Name: method for identifying small molecule inhibitors of the protein-protein interaction between hcn1 and trip8b
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This work was supported by National Institutes of Health Grant R21MH104471 and R01MH106511 (D.M.C.), Brain Research Foundation SG 2012-01 (D.M.C.), Northwestern University Clinical and Translational Sciences Institute 8UL1TR000150 (Y.H.), Chicago Biomedical Consortium HTS-004 (Y.H. and D.M.C.), and National Institutes of Health Grant 2T32MH067564 (K.L.). A part of this work was performed by the Northwestern University Medicinal and Synthetic Chemistry Core (ChemCore) at the Center for Molecular Innovation and Drug Discovery (CMIDD), which is funded by the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust and Cancer Center Support Grant P30 CA060553 from the National Cancer Institute awarded to the Robert H. Lurie Comprehensive Cancer Center. The high throughput screen work was performed in the High Throughput Analysis Laboratory which is also a core facility of the Robert H. Lurie Comprehensive Cancer Center.

1. TRIP8b（241-602）蛋白的纯化<ol><li>变换含TRIP8b（241-602）中的细菌蛋白表达载体pGS21 3到感受态质粒大肠杆菌用于根据制造商的说明蛋白的表达。板300微升上卢里亚肉汤（LB）文化 - 琼脂用5微克/毫升氯霉素和氨苄西林。孵育在37℃的板16小时。 </li><li>第二天，挑取单个菌落接种50毫升LB用50微克/毫升氯霉素和氨苄西林。孵育在37℃下培养16小时（振荡）。 </li><li> 50ml培养添加到1升的LB用50微克/毫升氨苄青霉素的。在37℃孵育振荡。 </li><li>一旦培养达到的OD 600读数的0.8-1.2，培养箱的温度改变到18℃，并添加IPTG（异丙基-β-D-硫代半乳糖苷）至1mM的终浓度。允许蛋白质表达进行16小时。 </li><li>降速E.大肠杆菌在4℃6000×g离心15分钟以沉淀细菌。从离心机取出管后，保持细菌在冰上的过程的剩余。重悬于36毫升缓冲液A的细菌与0.25毫米的氟苯甲（PMSF）。 </li><li>超声处理用扁平螺栓在冰上再悬浮细菌5-10分钟，"上"在高功率和30秒"关"30秒之间交替。 </li><li>离心机以12,000 xg离心在4℃下15分钟。离心额外15分钟，如果上清液尚不清楚。 </li><li>适用上清液至2ml的Ni-NTA珠的柱。孵育在4℃下轻轻摇动60分钟。 </li><li>使未结合的材料通过柱流动并用500ml缓冲液A洗</li><li>用250毫升缓冲液B洗柱</li><li>洗柱用125毫升缓冲液A中添加5 mM咪唑。 </li><li>洗脱蛋白质来回回米，20毫升洗脱缓冲液的柱。 </li><li>洗脱的蛋白添加至透析盒（分子量截留值 - 10 kDa）的使用注射器。透析的蛋白质在4℃下在4升冷磷酸盐缓冲盐水（PBS）中的60分钟。 </li><li>移动透析盒冷PBS的一个新的4升一桶。在4℃透析蛋白16小时</li><li>第二天早上，检查用Coomassie蛋白质检测试剂盒中的蛋白质浓度，按照制造商的说明进行操作。