Name: a rapid and quantitative fluorimetric method for protein-targeting small molecule drug screening
Uploaded: 2015-10-16T15:00:00.000+00:00
Duration: 8 min 34 s
Description: Watch this Scientific Journal Video about A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening at JoVE.com

Y.N.T. would like to acknowledge the Agency for Science, Technology and Research (A*STAR), Singapore for the financial support under the JCO CDA grant 13302FG063.

注意:使用前请咨询了所有相关化学品的安全数据表（SDS）。的药物筛选试验涉及纳米材料的合成和处理相比，他们散装对方也可以有额外的危害。整个实验过程中要实行请确保所有必要的管制措施，包括利用工程控制（通风柜）和个人防护装备（PPE， 例如 ，安全长裤，闭趾鞋，化学防护手套和安全眼罩）的。 1.前处理化学试剂用于药物筛选<ol><li>前体金NC的合成<ol><li>溶解30毫克的金（III），氯化溶液（99.99％的微量金属的基础上，30％（重量）在稀HCl中）在6.9 ml的超纯水中，制备金（III），氯化溶液15mM的。注意:氯化金溶液腐蚀性和刺激性。穿戴合适的个人防护装备，以避免与眼睛和皮肤直接接触。 </li><li>迪在1ml超纯水中ssolve 74毫克的HSA或BSA中，制备74毫克/毫升的蛋白储备溶液。 </li></ol></li><li>药物解决方案<ol><li>溶解药物的所需量，例如，1.9毫克（一）布洛芬，2.8毫克（二）华法林，2.3毫克（三）苯妥英，和1.5毫克（四）磺胺在20μlDMSO中以制备450毫药物储备溶液。 注意:二甲基亚砜被选择作为溶剂，因为这些药物是疏水性的，并具有在水中的溶解性差。 </li></ol></li><li>其他试剂<ol><li>溶解600毫克NaOH颗粒在10ml超纯水中，制备1.5 NaOH溶液微米。溶解2.4克尿素的2ml超纯水中，制备20的尿素溶液微米。 </li></ol></li></ol> 2.合成蛋白质模板化的Au纳米晶<ol><li> HSA为模板的Au纳米晶（HSA金NCS） <ol><li>放置两个玻璃小瓶，每个都包含在其中的微磁力搅拌棒，在两个独立的温度控制的磁力搅拌rers。 </li><li>设置一个磁搅拌器的温度升高到60℃，并标记在它上面的玻璃小瓶作为"60℃";而其他磁力搅拌器保持在室温（RT），其中在它的上面的玻璃小瓶被标记为"RT"。 </li><li>加入200微升的HSA溶液，加入200μl超纯水，和200微升金（III），氯化溶液，以在恒定搅拌下每个小瓶（除非另有规定设定为360转），以允许蛋白质模板中的Au离子的封装。 </li><li> 2分钟后，加入20微升NaOH溶液向每个小瓶，从而激活HSA的还原能力在形成的Au NCS和开始记录的反应时间为0分钟。 </li><li>每隔20分钟，吸取50微升溶液从每个样品至384孔黑色板，并测量用酶标仪的发射光谱。典型的扫描设置:λ= 前370，λEM = 410 - 850纳米。 </li><li> 100分钟后停止磁力搅拌器。 </li><li>降温下水槽自来水的玻璃小瓶。注意:玻璃瓶是热的。戴上耐热手套以避免燃烧。 </li><li>绘制光电子能谱在所有的时间对每个样品采集的Au纳米晶的形成动力学，在不同的温度条件。 </li></ol></li><li> BSA为模板的Au纳米晶（BSA金NCS） <ol><li>上放置装有磁力搅拌器的顶部的微磁搅拌棒的玻璃小瓶中。离开温度为室温。 </li><li>混合200微升BSA溶液，200微升的尿素，和200微升在不断搅拌下的玻璃小瓶金（III），氯化溶液。 </li><li> 2分钟后，加入20微升NaOH溶液来激活的BSA的还原能力，以形成金NCS和开始记录的反应时间为0分钟。测量反应混合物的光电发射光谱每小时。 注意:的BSA-Au纳米晶的光电发射光谱测量较不频繁，因为的BSA-Au NC的形成速度比HSA金在相同温度下的慢由于尿素少效力以展开蛋白质。 </li><li> 7小时后停止磁力搅拌器。绘制光电子能谱在所有的时间通过尿素变性看到金纳米晶形成动力学。 </li></ol></li></ol> 3.小分子药物筛选<ol><li>不同的小分子药物与HSA屏幕相对结合亲和力<ol><li>放置5的玻璃小瓶，每个包含在其磁力搅拌棒，在一个温度可控的多磁搅拌器的顶部。标注这些小瓶为a，b，c和分别对于每种药物，即布洛芬，华法令，苯妥英，和磺胺Ð。标签一个纯HSA作为对照。 </li><li>加入200μl的HSA向每个小瓶。加入1微升药液到四个相应的小瓶。加入1μlDMSO来控制。接通搅拌器并设置纺丝速度至360转，并培育1小时，以允许药物结合到HSA的完成。 </li><li> 1小时后，加入200微升Milli-Q水和200微升金（III），氯化溶液，以在恒定搅拌下的每个小瓶中。 10分钟设定在恒定搅拌下将温度至60℃。加入20微升1.5M的NaOH中的每个小瓶中，并开始记录反应时间为0分钟。 </li><li>快速绘制50微升从每个小瓶溶液至384孔黑色板和测量的发射光谱（结果标记为0分钟）。记录每个样品每10分钟的发射光谱。收集至少四个光谱在四个不同的时间，即是0，10，20，和30分钟。 </li><li>重复步骤3.1.1 - 3.1.4几次以获得具有一致的趋势的结果。停止磁力搅拌器。 </li><li>绘制每个样品的时间分辨光电子能谱（一 - d和控制）。确定所有样品和情节的峰值强度对抗中比较迪金纳米晶的形成动力学的时间fferent载药蛋白质的模板。 </li></ol></li><li>测量特定药物与HSA的结合常数<ol><li>重复步骤3.1.1 - 3.1.4。更换布洛芬解决方案的药液在四个不同的浓度（包括使用HSA只是没药量的对照样品）。根据药物浓度相应地标记该玻璃小瓶。 </li><li> 10分钟后，降温下水槽自来水的玻璃小瓶。注意:玻璃瓶是热的。戴上耐热手套以避免燃烧。 </li><li>吸取50μl的从每个小瓶上述解决方案的一个384孔黑色板和测量的发射光谱。 </li><li>重复步骤3.2.1 - 3.2.3两次以上，得到另一两组结果在不同的药物浓度。 </li><li>停止磁力搅拌器。画出光电子谱和分析结果。 <ol><li>对于每个单独的批次中获得的原始数据，绘制各光电子能谱在软件样本，如OriginPro软件，并找出峰强度。 </li><li>计算并绘制相对荧光强度[（I 0 - I）/ I（0）]对药物浓度，哪里I和I 0指的Au NC的形成在载药HSA和HSA的纯的荧光强度分别。 </li><li>计算的结合常数通过使用米氏方程的数据拟合到单点结合模型:Y = R 最大值 ×C /（K D + C）中，其中，R 最大值表示最大响应信号，C是配体的浓度和K D是结合常数。在软件中执行此如OriginPro。选择菜单分析拟合非线性曲线拟合，然后从生长/ S形类希尔功能。在设置:功能选择页上，单击适合显示拟合结果。 </li></ol></li></ol></li></ol>

