR5+) 使用完全相同的实验条件和硬件设置。在添加 CCL5 的串行稀释 (图 3C) 之后, 将显示与剂量相关的荧光 Ca2 +响应。此外, 与其对 CXCR4 的影响相比, maraviroc 100 nM (最终浓度) 的预孵化强烈抑制了此响应 (图 3D)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56780/56780fig1v2.jpg"> 图 1: 检测工作流的示意图概述.日0细胞表达 GPCR 的兴趣 (在这种情况下 CXCR4) 是种子在黑壁96井板, 有明确的底部, 并在37摄氏度和 5% CO2一夜之间增长。1天, 执行基于荧光的 Ca2 +检测。单元格首先加载一个荧光 Ca2 +敏感染料 (fluo-2 AM) 和孵化45分钟在 RT 在黑暗中。制备了 "趋化因子板" 和 "复合板" (PP = 聚丙烯)。在加载染料孵化后, 种子细胞用检测缓冲剂 (150 µL/井) 冲洗一次, 然后添加80µL/井。在开始化验之前, 所有的盘子都在37摄氏度的设备中孵化5分钟。然后, 感兴趣的化合物 (例如, 小分子) 被分配到所需浓度的测量板的水井, 并允许孵化10分钟, 而荧光连续测量。接下来, GPCR (此处为 CXCL12) 的内源性激动剂的固定浓度将被添加以唤起 Ca2 +的释放, 并随着时间的推移进一步记录荧光。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56780/56780fig1v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56780/56780fig2v2.jpg"> 图 2: CXCL12 对 CXCR4+单元格的激动剂活动和检测到的响应的特异性.(A) CXCL12 对 U87 中的 Ca2 +动员的剂量依赖性影响。CD4. CXCR4+单元格。(B) 基于度量值84和243之间基线的最大最小响应, 生成了剂量响应曲线, 并计算了 EC50值 (n = 4, 平均值 @ SD)。(C) CXCL12 在 U87 上被免除时, 不会引起任何反应。CD4 细胞缺乏 CXCR4。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56780/56780fig2v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56780/56780fig3v2.jpg"> 图 3: CXCR4 拮抗剂 (AMD3100) 和非活性化合物 (maraviroc) 对 CXCR4+细胞的影响及其内源性激动物 CCR5 激活 CCL5 的图示.(A) AMD3100 对 Ca2 +动员的剂量依赖性抑制效应, 通过将 6.4 nM CXCL12 添加到 U87。CD4. CXCR4+单元格。Maraviroc (10 µM 最终浓度) 对 CXCL12-induced Ca2 +响应没有显示任何抑制效果。(B) 基于测量84和243之间基线的最大最小响应, 生成了一个抑制剂量响应曲线, 而 IC50值被计算为 770.8 nM (n = 4, 平均值 * SD)。(C) 由其内源性激动剂 CCL5 的剂量依赖性活化 CCR5。(D) 通过对具有 100 nM maraviroc 的细胞进行预孵化, 抑制 CCR5-mediated Ca2 +响应。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56780/56780fig3v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators at JoVE.com

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

所描述的细胞检测是为鉴定 CXC 趋化因子受体 4 (CXCR4)-相互作用的代理, 抑制或刺激, 无论是竞争性或 allosterically, 细胞内 Ca2 +释放启动的 CXCR4 激活。

一种基于动力学荧光的 Ca2 +动员法, 用于识别 G 蛋白偶联受体激动剂、拮抗器和构调制器

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include ...

a-kinetic-fluorescence-based-ca2-mobilization-assay-to-identify-g

Universidad San Sebastian

Research

JoVE Journal

Medicine

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

8.8K 次观看.  KU Leuven. 所描述的细胞检测是为鉴定 CXC 趋化因子受体 4 (CXCR4)-相互作用的代理, 抑制或刺激, 无论是竞争性或 allosterically, 细胞内 Ca2 +释放启动的 CXCR4 激活。