使用蛋白浓缩，按照制造商的说明，如果观察到低于40μM的浓度浓缩蛋白质。等分试样并冷冻在-80℃下贮存。 </li></ol> 2.小​​型荧光偏振检测，以表征两个蛋白片段相互作用<ol><li>解冻将200μl40μM的TRIP8b（241-602），并把它添加到1.5ml管中。加入100微升的PBS至11追加15毫升管。 </li><li>通过从原始管中的一系列转印100μl的TRIP8b（241-602）的下管，上下吹打，并重复该过程执行连续稀释。这将产生从0.01-40微米，12点系列TRIP8b（241-602）的2倍稀释的。 </li><li>制备0.1μMHCN1 FITC（6.5微升10μM的等分试样的）和2mM二硫苏糖醇（DTT）在PBS中的650微升主混合物。 注:主结构为2倍。 </li><li>设立了12个新的1.5 mL管。加入50μl含HCN1 FITC每个管的主混合物，然后从每个12系列稀释的加入50微升。 注意:这将产生12管，各含0.05微米HCN1 FITC和TRIP8b（241-602）的浓度的一半从原来的连续稀释。 </li><li>稀释系列加入测定板，30微升每孔，一式三份。使用低结合实心黑色384孔板。 </li><li>旋转的托盘f或在RT 900 xg离心2分钟。 </li><li>用酶标仪，按照制造商的指示读板。使用以下测量参数测量荧光偏振:485nm激发，530nm处发射，在505nm分色镜，100毫秒的积分时间。 </li><li>绘制极化（MP）的值相对于TRIP8b对数标度（241-602）的浓度。与希尔方程来确定亲和力（K D）TRIP8b（241-602）的用于标记的HCN1肽拟合数据。 注意:为了制备系列稀释液，多孔板也可以用来代替管。 </li></ol> 3.检查蛋白质相互作用使用阳性对照<ol><li>加入200μl未标记HCN1肽（200μM）的到1.5ml管中。加入100微升的PBS至11追加15毫升管。通过从含有200μl的肽以在第一管的管转移100μl的执行连续稀释液的100微升PBS中。重复这个过程，产生12管与未标记HCN1肽的2倍稀释。 </li><li>准备0.2μMHCN1 FITC和4μMTRIP8b（241-602）的650μl反应混合液。 注:这是一个2倍的解决方案。 </li><li>加入50μl反应混合液到12新的1.5 mL管。 </li><li>加入50μl的每个系列稀释的到1.5ml管中。 </li><li>装载在一式三份的黑色384孔微量滴定板，每孔加入30微升。 </li><li>离心板在室温900 XG 2分钟。 </li><li>阅读使用能够FP的酶标仪板。如步骤2.7中所述使用相同的设置。 </li><li>计算使用Cheng-Prusoff方程为TRIP8b未标记的肽（241-602）的亲和力:K d 1 的 = IC 50 /（1+ [L] / K D 2），其中K d 1 的代表未标记的亲和肽为TRIP8b（241-602），IC 50是在前面的步骤确定的并代表未标记的配位体的置换标记的配体的能力，[L]为标记的配体的在步骤3.2的浓度，和K D 2是TRIPb（241-602）的该标记配体的亲和力。 </li></ol> 4.评估分析性能（计算Z参数） <ol><li>通过使FP缓冲的12 ml溶液与2微米TRIP8b（241-602），50nM的HCN1 FITC，和1mM DTT中的FP缓冲制备主混合物。 注:这将需要60微升40μMTRIP8b（241-602）的60微升10μMHCN1 FITC，而1080微升FP缓冲。 </li><li>添加30微升主混合物到一个黑色384孔微量滴定板的每个孔中。 </li><li>加入1微升1毫米未标记HCN1肽对192口井作为阳性对照。 </li><li>加入1μlFP缓冲液至192孔充当阴性对照。 </li><li>离心板在室温900 XG 2分钟。 </li><li>阅读使用MI板croplate读卡器。 </li><li>使用下列公式计算测定在Z因子Z = 1 - 3 *（σPOS-σ 负 ）/（μ 负 -μ 正 ）其中σPOS和σ 负是阳性和阴性对照孔的标准偏差，μPOS和μ 负表示在阳性和阴性对照孔的平均信号。 </li></ol> 5.高通量筛选<ol><li>解冻TRIP8b（241-602）和HCN1 FITC的等份，并保持他们在冰上。 </li><li>在FP缓冲准备10毫升TRIP8b（2μM）和HCN1 FITC（50纳米）。 </li><li>分配25微升混合物的进入低结合使用移液管384孔黑色微量滴定板的每个孔中。 </li><li>用于文库筛选中，添加化合物成使用声液体处理器列的各孔3〜22（40μM终浓度为每）。 注:由于relativel与此法观察ý低命中率，两种不同的化合物被汇集成一个单一的孔。从每个活动以及这两种化合物随后单独进行测试，以确定哪些所赋予的活性。 </li><li>添加100 NL二甲亚砜（DMSO）加入到1列和各板23作为阴性对照的每个孔中。 </li><li>添加未标记HCN1肽成2列和各板24作为阳性对照的各孔中。 </li><li>孵育在室温将板2小时。读板或阅读之前，在4℃孵育16小时。 </li><li>测量酶标仪荧光偏振。如步骤2.7中所述使用相同的设置。 </li><li>通过使用从每个平板为100％的平均阳性和阴性对照归一化信号计算抑制百分比。使用方程XN = 100％*（（X 负 - X）/（X 负 - X 正 ）），其中X，N是对应于信号X的归一化抑制百分率，X POS </sub&gt;是从阳性对照孔的平均信号，和X 负距离阴性对照孔的信号。 </li></ol> 6.点击确认使用TAMRA标记肽HCN <ol><li>验证命中使用TAMRA标记HCN1肽（HCN1 TAMRA）超过50％的抑制。 <ol><li>准备12×2微米TRIP8b（241-602）和50纳米HCN1 TAMRA在FP缓冲的主结构。 </li><li>分配25微升主混合物的成黑色384孔微量滴定板的每个孔中。 </li><li>转移的每个活性化合物的系列稀释液到相应的孔中。使两倍稀释，使得每个的浓度击中范围从0.2至200μm。 </li><li>为阴性对照的反应，转移100 NL DMSO中到每个孔中。为阳性对照添加100 NL系列稀释未标记的HCN1肽的终浓度范围从200μM至0.1μM。 </li><li>孵育在室温在板2小时。读板或在4孵育16小时 阅读前°以下。 </li><li>使用以下测量参数测量荧光偏振（MP）:535毫微米激发; 580 nm发射; 535纳米分色镜，20毫秒的积分时间。 </li><li>通过使用四参数非线性回归模型23拟合各化合物的浓度依赖的FP数据计算的IC 50值。 </li></ol></li></ol> 7.基于微珠的亲近试验的剂量 - 反应验证<ol><li>解冻1等分试样His-标记TRIP8b（241-602）（他-TRIP8b）和GST标签HCN1（GST-HCN1 C40）融合蛋白的，并保持他们在冰上。 </li><li>在400nm的His-TRIP8b和40纳米的GST HCN1 C40的浓度结合的His-TRIP8b与GST-HCN1 C40在测定缓冲液。制备300微升混合物的每种化合物进行测试。 注:化合物一式三份在11个不同浓度的加了DM运行SO控制（无化合物）。 </li><li>对于对照孔，准备30微升他-TRIP8b（200纳米），而GST-HCN1C40，然后分别在分析缓冲液无他，TRIP8b准备30微升GST-HCN1C40（20纳米）。 </li><li>使用16通道移液器的GST-HCN1 C40混合物（在上面的步骤7.2的方法制备）至36孔的板:为每个要测试的化合物中，添加7微升的His-TRIP8b的。为了测试单一化合物，安排行AL的12个不​​同浓度，并在列1-3三次重复。新增7微升了他的-TRIP8b溶液（7.3编写），以列排M的1-3和7微升GST-C40 HCN1溶液（7.3编写），以排N. 1-3列短暂离心板确保液体是在每个孔的底部，以除去任何气泡。 </li><li>分配的命中化合物待测试入盘，使得每个的浓度击中范围为200微米（在A行）向下至0.1微米（在排K）。分配的体积DMSO中以行LN匹配化合物的行的AK分配的量。 </li><li>摇板2分钟，在室温下孵育2.5小时。 </li><li>稀释抗GST受体珠1:50用测定缓冲液。 </li><li>添加3.5微升的稀释受体珠粒以16通道移液器板的各孔中，轻轻吹打，以避免产生气泡混合。 </li><li>将培养板在室温在黑暗中1小时。 </li><li>稀释镍螯合供体珠1:50与分析缓冲液。不要混合物置于阴暗处。 </li><li>添加3.5微升的稀释供体珠以16通道移液器板的各孔中，轻轻吹打，以避免产生气泡混合。 </li><li>将培养板在室温在黑暗中1.5小时。 </li><li>阅读酶标仪。使用具有以下测量参数酶标仪:40毫秒激发时间，100毫秒发射时间为0.1mm检测高度。 </li><li>由选管会数据拟合计算IC 50值使用四参数非线性回归模型23小时化合物。 </li></ol>