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The authors have nothing to disclose.

有需要在该方法中，以被突出显示的几个关键步骤。在筛选不同的小分子药物的相对结合亲和力，对协议的步骤3.1.2，3.1.3，3.1.4和关键是要取得良好的结果，显示了相对结合强度相一致的趋势。在这些步骤中，测量添加化学品和绘图反应液的行动应尽快以减少时滞效应和测量添加化学品和绘图反应液应实行以确保一致性的相同的序列。在测量布洛芬与HSA的结合常数的协议，步骤3.2.4朝向杀敌测量的成功是非常重要的。虽然更多的集中点可以选择，由于更多的样本，测量时间滞后效应变得更加深刻。为了最大限度地减少时间滞后效应，合成不同浓度可以分成几个各色nt的批次并联。在每批，应该包括没有药物的控制，以确保一致性和消除可能的系统错误。 在此视频，HSA被选择作为模型蛋白直接荧光的Au NC的合成药物筛选。仅四种不同的药物， 即 （一）布洛芬（二）华法林，（三）苯妥英，和（d）磺胺这里测试。作为事实上，目前的方法是通用的，并且可以被修改成几种不同的形式。从理论上讲，这种方法可以适用于其他药物，小分子如类固醇，脂肪酸，甲状腺激素，和装订肽，提供这些分子可以结合HSA和提高蛋白质的稳定性对展开到不同的程度。其它蛋白，不限于白蛋白如HSA和BSA作为这里所示，也可以，只要它们是能够直接荧光的Au NC的合成用作药物筛选功能性模板（inclus香港专业教育学院活性官能团的金NC的形成，如酪氨酸残基），而不会影响药物的结合位点。即使是金属的选择是灵活的;其他金属的NC诸如Ag的NC也可以用作用于药物筛选的荧光探针。 总之，相比于如X射线晶体学和NMR研究蛋白质 - 药物相互作用等更复杂的技术当前的方法是简单，快速和成本效益。它不需要同位素标记，并允许直接荧光检测蛋白药物均匀的溶液结合。在当前的工作中，我们已经证明使用绑定，提高蛋白稳定性的药物的例子的概念。这也可能是可能延长目前的方法来筛选药物，破坏蛋白质在作为未来研究的一个有趣的话题结合。

血清白蛋白例如人血清白蛋白（HSA）和牛血清白蛋白（BSA）是在血浆中最丰富的蛋白，并在维持血液舱的渗透压发挥至关重要的作用。他们也被确认为载体蛋白为低水溶性的小分子，如类固醇，脂肪酸，甲状腺激素，以及各种各样的药物。的结合性（例如，结合位点，结合亲和力或强度）这些分子与血清白蛋白的形成在药动学的一个重要课题。1-4几种分析方法已经开发，研究不同的药物的结合性质的血清白蛋白，如X射线晶体学，5,6-核磁共振（NMR），7-11和表面等离子体共振（SPR），12,13等然而，这些方法是通过任一个繁琐和耗时的分析处理（ 例如受限的，单晶的X-射线crystallo生长图形研究），专业和昂贵的设备要求（SPR），或需要昂贵的同位素标记（NMR）进行检测。因此，非常需要开发为小分子药物筛选的替代方法以快速，直接的，并且成本效益的方式。 金纳米簇金（Au NCS）是一种特殊类型的纳米材料，其包含几个到几十个金属原子的有尺寸小于2纳米。14-17他们已经吸引了广泛的研究兴趣较小的由于它们的离散和尺寸相关的电子结构，18个， 19和分子状吸收和排放。20-23这种独特的材料性质，特别是强的荧光，已经发现不同的应用，如感测和成像的生物系统。24-32超小型荧光灯的Au NC的可使用的功能的蛋白质合成，如血清白蛋白，如模板33在一个典型的蛋白质为模板合成<html>的Au NC的，一定量的Au盐的被封装的蛋白质内，随后通过蛋白质本身降低。该蛋白质的还原能力是由于构成功能的氨基酸残基（例如，酪氨酸），可以通过增加溶液的pH值至碱性激活。解折叠蛋白质结构被认为是对金NC的形成中的关键步骤。这是因为在未折叠蛋白，多种还原剂官能团可以暴露于封装的金盐。蛋白质解折叠，可以通过热处理或暴露于变性剂来实现的。小分子药物的引入也可影响展开的过程，也就是修改中点变性温度和展开的焓。34,35所有这些因素的影响，又可以通过荧光的Au纳米晶的形成动力学得到反映并表现在所得的Au NC的荧光强度。36 e_content"&gt;此视频通过在更高的温度（60℃）在载有药物的白蛋白的蛋白质合成的Au纳米晶或在变性剂（如尿素）所得的Au NC的的荧光强度的存在表明药物筛选的方法是信号读出。首先，金的NC被合成的温度为60℃或在尿素的存在下处理的HSA和BSA的模板，以展示如何蛋白解折叠（通过热处理或变性剂诱导的）影响的Au纳米晶二的形成动力学，的Au NC的合成蛋白质的模板预装不同的药物，及对所得的Au NC的相对荧光强度的载药效果进行了研究，它们提供了相对结合强度的量度。最后，在Au NC-药物筛选协议被修改为药物-蛋白结合常数（K D）的通过改变预装在固定浓度的蛋白质的药物含量定量测定。