视频: A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

基于细胞中钙含量TRP通道功能的高通量筛选，使用FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

科研

生物工程

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a major pharmacological target for multiple indications, including analgesia, blood pressure regulation, and the treatment of psychiatric disease. Upon ligand binding, GPCRs catalyze the activation of intracellular G-proteins by stimulating the incorporation of guanosine triphosphate (GTP). Activated G-proteins then stimulate signaling pathways that elicit cellular responses. GPCR signaling can be monitored by measuring the incorporation of a radiolabeled and non-hydrolyzable form of GTP, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTP&#947;S binding measures a proximal event in GPCR signaling and, importantly, can distinguish agonists, antagonists, and inverse agonists. The present protocol outlines a sensitive and specific method for studying GPCR signaling using crude membrane preparations of an archetypal GPCR, the &#181;-opioid receptor (MOR1). Although alternative approaches to fractionate cells and tissues exist, many are cost-prohibitive, tedious, and/or require non-standard laboratory equipment. The present method provides a simple procedure that enriches functional crude membranes. After isolating MOR1, various pharmacological properties of its agonist, [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO), and antagonist, naloxone, were determined.

Guanosine triphosphate (GTP) binding is one of the earliest events in G-Protein-Coupled Receptor (GPCR) activation. This protocol describes how to pharmacologically characterize specific GPCR-ligand interactions by monitoring the binding of the radio-labeled GTP analog, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), in response to a ligand of interest.

通过放射性标记的GTP结合测量G蛋白偶联的受体信号

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a ...

生物化学

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

一个钙生物发光检测蚊虫的功能分析（埃及伊蚊）和刻度（镰形扇头microplus）G蛋白偶联受体

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

免疫与感染

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

使用 PRESTO-Tango Assay 在 GPCR-宽范围范围内对 β-Arrestin2 进行配制筛查的平行审讯

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

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

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

<meta charset="utf8"></head><body>研究内皮细胞中的蛋白酶激活受体配体

Characterizing Modulators of Protease-Activated Receptors with a Calcium Mobilization Assay Using a Plate Reader

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding dyes and imaging instruments that can measure fluorescent emissions from up to 1,536 wells simultaneously. However, these instruments are much more expensive and can be challenging to maintain relative to widely available plate readers that scan wells individually. Described here is an optimized plate reader assay for use with an endothelial cell line (EA.hy926) to measure the protease-activated receptor (PAR)-driven activation of G&#945;q signaling and subsequent calcium mobilization using the calcium-binding dye Fluo-4. This assay has been used to characterize a range of PAR ligands, including the allosteric PAR1-targeting anti-inflammatory "parmodulin" ligands identified in the Dockendorff lab. This protocol obviates the need for an automated liquid handler and permits the medium-throughput screening of PAR ligands in 96-well plates and should be applicable to the study of other receptors that initiate calcium mobilization.

<meta charset="utf8"></head><body>作者聚焦：开发靶向蛋白酶激活受体的 Parmodulins 以控制炎症

An improved protocol for a calcium mobilization assay with endothelial cells, used to identify ligands of protease-activated receptors (PARs), has been developed. The new protocol reduces total assay time by 90-120 min and yields reproducible concentration-response curves.

使用读板器通过钙动员测定表征蛋白酶激活受体的调节剂

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

一个荧光识别GIRK通道调制器的筛选试验

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

生物学

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

G蛋白偶联受体的基于荧光的钙动员含量表征

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

一种基于动力学荧光的 Ca^{2 +}动员法, 用于识别 G 蛋白偶联受体激动剂、拮抗器和构调制器

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此视频中的章节

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一个钙生物发光检测蚊虫的功能分析（埃及伊蚊）和刻度（镰形扇头microplus）G蛋白偶联受体

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使用读板器通过钙动员测定表征蛋白酶激活受体的调节剂

使用读板器通过钙动员测定表征蛋白酶激活受体的调节剂

一个荧光识别GIRK通道调制器的筛选试验

G蛋白偶联受体的基于荧光的钙动员含量表征