Name: development of inhibitors of protein-protein interactions through replace: application to the design and development non-atp competitive cdk inhibitors
Uploaded: 2015-10-26T14:00:00.000+00:00
Duration: 10 min 33 s
Description: Watch this Scientific Journal Video about Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors at JoV

We thank Dr&#8217;s. Douglas Pittman and Michael Wyatt for their assistance with cell culture and Dr Wyatt and Ms. Erin Anderson for help in development of the binding assays. We acknowledge Mike Walla and Bill Cotham in the Department of Chemistry and Biochemistry at the University of South Carolina for assistance with Mass Spectrometry, Helga Cohen and Dr. Perry Pellechia for NMR spectrometry. This work was funded by the National Institutes of Health through the research project grant, 5R01CA131368.

潜在的小分子封端基团的1计算识别注意:原则上，各种各样的对接或药效搜索方法可用于预测潜在封盖基。计算研究中REPLACE的主要目的是确定的小分子，其保留的被取代的氨基酸的功能和相互作用。 <ol><li>在LigandFit对接协议14的验证</li></ol>注意:在以前的研究中，对接方法（LigandFit 15，在分子建模程序套件的模块，发现工作室3.0）进行验证，以确保该算法足以重现已知NCAPS的结合方式，并表明，该结果获得的未知化合物的预测14。 <ol style="margin-left: 40px;"><li>通过以下步骤（1.2 - 1.5），识别LigandFit最佳条件为:PLP1恩ERGY网格姿势代，最小化产生的姿势和PLP1打分函数姿势的分析。 </li><li>为了限制所得到的构象，并在封端的肽适当地定位的配体形成共价键，设置Trp217和Gln254的吲哚氮与酰胺氮原子分别作为相互作用滤波器氢键。 </li><li>设计和码头的片段替代品库到CDK2 /细胞周期蛋白受体（2UUE）。优先潜力封盖组​​进行综合和基于PLP1得分，以互动的过滤器和互补的CBG互动的商业采购。是所需LigandFit对接的各个步骤如下。 </li></ol><ol start="2"><li>的受体结合位点14制备 <ol><li>导入CDK2 /细胞周期蛋白晶体结构（PDB ID:2UUE）到软件中的一个可视化窗口。这个结构包含先前标识FLIP 5-甲基-1-苯基-1H-1，2,4-三唑-3-甲酰（3,5- DCPT）RLIF结合于细胞周期蛋白槽（图3）。 </li><li>删除或守在原地的残留RLIF（C端）。当被保持在肽序列中，使用肽的N末端氨基作为相互作用滤波器的配位体。从"受体 - 配体相互作用"工具，创建一个围绕的N帽3,5- DCPT来自显示/隐藏部位球的球体。球体被创建以限定在配体 - 受体相互作用允许发生期间结合所述结合位点的活性区。 </li><li>定义的结合位点进一步从"找到站点作为选择的配体的量"，并从蛋白质删除配体3,5- DCPT。执行此步骤来定义创建的范围内具有约束力的体积。 </li><li>设置残基Trp217和Gln254作为相互作用滤波器氢键的吲哚氮与酰胺氮原子。设置的过滤器相互作用，使得只有那些INTE姿势RACT与集残留的原子将在对接过程中，因此保留在停靠姿势重要的相互作用进行过滤。 </li></ol></li><li>配14的制备<ol><li>设计的100潜在封盖基根据标准的库包括分子量小于500时，存在羧酸酯基的结扎用截短的肽和夹杂物的范德华相互作用适当疏水性基团（取代的苯基）在二次口袋作为表 1所示。 </li><li>选择杂环用于模仿肽氢键相互作用与Trp217和取代基包括更换HAKRRLIF的碱性侧链的离子配对相互作用。停靠的配位体作为相应醛分子，以使羰基氧可以与Gln254的酰胺氮原子的母体肽（表1）相似的肽主链相互作用。 </li><li>此前DOC王，最大限度地减少设计的配体低能量构象，并使用"准备配体"协议，在适当的电离状态的准备。绘制和中的ChemDraw .sdf文件格式的电子表格之前导出这些潜在的N帽被导入到软件中。 注意:准备配位体协议用于最小化所有的配体停靠到它们的最低能量状态和电离指控被施加到适用原子。默认参数用于此步骤。 </li></ol></li><li>对接的配体进入细胞周期蛋白A槽14 <ol><li>选择从受体 - 配体相互作用协议的软件的集合的LigandFit例行程序。使用受体和配体准备作为输入此协议。 </li><li>这些试验选择PLP1作为能量网格，指定构成是能量最小化，并生成姿势为10的数目，保持所有其他参数为默认值。 注:LigandFit是对接方法，它可以识别并产生高的互补性和潜力的亲和姿势与使用被结合蒙特卡洛构象搜索算法基于形状进行比较滤波器的蛋白质的活性位点。使用对接程序的能量网格是力场用于产生配体 - 受体相互作用和潜在姿势。 PLP1是特异性优先氢键相互作用和分段线性电位，其中所述PLP原子类型分配每个非氢原子的配体或非氢原子受体。 </li></ol></li><li>结果的分析<ol><li>在第一个实例，显示由降PLP1得分值带来的可视化程序，然后排序。使用计分函数如PLP1来估计根据一个候选配体对接的配体的结合亲和力姿势几何和非共价相互作用与靶受体结构。 注:在这里，PLP1打分函数被发现与G香港专业教育学院最好的结果，因为它包括的氢键的估计。 </li><li>目测分析打进姿势为1的顶部25％）superimposability与已知封端基团（具有相对均方偏差（RMSD）值小于2的），2）满足相互作用的过滤器和3）视觉的互补与细胞周期蛋白槽。它接受一个正确的停靠姿势（相较于实验结构）具有&lt;2的RMSD值。 </li><li>检查姿势为视觉的互补性，其被定义为具有的方式，是与已知的结构 - 活性关系一致的结合口袋的有效填充。相互作用滤波器被设置为包括需要已知可用于结合和/或需要在用于酰胺键形成正确的几何定位的电位加帽基临界分子间接触原子约束。潜在的N-端封基团使氢键与interactio停靠姿势三个例子N个滤波器示于图4。 </li></ol></li></ol> 2.合成和潜在的N -capping组表征<ol start="1"><li>合成的N-封端基团。 </li><li>对于合成，使用所有的商业原料，溶剂和试剂而获得。通过常规有机合成化学（图5 12,13的例子）合成潜在的N-封端基团。 </li><li>用于监测反应进行硅胶薄层层析（TLC）。 <ol><li>溶解起始原料和反应混合物（1毫克）中的流动相，发现它们对使用细管的TLC板上。 </li><li>将TLC板中含有流动相（乙酸乙酯和己烷以35:65的比例）的腔室。一旦流动相达到90％的薄层板上，捞出晾干板。 </li><li>使用UV光来检测起始材料和反应混合物作为可见的斑点。卡尔culate所有部位中的R F（距离由点和流动相行进的比）。 注意:该反应完成时，没有光斑见于起始材料中的R F。 </li></ol></li><li>在反应完成后通过放置在分液漏斗和洗涤用含水酸或碱溶液适当工作了反应混合物中。收集有机溶剂，在旋转蒸发器中蒸发，并在真空下干燥所得到的粗产物。 </li><li>溶解500毫克粗产物在3-5毫升合适的溶剂，并加入到一个1克的二氧化硅或RP18 samplet和下空气干燥。放置含粗产物在二氧化硅/ RP SNAP闪光灯墨盒samplet和使用自动高性能快速色谱法（根据制造商的协议）采用SNAP 100个克柱和梯度运行的6％乙酸乙酯开始纯化粗物质:94 ％己烷至50％乙酸乙酯和50％己烷以上15个柱体积。 </li><li>通过蒸发所有溶剂至干干燥使用旋转蒸发器收集在溶剂从快速色谱纯化产物，并进一步干燥该产物在真空下除去所有的残留溶剂。通过NMR，MS和分析型HPLC进行纯化产物的表征。 <ol><li> 1 H NMR和13 C-NMR，重约10毫克的纯化样品，将其溶解在适当的氘代溶剂中，并将内容物转移到干燥NMR管，用于记录NMR谱2,14。 </li><li>记录1 H NMR和 13 C NMR谱与高场核磁共振仪（最小300兆赫）。收购质谱使用QTOF（串联四-1飞行时间质谱仪），电喷雾离子化（ESI）或VG 70S（双聚焦扇形磁场质谱仪，EI）2,14。 </li><li>通过HPLC用二极管阵列检测器分析产品的纯度和配备在C18（2）100，250×4.6毫米，5微米柱进行分析。使用的梯度运行从100％水（0.1％三氟乙酸）开始至60％乙腈（0.1％三氟乙酸）在30分钟内并保持梯度为4分钟。提取色谱图在226和254纳米2,14。 注:评价化合物的分析纯度分别&gt; 95％。 </li></ol></li></ol> 3.固相合成的翻转2代<ol start="1"><li>正封顶翻转合成<ol><li>装配通过使用以下步骤的标准固相合成方法的N-封端的肽化合物。 <ol><li>在2ml激活5当量的C-末端氨基酸（的Fmoc-Phe的生产）的，在O3 -Benzotriazole- N，N，N&#39;，N&#39;四甲基脲六氟磷酸盐（HBTU，221.93毫克）4.4当量DMF 5分钟。装载的Fmoc-PHE HBTU混合到使用6当量二异丙基胺的溜冰场树脂（DIPEA，103.13毫克）处理1小时，在室温。 </li><li>使用20％哌啶在3ml DMF中的10分钟的C-末端残基除去Fmoc保护基。夫妇随后的氨基酸（ 如 ，FMOC，异亮氨酸，FMOC亮氨酸，FMOC-精氨酸- PMC）一步一步来。在每一步，一对5当量使用6当量DIPEA（103.13毫克）和HBTU（221.93毫克）在2毫升DMF中4.4当量的1小时在室温下一个氨基酸。 </li><li>适用的洗涤周期（5×10毫升DMF + 5×10ml DCM中的）上述氨基酸偶联和Fmoc去保护步骤后。肽装配后，夫妇N-二封盖使用6当量DIPEA（103.13毫克）和HBTU（221.93毫克）的4.4当量的群体在2ml DMF中进行1小时，在室温。 </li><li>在肽组装完成后，治疗用2ml的裂解混合物的反应混合物（90:5:5的TFA / H 2 O / TIPS）O / N以除去侧链保护基团，并从树脂裂开翻转。用冷乙醚沉淀和我磨碎所得到的产物˚F必要的浓缩在旋转蒸发器。 </li></ol></li></ol></li><li>的N-封顶-C皑皑翻转的合成<ol><li>称重并溶解N-末端封端的和受保护的精氨酸-亮氨酸肽（16.1毫克，0.02毫摩尔）在二氯甲烷中，并添加[4-（3-氯苯氧基）吡啶-2-基]甲胺（C-帽）沿带 O -苯并三唑-N，N，N&#39;，N&#39;四甲基脲六氟磷酸盐（HBTU; 16.7毫克，0.02毫摩尔）和N，N-二异丙基 （DIPEA; 6.5毫克，0.02毫摩尔）。 </li><li>搅拌并监测在RT反应直至TLC / HPLC显示起始材料的完全消耗。反应完成后，浓缩，用乙酸乙酯和水之间在旋转蒸发器然后分区粗反应混合物。 </li><li>分离水和有机层;用1N NaOH，1N HCl和盐水洗涤洗涤有机层。与约1克硫酸钠干燥有机层并浓缩产物在旋转蒸发器中。菲娜LLY治疗产品O / N与裂解混合物（95:2.5:2.5三氟乙酸/ H 2 O / TIPS）进行脱保护。 </li><li>用冷的二乙醚，并在必要的浓缩在旋转蒸发至完全干燥以除去所有的溶剂磨碎所得到的产物。进一步在真空下干燥产物以除去所有的残留溶剂。 </li></ol></li><li>纯化和翻转的特性<ol><li>使用半制备反相HPLC纯化法粗翻转。冻干纯化的翻转和用质谱2,14描述他们的分子质量。 </li><li>通过HPLC用二极管阵列检测器确定所述翻转的分析纯度和配备有C18（2）100，250×4.6毫米，5微米柱。使用从95％的H 2 O（0.1％三氟乙酸）/ 5％乙腈（0.1％三氟乙酸）到35％ 的 H 2 O（0.1％三氟乙酸）/ 65％乙腈（0.1％trifluoroace开始梯度法抽动酸）在30分钟内并保持梯度为4分钟。提取色谱在226和254纳米。 </li></ol></li></ol> 4.荧光偏振结合分析的竞争性结合2,14的测定<ol start="1"><li>请在黑色384孔（138微升）使用以下步骤板的测定法。 </li><li>稀释的样品中，示踪肽（4纳米），和激酶复合物（CDK2 /细胞周期蛋白A - 18微克/毫升，CDK4 /细胞周期蛋白D - 37微克/毫升），以所需浓度在测定缓冲液（25mM HEPES pH为7，10毫氯化钠，0.01％的Nonidet P-40，1mM的二硫苏糖醇（DTT）。 </li><li>向384孔板的各孔中，添加:5微升CDK4D1或CDK2CA（0.3微克/孔纯化重组人激酶复合物），5微升化合物溶液，5微升30nM的示踪肽（荧光素AHX-亲缬氨酸的-Lys精氨酸基-Arg-Leu-（3ClPhe）-Gly或荧光素AHX-PRO-VAL-赖氨酸 - 精氨酸 - 精氨酸 - 亮氨酸 - 苯丙氨酸 - 甘氨酸示踪肽）。使用每个DMSO（6％），buffe的5微升R，示踪和5μlDMSO（6％），激酶复合物，示踪物作为对照孔</li><li>在加入所有组分后，离心平板在500rpm下1分钟（41.16 XG）在RT，然后孵育该板与搅拌在室温45分钟。使用多模板读取器和检测器装有485毫微米/ 535毫微米的激发/发射滤​​光片和分色镜读取各孔在板的荧光强度。 </li><li>计算相对平均偏振（熔点）使用该方程表示下面的所有测试样品。确定IC 50值用对数回归通过关联相对MPS和测试浓度。 </li></ol><img alt="式（1）" src="/files/ftp_upload/52441/52441eq1.jpg" />