<table><tbody><tr><td>Gold (III) chloride solution, 30%</td><td>Sigma-Aldrich</td><td>484385</td><td>Corrosive, irritant</td></tr><tr><td>Human serum albumin, 96%</td><td>Sigma-Aldrich</td><td>A1887</td><td></td></tr><tr><td>Bovine Serum albumin, 96%</td><td>Sigma-Aldrich</td><td>A2153</td><td></td></tr><tr><td>Ibuprofen, 98%</td><td>Sigma-Aldrich</td><td>I4883&amp;nbsp;</td><td></td></tr><tr><td>warfarin, 98%</td><td>Sigma-Aldrich</td><td>A2250</td><td></td></tr><tr><td>phenytoin</td><td>Sigma-Aldrich</td><td>PHR1139</td><td></td></tr><tr><td>sulphanilamide, 99%</td><td>Sigma-Aldrich</td><td>S9251</td><td></td></tr><tr><td>dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>urea</td><td>Sigma-Aldrich</td><td>U5128</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>221465</td><td></td></tr><tr><td>Magnetic stirrer</td><td>IKA</td><td>RT5</td><td></td></tr><tr><td>Microplate reader</td><td>Tecan</td><td>Infinite M200</td><td></td></tr><tr><td>384-well plate</td><td>Corning</td><td></td><td></td></tr><tr><td>5 ml air displacement pipette</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>1,000 &amp;#956;l air displacement pipette</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>100 &amp;#956;l air displacement pipette</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>5,000 &amp;#956;l Eppendorf tips</td><td></td><td></td><td></td></tr><tr><td>1,000 &amp;#956;l Eppendorf tips</td><td></td><td></td><td></td></tr><tr><td>100 &amp;#956;l Eppendorf tips</td><td></td><td></td><td></td></tr><tr><td>1.5 ml micro tube</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>20 ml glass vial with screw cap</td><td></td><td></td><td></td></tr><tr><td>4 ml glass vial with screw cap</td><td></td><td></td><td></td></tr></tbody></table>

a rapid and quantitative fluorimetric method for protein-targeting small molecule drug screening

我们表明，用于确定小药物分子的结合亲和力与靶蛋白通过形成荧光金纳米簇金（Au NCS）加载药物的蛋白质内，基于由在Au纳米晶发射的差分荧光信号一个新的药物筛选方法。白蛋白蛋白质如人血清白蛋白（HSA）和牛血清白蛋白（BSA）被选择作为模型蛋白质。四个小分子药物 （如布洛芬，华法令，苯妥英，和磺胺）不同的结合亲和力到白蛋白的蛋白质进行测试。已经发现，荧光金NC的药物加载白蛋白蛋白质内的变性条件（即60℃或在尿素的存在下）下的形成速度比形成在原始蛋白（不含药物）慢。而且，这样形成的NC的荧光强度被发现负相关对这些药物的结合亲和力到白蛋白的蛋白质。特别是，越高药物 - 蛋​​白结合亲和性，较慢的凹NC的形成速率，并因此所得的Au NC的低级荧光强度观察。所得的Au NC的荧光强度因此提供测试了不同药物的相对结合强度的简单量度。这种方法也可延伸通过简单地改变在固定的蛋白质浓度预装在蛋白质的药物含量测定的具体药物-蛋白结合常数（K D）。测量结果与使用其它声望，而是更复杂的方法获得的值相匹配良好。