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Y., Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+dorsal+hippocampal+activity+by+knockdown+of+HCN1+channels+leads+to+anxiolytic-+and+antidepressant-like+behaviors.">Enhancement of dorsal hippocampal activity by knockdown of HCN1 channels leads to anxiolytic- and antidepressant-like behaviors.</a> Neuron. 75 (3), 503-516 (2012).</li><li>Wahl-Schott, C., Biel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HCN+channels:+structure,+cellular+regulation+and+physiological+function.">HCN channels: structure, cellular regulation and physiological function.</a> Cell Mol Life Sci. 66 (3), 470-494 (2009).</li><li>DeBerg, H. A., Bankston, J. R., Rosenbaum, J. C., Brzovic, P. S., Zagotta, W. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hill+equation+revisited:+uses+and+misuses.">The Hill equation revisited: uses and misuses.</a> FASEB J. 11 (11), 835-841 (1997).</li><li>Prinz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hill+coefficients,+dose-response+curves+and+allosteric+mechanisms.">Hill coefficients, dose-response curves and allosteric mechanisms.</a> J Chem Biol. 3 (1), 37-44 (2009).</li><li>Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Guide+to+Simple+and+Informative+Binding+Assays.">A Guide to Simple and Informative Binding Assays.</a> Mol Biol Cell. 21 (23), 4061-4067 (2010).</li><li>Rossi, A. M., Taylor, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein-ligand+interactions+by+fluorescence+polarization.">Analysis of protein-ligand interactions by fluorescence polarization.</a> Nat Protoc. 6 (3), (2011).</li><li>Ryan, A. J., Gray, N. M., Lowe, P. N., Chung, C. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Detergent+on+"Promiscuous"+Inhibitors.">Effect of Detergent on "Promiscuous" Inhibitors.</a> J Med Chem. 46 (16), 3448-3451 (2003).</li><li>McGovern, S. L., Caselli, E., Grigorieff, N., Shoichet, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+mechanism+underlying+promiscuous+inhibitors+from+virtual+and+high-throughput+screening.">A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening.</a> J Med Chem. 45 (8), 1712-1722 (2002).</li><li>Hertzberg, R. P., Pope, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening:+new+technology+for+the+21st+century.">High-throughput screening: new technology for the 21st century.</a> Curr Opin Chem Biol. 4 (4), 445-451 (2000).</li><li>Sundberg, S. 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The authors have nothing to disclose.