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M., Jiang, X., Oldfield, T., Waldman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LigandFit:+a+novel+method+for+the+shape-directed+rapid+docking+of+ligands+to+protein+active+sites.">LigandFit: a novel method for the shape-directed rapid docking of ligands to protein active sites.</a> J Mol Graph Model. 21, 289-307 (2003).</li><li>Mendoza, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+cyclin-dependent+kinase+2/cyclin+A+antagonists+that+differ+from+ATP+site+inhibitors+block+tumor+growth.">Selective cyclin-dependent kinase 2/cyclin A antagonists that differ from ATP site inhibitors block tumor growth.</a> Cancer Res. 63, 1020-1024 (2003).</li><li>Denizot, F., Lang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+colorimetric+assay+for+cell+growth+and+survival.+Modifications+to+the+tetrazolium+dye+procedure+giving+improved+sensitivity+and+reliability.">Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability.</a> J Immunol Methods. 89, 271-277 (1986).</li><li>Zheleva, D. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+potent+p21(WAF1)-derived+peptide+inhibitors+of+CDK-mediated+pRb+phosphorylation:+delineation+and+structural+insight+into+their+interactions+with+cyclin+A.">Highly potent p21(WAF1)-derived peptide inhibitors of CDK-mediated pRb phosphorylation: delineation and structural insight into their interactions with cyclin A.</a> The journal of peptide research : official journal of the American Peptide Society. 60, 257-270 (2002).</li><li>Ivanov, A. A., Khuri, K. F. R., Fu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+protein-protein+interactions+as+an+anticancer+strategy.">Targeting protein-protein interactions as an anticancer strategy.</a> Trends in Pharmacological Sciences. 34, (2013).</li><li>Thayer, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+peptides.">Improving peptides.</a> Chemical and Engineering News. 89, 13-20 (2011).</li><li>Yin, H., Hamilton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+targeting+protein-protein+interactions+with+synthetic+agents.">Strategies for targeting protein-protein interactions with synthetic agents.</a> Angew Chem Int Ed Engl. 44, 4130-4163 (2005).</li><li>Jubb, H., Higueruelo, A. P., Winter, A., Blundell, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+biology+and+drug+discovery+for+protein-protein+interactions.">Structural biology and drug discovery for protein-protein interactions.</a> Trends Pharmacol Sci. 33, 241-248 (2012).</li><li>Cannon, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmaceutics+and+drug+delivery+aspects+of+heme+and+porphyrin+therapy.">Pharmaceutics and drug delivery aspects of heme and porphyrin therapy.</a> J Pharm Sci. 82, 435-446 (1993).</li></ol>