蛋白折叠为蛋白质为模板的金NC的形成的重要过程，因为蛋白质的多反应性官能团（例如，酪氨酸残基）可以暴露于降低封装的金离子，从而加速的Au纳米晶的形成速率。加热和外部变性剂是促进蛋白质折叠过程中的两个共同的装置。 图1展示了加热的影响，并增加外部变性剂对Au NC的形成动力学，使用HSA作为模型蛋白质。加热对Au NC的形成动力学的影响，首先，通过测量光致发光光谱的时间过程变化（图1A和1B）的影响。当反应是在60℃，红色发光的Au NC的迅速形成在时间（100分钟）的短周期，证实了明显的峰值中心的光电子能谱33,36为670nm，增加progressively随着时间（图1B）。作为对比，金NC的在670nm处不发光峰观察到的时间（图1A）同期地在室温下反应。 引入外部变性剂对Au NC的形成动力学的影响还研究使用BSA作为模板和尿素作为变性剂。 图1C示出了所合成的Au NC的光致发光光谱的时间过程变化。的Au NC的在670nm处的类似发射峰，也观察到，这表明BSA也适用于荧光的Au NC的外部变性剂的存在下的合成。然而，所得的Au NC的荧光强度比，即使经过的反应时间（7小时）长时间形成在60℃下低得多。这些结果表明，相比于使形成在凹NC的在尿素的存在下形成是慢得多60℃，由于蛋白质的低效率解折叠诱导的在室温下的外部变性剂。因此，加热在60℃下被选择为变性的优选的装置来筛选小分子药物与考虑的快速检测时间以后的实验。 图2示出了药物筛选的基础上的Au NC的与载药的HSA的形成动力学的结果。 如图2A所示，凹NC的与纯HSA模板所形成的荧光强度是始终如一最高贯穿的化验时间（30分钟）的全部时间，其次是与磺胺（四），苯妥英（c）中，华法林（二），和布洛芬（一）中的序列，这表明金NC的在不同模板的形成速率如下控制&gt; D&gt; C&gt; B&gt; A的趋势。因此，为了缩短测定时间，所有的所得的Au NC的在10分钟只有光电发射光谱是COMPAR编，以确定药物与HSA（图2B）的相对结合亲和力。实验的多次运行已经进行，以确认这个趋势（图2C）的一致性。频谱强度差可以从数码照片所得的Au NC的容易地可视化，如图2C所示 （插图）。所得的Au纳米晶中的不同载药蛋白模板存在下的差是荧光强度成反比这些药物与HSA的结合强度作为在以往的研究36确定的，这表明药物的结合提高蛋白质的稳定性对金纳米晶的形成过程中展开。因此，这些药物与HSA的结合亲和性可以容易地区别通过比较这样形成的Au NC的药物加载的HSA的荧光强度。 最后，目前的药物筛选方法应用于测量的结合定一个特定的药物蛋白质。布洛芬被选择来演示如何测量一个小分子药物与HSA的结合常数。不同浓度（0 - 450毫摩尔）的布洛芬溶液预孵育的HSA（74毫克/毫升，200微升）以形成凹NC的合成之前布洛芬-HSA模板图3示出了在Au NC的形成的荧光光谱。在HSA的模板预先加载不同浓度的布洛芬在单个批次的反应时间10分钟。由此可以看出，荧光强度与药物加载到蛋白质模板的增加量减小。以确定布洛芬的结合常数于HSA蛋白，相对荧光（I 0 -I）/ I 0从几个不同批次获得的各金NC的样品的计算，其中I 0是NC的纯合成的荧光强度HSA和I是在Au NC的合成的荧光强度<html>在布洛芬加载的HSA（ 表1）。第（I 0 -I）/ I 0的值绘制对药物浓度（图4，虚线）。该数据是进一步装配到使用米氏方程的单个点结合模型:Y = R 最大值 ×C /（K D + C）中，其中，R 最大值表示最大响应信号，C是配体和K D的浓度是结合常数（图4，实线）。这件给人布洛芬A K D值的0.74±0.1微米到HSA。这个值非常接近（0.5±1.0微米）使用NMR技术测定37从而进一步证实了当前方法来测量的小分子药物在均相溶液中结合常数的可靠性，而无需使用复杂的设备。 3261fig1.jpg"/&gt; 图对Au纳米晶的形成动力学不同的变性条件1.效应。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53261/53261fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 在60℃下形成的（A）中的HSA在室温下，（B）中的HSA时间分辨光电子的A​​u NC的光谱，和（C）的BSA与6.4尿素。 <img alt="图2" src="/files/ftp_upload/53261/53261fig2.jpg" /> 图2. HSA-目标的基础上形成的载药蛋白质模板金NC的荧光强度药物筛选。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53261/53261fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> （A）的Au纳米晶的形成（一）布洛芬-HSA，（B）华法林HSA，（C）苯妥英-HS形成动力学A，（D），磺胺-HSA，和纯粹的HSA（控制）。 （B）的光电发射HSA金NC的光谱中不同药物的存在下制备在60℃下:（1）布洛芬-HSA，（二）华法林-HSA，（三）苯妥英-HSA，（四）磺胺-HSA，和纯粹的HSA（控制）。 （C），HSA药物 - 金NC的抗HSA金NC的（控制）的标准化光电子强度。这两个光谱和数码照片（C ​​的插图）的加入NaOH在60℃之后，采取10分钟，36转载化学皇家学会的许可。 <img alt="图3" src="/files/ftp_upload/53261/53261fig3.jpg" /> 图上的HSA蛋白质载药量3的浓度依赖性研究。 形成HSA金NC的光电子能谱装载布洛芬在不同浓度的单批过的反应时间为10分钟。36改编化学皇家学会的许可。 图4.测定从实验数据蛋白质-药物结合常数。 布洛芬结合HSA的杀敌基于HSA-布洛芬金NC的相对荧光强度合成在60℃下以增加药物量来确定。实验数据拟合使用米氏方程的单个点结合模型:Y = R 最大值 ×C /（K D + C）中，其中，R 最大值表示最大响应信号，C是配体和K D的浓度是结合常数。36转载化学皇家学会的许可。 <img alt="表1" src="/files/ftp_upload/53261/53261table1.jpg" /> 表1.相关的计算形成蛋白质的模板金NC的荧光强度用不同浓度的药物。 相对荧光强度[（I 0 - I）/ I 0，我和我0指的Au纳米晶中形成的载药HSA和纯HSA的荧光强度，分别]的Au纳米晶形成HSA的装载布洛芬不同浓度在10分钟的反应时间。样品号XY是指在X 个批次编制的第 y的样本。36改编化学皇家学会的许可。