因为其在MDD 24的治疗目标电位的，出现了在拮抗HCN通道功能在中枢神经系统4药理学方法相当大的兴趣。然而，这些努力已经被HCN通道在心脏起搏中的重要作用和心律不齐25的风险停滞不前。我们推断，破坏HCN和其大脑特定辅助亚基，TRIP8b 8之间的相互作用可能是足以产生，而不会影响心脏HCN通道3抗抑郁样作用。这一假说是由缺乏的小鼠表现出TRIP8b抗抑郁样...

超极化激活环核苷酸门控（HCN）频道在心脏和中枢神经系统，其中它们起到调节膜兴奋1中起重要作用表示。 HCN通道有牵连抑郁症（MDD）2，这导致几个组提议限制性HCN通道功能的药理学可以有效地作为一种新型治疗MDD 3的发病机制。然而，直接针对HCN通道是因为它们在心脏动作电位4重要的作用并不可行。伊伐布雷定，唯一的FDA批准的HCN通道拮抗剂，用于心脏衰竭，以产生一个心动过缓作用5的治疗。因此，有必要为限制HCN通道在中枢神经系统专门功能的药理学试剂。 Rab8b相互作用蛋白（TRIP8b）三十四肽重复含，是一种大脑特定FIC HCN通道控制HCN通道6,7的表面表达和定位的辅助亚基。 TRIP8b基因敲除导致而不影响心脏HCN 8表达降低大脑HCN通道7。有趣的是，TRIP8b基因敲除小鼠花强迫游泳任务，尾吊任务7，两种常用的筛选试验的抗抑郁疗效9-11时间少动。这些结果表明，而不是直接靶向HCN通道与HCN通道功能的小分子拮抗剂，扰乱TRIP8b和HCN之间的相互作用可以是足以产生抗抑郁样行为。 TRIP8b结合氰化氢在两个不同的结合位点。 HCN的环核苷酸结合结构域（CNBD）与TRIP8b位于N端的到的TRIP8b 12,13的TPR结构域的保守结构域相互作用。虽然所涉及在此CNBD的残相互作用已经映射14，即所涉及的TRIP8b的区域还没有被缩小之外的80个氨基酸的片段13。 TRIP8b的三十四肽重复（TPR）结构域和HCN的C末端三肽（&#39;SNL&#39;在HCN1，HCN2和HCN4，而是"ANM"在HCN3）3,12之间发生的第二交互作用。本C尾相互作用的最近解析晶体结构15揭示实质结构相似性的过氧化物酶体导入受体之间的相互作用，peroxin 5（PEX5），和其相互作用伙伴，含有1型的过氧化物酶体靶向序列（PTS1）16。 虽然两者相互作用位点所必需的HCN通道功能，TRIP8b的TPR结构域和HCN1的C-末端三肽之间的相互作用作为主导结合位点和调节HCN表面表达。因此，这种相互作用被选为本研究中的定位点。为原稿，当提及到TRIP8b和HCN之间的相互作用制成的其余部分，它是这种相互作用正在提及。这种相互作用是由TRIP8b的对应于含有它的保守C端结合的HCN的C-末端尾部（残基的第1a-4同种型TRIP8b的241-602）3所需的TPR结构域的高可溶性片段概括。 为了开发高通量筛选，以确定能够破坏这种互动的小分子，荧光偏振（FP）为基础的测定采用17。荧光偏振是基于与偏振光，并测量发射的荧光18的偏振度的荧光团标记的配体的激励。在结合伴侣的存在，所述荧光配体的旋转运动被约束和偏振光发射19。在没有一个结合伴侣，T他的配位体的旋转运动导致去极化光的发射。 在封闭的协议，提出了N末端标记（的6xHis）TRIP8b使用镍氮基三乙酸（的Ni-NTA）珠粒纯化（241-602）的方法。一个类似的协议是用来净化该协议的步骤7中使用的谷胱甘肽-S-转移酶（GST）-taggedçHCN1端40个氨基酸（HCN1 C40）。出于空间考虑，省略该步骤的详细描述。 在步骤2到协议的7，高通量筛选工作流提出了（ 见图1）。蛋白质-蛋白质相互作用的高通量筛选出了名的困难的目标，并建议读者去寻找关于这个专题的20个额外的资源。 步骤2和步骤3表征纯化TRIP8b（241-602） 的体外构建亲和力FO岭异硫氰酸荧光素（FITC）-tagged对应HCN1（HCN1 FITC）的C-末端尾部11个氨基酸的肽。基于所述TRIP8b-HCN络合物15的晶体结构该11氨基酸片段是足以产生与TRIP8b（241-602）的结合。在步骤2中，相互作用的K个d由滴定TRIP8b（241-602）插入HCN1 FITC的固定浓度测定。在步骤3，在步骤2中使用的氰化氢肽的未标记的版本滴定到两个TRIP8b（241-602）和HCN1 FITC的固定浓度如果FITC标记具有结合干扰检查。这些实验都是在选择高通量筛选使用TRIP8b（241-602）和HCN1 FITC的适当浓度是必不可少的。 高通量筛选的前提是能够扰乱TRIP8b（241-602）和HCN1 FITC之间的相互作用将产生一个分解的小分子rease在偏振光。在步骤4中，测定的Z因子计算21 TRIP8b（241-602）和HCN1 FITC的给定浓度，以确保该测定是适当的高通量筛选（步骤5）。步骤6和7是验证实验，以确认在初级高通量筛选中鉴定的命中通过破坏TRIP8b（241-602）和HCN1 FITC之间，而不是通过非特异性机制的交互作用。在步骤6中，carboxytetramethylrhodamine（TAMRA）标记的HCN1肽（HCN1 TAMRA）是在其它方面相同的荧光偏振测定法用于筛选危及使用FITC标记的FP试验的荧光化合物。步骤7使用较大HCN1 C端片段（HCN1 C40）并采用基于珠子的亲近测定，这是基于从供体珠子单线态氧的"隧道"由相互作用的蛋白质带到彼此接近的受体珠22。