The authors declare that they have no competing financial interests.

Targeting protein-protein interactions (PPI) in drug discovery is highly challenging as these typically involve a large shallow contact interface comprised of numerous and diffuse contacts19. Furthermore, peptidic compounds which inhibit PPI&#8217;s that are amenable to drug discovery are problematic due to their higher molecular mass, metabolic instability and poor bioavailability20. Current strategies that have been applied for the development of PPI inhibitors include design of proteomimetics and...

在这篇文章中，应用REPLACE（更换使用计算富集部分配体的替代品）策略，以蛋白质-蛋白质相互作用的肽酶抑制剂转换成多种药物相关的分子为例进行说明1-3。虽然生产者价格指数代表了丰富，但开发不足潜在的药物靶点的来源，现有方法在很大程度上不足以让这些普及。当前策略包括基于片段设计4，高通量筛选5和6提供了进步装订肽，但是这些在许多情况下是无效的。其结果是，更多的进展和更有效的方法是必需的。 REPLACE已激酶抑制剂具有改善的类药性的属性和有潜力的进一步发展为抗肿瘤治疗剂的开发已充分验证。这种策略是例示的电池的非ATP酶抑制剂的开发周期CDK的并涉及如下:1）获得关于HAKRRLIF / RRLIF与细胞周期蛋白结合槽相互作用三维结构信息; 2）确定的重要结合决定肽相互作用;含有一种或多种结合决定簇的肽的N-末端的3）截断; 4）计算确定可能的小分子的替代品（局部的配位体的替代品，聚乳酸类）的肽和其中的截断部分保留母体肽的关键相互作用; 5）合成或PLA中的商业采购预测与先前所删除的肽残基所占据的子站点贪婪地结合; 6）的合成通过使用固相合成截短的肽最好PLA中的结扎翻转的; 7）检测结合在体外翻转或功能测定（在CDK /细胞周期蛋白上下文荧光偏振），随后在细胞生存力测定法进一步表征。更换的战略中一个示意图y为如图 1所示。在这篇文章中，REPLACE策略的迭代被讨论，并应用到CDK2 /细胞周期蛋白A进行详细说明。激酶被认为是直接或间接地解除管制在大多数肿瘤中，因此被视为适当的抗癌药物靶 7。 CDK的要求协会细胞周期的完全激活，随后磷酸化参与细胞周期调控8关键蛋白。激酶的两个主要群体是控制细胞周期检查站同型[G1 / S（CDK4 /细胞周期蛋白D，CDK6 /细胞周期蛋白D和CDK4 / cyclin E的），S期（CDK2 /细胞周期蛋白A）和G2 / M（CDK1 /细胞周期蛋白B）]和RNA聚合酶通过磷酸化调节（CDK7 /细胞周期蛋白H，CDK8 /周期蛋白C，CDK9 /周期蛋白T）。当E2F1转录因子形成与DP蛋白质然后与DNA结合，并启动基因转录的复合S期进展中的关键步骤发生。 CDK2 /细胞周期蛋白A需要中和E2F1转录通过磷酸化活性，从而导致释放E2F1-DP的复杂和其随后的退化。 CDK2 /细胞周期蛋白A的抑制被认为是维持E2F1在其DNA绑定状态，导致持续激活。的E2F-1的活性得到的水平将超过诱导的p53依赖的细胞凋亡，因此暗示治疗策略所需的阈值。由于E2F-1的失调p53和pRb的通路，高含量经常发生在肿瘤细胞中和抑制CDK2 /细胞周期蛋白A的应导致选择性凋亡的肿瘤和可以被认为是一个有效的癌症靶7。 临床研究CDK抑制剂靶向高度保守的ATP结合位点，导致跨越大于500蛋白激酶在人激酶组之间的反应性和潜在地引起副作用和毒性9。另一种方法是非ATP竞争抑制通过在CBG靶向性底招聘存在于细胞周期正调控亚基并因此显着，并从遥远的ATP结合位点10,11。所述CBG主要是存在于细胞周期蛋白A，细胞周期蛋白D和cyclin E疏水槽并已被证明为识别在基片和肿瘤抑制发现的共有序列。作为一种分离的肽，细胞周期蛋白结合基序（CBM）的结合到CBG，并已显示出抑制的细胞周期的CDKs激酶活性。煤层气已经被优化到八肽（HAKRRLIF，CDK2 /细胞周期蛋白A的IC 50 0.07±0.02μM，CDK4 /细胞周期蛋白D，IC 50 0.88±0.34μM），进而截断为代表的分子量与毒品之间的良好折衷五肽肖像和效力（RRLIF，CDK2 /细胞周期蛋白A的IC 50 1.01±0.17μM， CDK4 /细胞周期蛋白D，IC 50 25.12±2.97μM）12,13。该CBGs由一个大的初级和二级小疏水口袋它是由一个酸化桥接的C区（包括Glu220，Glu224和Asp283）。 HAKRRLIF的关键结合决定簇包括ALA2与Lys3，精氨酸4和Arg5的二次疏水口袋，离子配对和氢键与酸性区域和高度Leu6和Phe8的互补性与亲脂性主要部位的相互作用。此外，大量的氢键被从肽骨架贡献而Ile7充当间隔物残基允许与主口袋最佳的接触。装订模式和HAKRRLIF与CBG的相互作用如图2。 靶向CBM / CBG蛋白-蛋白相互作用将抑制CDK2 /细胞周期蛋白A，CDK2 /细胞周期蛋白E与CDK4 /细胞周期蛋白D的激酶活性，这一点应引起E2F1癌细胞介导的凋亡，而不影响正常细胞7。虽然煤层气衍生肽是细胞周期CDK的有效抑制剂，这是不可能的，他们将作为药物有用，因为它们的代谢不稳定和普遍缺乏的细胞通透性。为此，我们已经为这些有效的肽酶抑制剂转化为更多的类似药物的化合物的抗肿瘤治疗通过CDK2 /细胞周期蛋白利用放松管制E2F1一个抑制进一步的发展应用替换策略。下面的协议，总结了已更换的周期槽的应用程序已经完成的工作。在第一个实例，类药性封端基团替代HAKRRLIF的N末端四肽进行鉴定。此外改善这些群体进行了调查，一个额外的验证研究更换。从这些研究中有代表性的结果也列。