Watch this Scientific Journal Video about A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening at JoVE.com

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

一种快速定量荧光方法蛋白靶向小分子药物筛选

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

a-rapid-quantitative-fluorimetric-method-for-protein-targeting-small

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

10.0K 次观看.  Agency for Science, Technology and Research (A*STAR). A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

视频: A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe validation. Methods that measure this phenomenon without modification of the protein target or small molecule are particularly valuable though technically challenging. The cellular thermal shift assay (CETSA) is one technique to monitor target engagement in living cells. Here, we describe an adaptation of the original CETSA protocol, which allows for high throughput measurements while retaining subcellular localization at the single cell level. We believe this protocol offers important advances to the application of CETSA for in-depth characterization of compound-target interaction, especially in heterogeneous populations of cells.

Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA).

利用高含量成像对增强细胞中的目标参与进行量化

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

科研

生物化学

A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known small molecule-protein interactions and to find potential protein targets for natural products. Compared with other methods, DARTS uses native, unmodified, small molecules and is simple and easy to operate. In this study, we further enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The protein-ligand interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used the mTOR-rapamycin interaction as an exemplary case for establishment of our protocol. From the proteolytic curve we saw that the proteolysis of mTOR by pronase was inhibited by the presence of rapamycin. The dose-dependency curve allowed us to estimate the binding affinity of rapamycin and mTOR. This method is likely to be a powerful and simple method for accurately identifying novel target proteins and for the optimization of drug target engagement.

A Semi-Quantitative Drug Affinity Responsive Target Stability DARTS assay for studying Rapamycin/mTOR interaction

In this study, we enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used mTOR-rapamycin interaction as an exemplary case.