<table><tbody><tr><td>Ni-NTA agarose&amp;nbsp;</td><td>Qiagen</td><td>30210</td><td></td></tr><tr><td>Dialysis cassette&amp;nbsp;</td><td>ThermoFisher</td><td>66456</td><td></td></tr><tr><td>Isopropyl b-D-1-thiogalactopyranoside&amp;nbsp;</td><td>Sigma-Aldrich</td><td>I5502 - 1 gr</td><td></td></tr><tr><td>384-Well Black plate&amp;nbsp;</td><td>Corning</td><td>3820</td><td></td></tr><tr><td>Proxiplate&amp;nbsp;</td><td>Perkin-Elmer&amp;nbsp;</td><td>6008289</td><td></td></tr><tr><td>Anti-GST Acceptor beads&amp;nbsp;</td><td>Perkin-Elmer&amp;nbsp;</td><td>6760603C</td><td></td></tr><tr><td>NiChelate</td><td>Perkin-Elmer&amp;nbsp;</td><td>AS101D</td><td></td></tr><tr><td>pGS21-a</td><td>Genscript</td><td>SD0121</td><td></td></tr><tr><td>PMSF</td><td>Sigma-Aldrich</td><td>10837091001</td><td></td></tr><tr><td>Coomassie Kit</td><td>ThermoFisher</td><td>23200</td><td></td></tr><tr><td>Protein concentrator</td><td>ThermoFisher</td><td>88527</td><td></td></tr><tr><td>Perkin Elmer Enspire Multimode Plate reader</td><td>Perkin-Elmer&amp;nbsp;</td><td>#2300-001M</td><td></td></tr><tr><td>BL21 (DE3) Competent Cells</td><td>Agilent</td><td>200131</td><td></td></tr></tbody></table>

method for identifying small molecule inhibitors of the protein-protein interaction between hcn1 and trip8b

超极化激活环核苷酸门控（HCN）频道整个大脑，在那里它们起作用来调节神经元的兴奋遍在表达。海马CA1区锥体细胞这些通道的亚细胞分布由三十四肽含有重复-Rab8b相互作用蛋白（TRIP8b），辅助亚基调节。 HCN的遗传敲除孔形成亚基或TRIP8b，既导致增加抗抑郁样行为，这表明限制HCN通道的功能可以是作为用于抑郁症（MDD）的治疗是有用的。尽管显著治疗意义，HCN通道也都在心脏，在那里它们调节节律性表达。规避与阻断心脏HCN通道相关联的脱靶的问题，我们的实验室最近提出靶向为了专门破坏HCN通道功能在大脑HCN和TRIP8b之间的蛋白质 - 蛋白质相互作用。TRIP8b结合HCN孔形成在两个不同的相互作用位点亚基，尽管这里的重点是TRIP8b的三十四肽重复（TPR）结构域和HCN1的C-末端尾部之间的相互作用。在这个协议中，一种方法的用于纯化TRIP8b并执行高通量筛选，以鉴定HCN和TRIP8b之间的相互作用的小分子抑制剂的扩展的说明中，进行说明。用于高通量筛选的方法，利用荧光偏振（FP）为基础的测定以监测大TRIP8b片段荧光团标记十一结合氨基酸对应于HCN1 C端尾酸肽。基于在发射的光的偏振的变化，以确定该方法允许&#39;命中&#39;的化合物。然后进行验证试验，以确保&#39;打&#39;的化合物不是人为的。

以避免与我们先前的出版物的版权问题，一个TAMRA标记的探针HCN1 TAMRA用于生成图2和3。需要注意的是，这种替代并没有使在结果有明显的差别，并且这些协议是相同的上述与HCN1 FITC。来评估与HCN1 TAMRA，TRIP8b（241-602）的相互作用滴定成使用在步骤2（ 图2）中概述的协议HCN1 TAMRA的固定浓度。接着，进行在...