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Computational Chemistry&lt;/strong&gt;</td></tr><tr><td>Accelyrs Discovery studio 3.0</td><td></td><td></td><td></td></tr><tr><td>Dell Optiplex Workstations</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Synthetic Organic Chemistry&lt;/strong&gt;</td></tr><tr><td colspan="4">Silica gel (GF-254 plates) for TLC, Biotage (Uppsala, Sweden) for flash chromatography, Waters Alliance 2695 HPLC with a 2996 diode-array detector and equipped with a C18 (2) 100 A, 250 x 4.6&amp;nbsp;mm, 5&amp;nbsp;&amp;mu;m column (Phenomenox Luna) for purity determination, &lt;sup&gt;1&lt;/sup&gt;H NMR and &lt;sup&gt;13&lt;/sup&gt;C NMR spectra were recorded with a Varian Mercury 300 and 400 Spectrometer, respectively. Mass spectra were measured with a Micromass QTOF (Tandem quadruple-1 time of flight mass spectrometer), electrospray ionization (ESI) and VG 70S (Double-focusing magnetic sector mass spectrometer, EI).</td></tr><tr><td colspan="4">&lt;strong&gt;Flourescence Polarization Assay&lt;/strong&gt;</td></tr><tr><td>384 micro well plates, Micro pipets</td><td>Grenier Bio-one</td><td>110256602</td><td></td></tr><tr><td>CDK4D1 and CDK2CA (well purified recombinant human kinase complex)</td><td>BPS Bio Sciences</td><td>40094(CDK4/Cyclin D), 41101(CDK2/Cyclin A)</td><td></td></tr><tr><td colspan="4">assay buffer (25 nM HEPES pH 7, 10 mM NaCl, 0.01% Nonidet P-40, 1&amp;nbsp;mM dithiothretiol (DTT))</td></tr><tr><td>25 nM HEPES</td><td>CALBIOCHEM</td><td>375368</td><td></td></tr><tr><td>NaCl</td><td>Fisher</td><td>127838</td><td></td></tr><tr><td>Nonidet P-40</td><td>US Biological</td><td>N3500</td><td></td></tr><tr><td>DTT</td><td>Aldrich</td><td></td><td></td></tr><tr><td>-70 &amp;#176;C freezer</td><td>Revco (Ultima II)</td><td></td><td></td></tr><tr><td>DTX880 multimode detector fitted with 485&amp;nbsp;nm/535&amp;nbsp;nm excitation/emission filters and a dichroic mirror suitable for fluorescein</td><td>Beckman Coulter, Brea, CA</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Cell Culture&lt;/strong&gt;</td></tr><tr><td>96 well plates</td><td>Fisher</td><td></td><td></td></tr><tr><td>Frozen stocks of U2OS (osteosarcoma) and DU145 (prostate cancer) cell lines</td><td>ATCC</td><td></td><td></td></tr><tr><td>NU serum, DMEM media, trypsin, PEN/STRIP, MTT reagent</td><td>Fisher, Life technology, Alfa Aesar</td><td></td><td></td></tr><tr><td>Heamocytometer</td><td>VWR</td><td></td><td></td></tr><tr><td>-70 &amp;#176;C freezer</td><td>Revco (Ultima II)</td><td></td><td></td></tr><tr><td>Incubator</td><td>Thermo electron corporation</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804 R</td><td></td></tr><tr><td>Refrigerator 4-8 &amp;#176;C</td><td>Isotemp Fisher</td><td></td><td></td></tr><tr><td>DTX880 multimode detector fitted with 595&amp;nbsp;nm filter</td><td>Beckman Coulter, Brea, CA</td><td></td><td></td></tr></tbody></table>