用于研究拉帕霉素/mTOR相互作用的半定量药物亲和反应目标稳定性 （DARTS） 测定

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

纳米微分扫描荧光仪，用于在基于碎片的铅发现中筛选

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

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 ...

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different tags, truncations, deletions, fusion partner insertions, and stabilizing mutations to find one that is not aggregated after extraction from the membrane. Furthermore, buffer screening to determine the detergent, additive, ligand, or polymer that provides the most stabilizing condition for the membrane protein is an important practice. The early characterization of membrane protein quality by fluorescent size exclusion chromatography provides a powerful tool to assess and rank different constructs or conditions without the requirement for protein purification, and this tool also minimizes the sample requirement. The membrane proteins must be fluorescently tagged, commonly by expressing them with a GFP tag or similar. The protein can be solubilized directly from whole cells and then crudely clarified by centrifugation; subsequently, the protein is passed down a size exclusion column, and a fluorescent trace is collected. Here, a method for running FSEC and representative FSEC data on the GPCR targets sphingosine-1-phosphate receptor (S1PR1) and serotonin receptor (5HT2AR) are presented.

The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are presented.

通过荧光体积排阻色谱快速评估膜蛋白质量

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

免疫与感染

<meta charset="utf8"></head><body>定量-不可逆栓系:高效筛选亲电配体

Covalent Fragment Screening Using the Quantitative Irreversible Tethering Assay

Compounds that form covalent bonds with specific target proteins offer a variety of advantages&#160;as chemical probes and therapeutic agents. Most commonly, mildly reactive, electrophilic small molecules are employed to form covalent bonds with select cysteine side chains in specific proteins. Electrophile-first approaches of ligand discovery, whereby a library of electrophilic small molecules are screened against a protein target, have become popular as they avoid the need for time-consuming downstream installation of an electrophilic warhead. Such screening is complicated, however, as electrophilic ligands can exhibit a wide range of different rates of spontaneous reaction with cysteines. Quantitative-irreversible tethering (qIT) offers a fluorescence-based method for hit identification and development that normalizes data for these differences in intrinsic compound reactivity. Rates of reaction of individual compounds with a target protein are determined and compared to compound reactivity with the unstructured tripeptide glutathione (this being a proxy for spontaneous compound reaction), enabling the identification of compounds that preferentially react with the protein of interest. This methodology has been successfully applied to identify selective covalent fragments against several drug targets, including SARS-CoV-2 main protease, cyclin-dependent kinase 2, and RAP27A. Here, we demonstrate the application of qIT to a target protein to generate a quantitative and robust data set, allowing prioritization of hit ligands for future development.

<meta charset="utf8"></head><body>使用定量不可逆栓系测定法进行共价片段筛选

The quantitative irreversible tethering (qIT) assay is a fluorescence-based method of identifying covalent fragments that selectively engage with a target protein. Here, a detailed protocol is outlined to describe the qIT assay, with example results included.

使用定量不可逆栓系测定法进行共价片段筛选

Compounds that form covalent bonds with specific target proteins offer a variety of advantages&#160;as chemical probes and therapeutic agents. Most ...

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

基于生物传感器的高通量生物跨越和生物信息学分析战略，用于全球药物-蛋白质相互作用验证

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

一种快速定量荧光方法蛋白靶向小分子药物筛选

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利用高含量成像对增强细胞中的目标参与进行量化

用于研究拉帕霉素/mTOR相互作用的半定量药物亲和反应目标稳定性（DARTS）测定

纳米微分扫描荧光仪，用于在基于碎片的铅发现中筛选

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

通过荧光体积排阻色谱快速评估膜蛋白质量

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

使用定量不可逆栓系测定法进行共价片段筛选

使用定量不可逆栓系测定法进行共价片段筛选

使用定量不可逆栓系测定法进行共价片段筛选

基于生物传感器的高通量生物跨越和生物信息学分析战略，用于全球药物-蛋白质相互作用验证

一种快速定量荧光方法蛋白靶向小分子药物筛选

摘要

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利用高含量成像对增强细胞中的目标参与进行量化

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纳米微分扫描荧光仪，用于在基于碎片的铅发现中筛选

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