Watch this Scientific Journal Video about Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b at JoVE.com

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

HCN通道和其辅助亚基之间的相互作用已被确定为在抑郁症的治疗靶点。这里，用于识别这种蛋白质 - 蛋白质相互作用的小分子抑制剂荧光基于偏振的方法，提出。

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

method-for-identifying-small-molecule-inhibitors-protein-protein

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

8.5K 次观看.  Feinberg School of Medicine, Northwestern University.  HCN通道和其辅助亚基之间的相互作用已被确定为在抑郁症的治疗靶点。这里，用于识别这种蛋白质 - 蛋白质相互作用的小分子抑制剂荧光基于偏振的方法，提出。 

视频: Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

通过亲和色谱与西布洛特或质谱结合的亲致色谱,识别磷酸磷酸磷酸磷酸醇或磷酸蛋白

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

科研

生物化学

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular processes. The conserved MEEVD pentapeptide at the C-terminus of Hsp90 is responsible for the interaction with the tetratricopeptide repeat (TPR) motif of co-chaperones. FK506-binding protein (FKBP) 51 and FKBP52 are two similar TPR-motif co-chaperones involved in steroid hormone-dependent diseases with different functions. Therefore, identifying molecules specifically blocking interactions between Hsp90 and FKBP51 or FKBP52 provides a promising therapeutic potential for several human diseases. Here, we describe the protocol for an amplified luminescent proximity homogenous assay to probe interactions between Hsp90 and its partner co-chaperones FKBP51 and FKBP52. First, we have purified the TPR motif-containing proteins FKBP51 and FKBP52 in glutathione S-transferase (GST)-tagged form. Using the glutathione-linked donor beads with GST-fused TPR-motif proteins and the acceptor beads coupled with a 10-mer C-terminal peptide of Hsp90, we have probed protein-protein interactions in a homogeneous environment. We have used this assay to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

This protocol presents a technique for probing protein-protein interactions using glutathione-linked donor beads with GST-fused TPR-motif co-chaperones and acceptor beads coupled with an Hsp90-derived peptide. We have used this technique to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

使用同质珠为基础的检测的查佩龙-科查佩龙相互作用研究

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular ...

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).

一种通过牛磺胆酸钠共转运多肽作为治疗靶点检查乙型肝炎病毒进入的感受态肝细胞模型

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...

使用生物物理测定表征 PROTAC 中三元复合物的形成

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the &#39;hook effect&#39; on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 &#181;M). In that case, the binding for CRBN in SPR was not saturable, resulting in a &#34;hook-effect&#34;. Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

作者聚焦：评估用于表征 PROTACS 三元复合物的生物物理检测 

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

生物学

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

鉴定蛋白质 - 蛋白质相互作用的<em&gt;果蝇</em&gt;成人头由串联亲和纯化（TAP）

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently inhibit viral replication, they have no direct effect on the expression of viral proteins transcribed from a viral covalently closed circular DNA (cccDNA). As high viral antigen load may play a role in this chronic and HBV-related carcinogenesis, the goal of HBV treatment is to eradicate viral proteins. HBV regulatory protein X (HBx) binds to the host DNA damage-binding protein 1 (DDB1) protein to degrade structural maintenance of chromosomes 5/6 (Smc5/6), resulting in activation of viral transcription from cccDNA. Here, using a split luciferase complementation assay system, we present a comprehensive compound screening system to identify inhibitors of the HBx-DDB1 interaction. Our protocol enables easy detection of interaction dynamics in real time within living cells. This technique may become a key assay to discover novel therapeutic agents for treatment of HBV infection.

Here, we present a method for screening anti-hepatitis B viral agents that inhibit the HBx-DDB1 interaction using a split luciferase assay system. This system allows easy detection of protein-protein interactions and is suitable for identifying inhibitors of such interactions.

使用分体路西酶测定系统识别 HBx-DDB1 相互作用的抑制剂

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently ...

医学

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

鉴定蛋白质相互作用的网站使用多肽阵列

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

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通过亲和色谱与西布洛特或质谱结合的亲致色谱,识别磷酸磷酸磷酸磷酸醇或磷酸蛋白

使用同质珠为基础的检测的查佩龙-科查佩龙相互作用研究

一种通过牛磺胆酸钠共转运多肽作为治疗靶点检查乙型肝炎病毒进入的感受态肝细胞模型

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定

鉴定蛋白质 - 蛋白质相互作用的

使用分体路西酶测定系统识别 HBx-DDB1 相互作用的抑制剂

鉴定蛋白质相互作用的网站使用多肽阵列