development of inhibitors of protein-protein interactions through replace: application to the design and development non-atp competitive cdk inhibitors

REPLACE是发展到更有效地针对蛋白质 - 蛋白质相互作用（质子泵抑制剂）一个独特的策略。其目的是通过对抑制剂的识别这样的结合位点并提供改进的方法代表了大多数潜在的药物靶点，扩大可用药物靶点的空间。本文的主要目的是提供替换策略，涉及的计算和合成化学方法的使用和应用的方法概述。取而代之的是通过其应用到非ATP竞争性细胞周期蛋白依赖性激酶（CDK）抑制剂作为抗肿瘤治疗学的发展例举。激酶经常放松管制在癌症，因此被认为是药物开发的重要目标。在S期CDK2 /细胞周期蛋白A的抑制已报道通过E2F1途径促进癌细胞中p53的独立的方式选择性凋亡。靶向蛋白质 - 蛋白质相互作用的细胞周期蛋白结合蛋白摹槽（CBG）是一种方法，这将使细胞周期在转录激酶特异性抑制。该CBG是由CDK底物和肿瘤抑制蛋白的共识序列中，识别被称为细胞周期蛋白结合基序（CBM）。煤层气先前已优化，从P21WAF（HAKRRIF），然后八肽进一步截短为五肽保持足够的活性（RRLIF）。在一般不细胞渗透性肽，是代谢不稳定，因此，策略REPLACE（通过计算富集代用偏配体替代品）已被以产生更多的类药性的抑制剂的应用。策略开头片段结扎抑制肽（翻转），其选择性地抑制细胞周期CDK /细胞周期蛋白复合物的设计。通过迭代地更换HAKRRLIF / RRLIF的残基片段像小分子（封端基团），从N末端（NCAPS）开始生成翻转，随后更换上C末端。这些化合物开始的非ATP竞争性CDK抑制剂作为抗肿瘤疗法的产生点。

HAKRRLIF与细胞周期蛋白槽的相互作用示于图2。表示键结合决定的肽残基包括ALA2，ARG4，Leu6和Phe8与其他残基提供较小的贡献12,13,18。在这种情况下，研究替换策略已被用于为了找到残留HAKRRLIF的N末端四肽片段的替代品，主要是模仿ALA2和ARG4的相互作用。潜在NCAP片段（ 表1）库是基于标准构建。每个分子停靠到由N个封端基的截断其晶体结构与细胞周期蛋白A（PDB ID:2UUE...

Watch this Scientific Journal Video about Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors at JoV

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

的蛋白质 - 蛋白质相互作用抑制剂的发展，通过替换:应用程序的设计和开发非ATP竞争性CDK抑制剂

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

development-inhibitors-protein-protein-interactions-through-replace

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.2K 次观看.  University of South Carolina. We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

视频: Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

科研

生物化学

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

利用多功能生化方法鉴定新型 ck2 激酶基材

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

自组装的人类蛋白质微阵列中的激酶抑制剂筛选

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small molecule drug discovery. This class of compounds induces protein degradation by bringing into proximity the target protein and the E3 ligase machinery proteins required to ubiquitinate and ultimately degrade the target protein through the ubiquitin-proteasomal pathway (UPP). Profiling of target protein degradation in a high-throughput fashion, however, remains highly challenging given the complexity of cellular pathways required to achieve degradation. Here we present a protocol and screening strategy based on the use of CRISPR/Cas9 endogenous tagging of target proteins with the 11 amino acid HiBiT tag which complements with high affinity to the LgBiT protein, to produce a luminescent protein. These CRISPR targeted cell lines with endogenous tags can be used to measure compound induced degradation in either real-time, kinetic live cell or endpoint lytic modes by monitoring luminescent signal using a luminescent plate-based reader. Here we outline the recommended screening protocols for the different formats, and&#160;also describe the calculation of key degradation parameters of rate, Dmax, DC50, Dmax50, as well as multiplexing with cell viability assays. These approaches enable rapid discovery and triaging of early stage compounds while maintaining endogenous expression and regulation of target proteins in relevant cellular backgrounds, allowing for efficient optimization of lead therapeutic compounds.

This protocol describes the quantitative luminescent detection of protein degradation kinetics in living cells that have been engineered using CRISPR/Cas9 to express antibody free endogenous protein detection tag fused to a target protein. Detailed instructions for calculating and obtaining quantitative degradation parameters, rate, Dmax, DC50, and Dmax50 are included.

使用 HiBiT CRISPR 细胞系对靶向蛋白质降解化合物进行高通量细胞分析

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small ...

癌症研究

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

化学抑制剂库筛选 t 细胞受体信号的中介识别

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

免疫与感染

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.

蛋白质智慧：一个工作台<em&gt;硅片</em&gt;<em&gt;从头</em&gt;生物分子设计

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific ...

生物学

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

对于耐药性突变对激酶抑制剂的筛选和验证方法

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Developing and Testing Methylated Nano-Structured Dipeptides that Inhibit Src Kinase Activity In Vitro for Anti-Cancer Applications

Here, with the aim of developing a novel anti-cancer treatment, seven dipeptides were designed that contained methylated tryptophan and/or methylated arginine and were produced using Fmoc solid-phase peptide synthesis. Overexpression of the Src tyrosine kinase enzyme has been implicated in the development of different cancers. Dipeptides containing unnatural amino acids such as methylated arginine (RCH3), dimethylated arginine (R(CH3)2), and/or methylated tryptophan (WCH3) residues have earlier been shown to inhibit Src kinase. In this study, three such dipeptides, W-RCH3, WCH3-RCH3, and W-R(CH3)2, were tested using acellular assays and were found to have IC50 values (the concentration at which 50% inhibition occurs) of 510 nM, 916 nM, and 1 &#181;M, respectively. These values were comparable to those obtained for cyclic penta- to nano-W-R peptides ([W-R]5-[W-R]9) synthesized in previous studies. However, the unmethylated versions of the dipeptides did not show any inhibitory activity against Src kinase. All of these dipeptides (50 &#181;M) did not show any cytotoxicity after incubation up to 72 h with three different cancer cell lines, including leukemia (CCRF-CEM), breast adenocarcinoma (MDA-MB-231), and ovarian adenocarcinoma (SK-OV-3) cell lines, indicating the limited permeability of the peptides through the cell membrane. Therefore, further study is needed to improve the permeability of these peptides for anticancer applications, such as by using a peptide carrier or additional peptide functionalization. In summary, this study provides a protocol to synthesize and test peptides that inhibit Src kinase activity, and thus possess promising anticancer ability, as demonstrated using acellular and cellular assays.

Developing and Testing Methylated Nano-Structured Dipeptides that Inhibit Src Kinase Activity In Vitro for Anti-Cancer Applications

Presented here is a protocol for designing and testing dipeptides that can inhibit Src kinase enzyme activity using acellular and cellular assays for anti-cancer applications. The peptides formulated here (W-RCH3, WCH3-RCH3, and W-R(CH3)2)&#160;inhibited Src kinase with IC50 values of 510 nM, 916 nM, and 1 &#181;M, respectively.

Here, with the aim of developing a novel anti-cancer treatment, seven dipeptides were designed that contained methylated tryptophan and/or methylated ...

的蛋白质 - 蛋白质相互作用抑制剂的发展，通过替换:应用程序的设计和开发非ATP竞争性CDK抑制剂

摘要

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此视频中的章节

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

利用多功能生化方法鉴定新型 ck2 激酶基材

自组装的人类蛋白质微阵列中的激酶抑制剂筛选

使用 HiBiT CRISPR 细胞系对靶向蛋白质降解化合物进行高通量细胞分析

化学抑制剂库筛选 t 细胞受体信号的中介识别

蛋白质智慧：一个工作台

对于耐药性突变对激酶抑制剂的筛选和验证方法

激酶底物对鉴定使用高通量筛选

Developing and Testing Methylated Nano-Structured Dipeptides that Inhibit Src Kinase Activity In Vitro for Anti-Cancer Applications

抑制 CDK 活性