Cell-free assay technologies for the identification of active compounds

该项目导致开发高通量筛选目标粘膜相关淋巴组织淋巴瘤易位蛋白1 （MALT1）和polo样激酶1 （Plk1）。Louis Staudt和Kyung Lee （CCR）。此外，我们与Yves Pommier博士（CCR）合作，对酪氨酸- dna磷酸二酯酶-1 （Tdp1）抑制剂进行了动力学表征。Plk1属于丝氨酸/苏氨酸蛋白激酶的polo样激酶家族，与周期蛋白依赖性激酶相似，在细胞增殖中起重要作用。它在80%的人类癌症中过度表达。相反，两个最密切相关的激酶，Plk2和Plk3，似乎在检查点介导的细胞周期阻滞中发挥作用，以确保遗传稳定性。特异性抑制Plk1，而不是Plk2或Plk3，是抗plk癌症治疗的关键。Plk蛋白有两个功能上至关重要的靶点，一个是与其他几种蛋白激酶密切相关的激酶结构域，另一个是独特的polo-box结构域（PBD），它已被证明具有特异性。因此，靶向激酶结构域的化合物可能对Plk1的特异性较低，而靶向PBD结构域的化合物可能更具特异性。该项目的目标是鉴定Plk1的抑制剂。与Dr. Kyung Lee （LM）合作，我们设计了一种具有竞争力的结合试验，用于抑制pT78肽与Plk1的PBD之间结合的天然产物提取物。将酶联免疫吸附试验修改为384孔板，对MTL纯化合物文库和MTL预分离天然产物提取物文库进行筛选。实验从涂膜步骤开始，将生物素化的pT78肽（9-mer PBIP序列）添加到链霉亲和素涂膜的黑色384孔板的底部。实验进展到结合步骤，将全长GST-Plk1（单独和在测试样品存在的情况下）添加到表面结合的pT78肽上。结合Plk1的水平通过ELISA测定，使用Plk1特异性一抗，通过比色检测可视化。共筛选了97,504个预分馏天然产物提取物样品，产生了1573个初始命中。这些经过筛选，优先考虑440个Z分数为&gt；4的提取物，用于确认分析（0.45%的初始命中率）。确认试验产生185个天然产物提取物组分，然后根据NPCS标准对其进行优先排序，并在剂量反应试验中进行测试。剂量反应结果确定了72个组分（0.07%的最终命中率），用于进一步的生物测定指导分离。在72个铅馏分样品中，24个样品的IC50值为20 mg/ml， 48个样品的IC50值在20-50 mg/ml之间。MALT1是一种含有824个氨基酸的多结构域蛋白，具有能够切割含精氨酸底物的蛋白水解副半乳糖酶结构域。MALT1已被证明可以切割多个参与NF-kappa B激活和Jun n -末端激酶激活的信号分子。此外，MALT1已被证明可以调节T细胞对纤维连接蛋白的粘附并激活caspase-8。特异性MALT1小分子蛋白酶抑制剂的发现将影响癌症和免疫学研究的许多方面。最近的研究表明，RNAi和肽抑制剂抑制MALT1蛋白酶活性导致活化的B细胞样弥漫性大B细胞淋巴瘤细胞系的存活率降低，为分子靶向MALT1治疗提供了原理证明。通过与Staudt实验室的合作，我们开发并验证了MALT1蛋白酶活性抑制剂的HTS检测。PCMBS承担的第一项任务是产生活性MALT1。构建了谷胱甘肽- s -转移酶（GST）标记的全长人MALT1亚型A表达载体。用BL21（DE3）大肠杆菌表达GST-MALT1酶。MALT1通过亲和、TEV蛋白酶裂解/ gst再吸附和大小排斥层析纯化至接近均匀性。通过切割Bcl-10衍生的荧光肽底物acetyl-Leu-Arg-Ser-Arg-4-methyl-coumaryl-7-amide （ac - lrs - mca）来检测纯化的重组MALT1的蛋白酶活性。优化的MALT1缓冲液为50 mM Tris-HCl， 1 mM DTT, 0.05% CHAPS, 0.1 mM EGTA， 0.8 M柠檬酸钠，pH = 7.5。最初，用MALT1不可逆抑制剂Z-VRPR-FMK滴定测定活性MALT1的浓度。得到的酶活性浓度为497 nM，占总蛋白的31%。对MALT1的线性和稳态动力学参数进行了表征。测定了MALT1反应的Km为103 uM。为了鼓励竞争性和非竞争性抑制剂的发现，底物浓度被设置为100 uM，以接近MALT1的Km。100 nM MALT1和100 uM Ac-LRSR-MCA的组合在130分钟内产生了线性分析响应，并且在60分钟内信号与背景的可接受比率（4.9）。在蛋白酶反应中加入0.1%的SDS可使MALT1蛋白酶活性停止。MALT1抑制化合物的HTS正在进行中，将包括MTL合成化合物和天然产物提取物文库的测试。介导免疫反应的一类主要免疫细胞是t细胞。t细胞活化的一种机制是通过t细胞受体（TCR）的参与，它启动细胞内激酶信号级联，最终导致促炎细胞因子的产生和炎症反应的诱导。Ashwell实验室现在已经证明，通过激活TCR近端激酶ZAP70， TCR接合可以介导p38激活，而无需经典的信号级联，从而导致p38在酪氨酸323 （Y323）处的新型磷酸化。与经典激活的p38不同，Y323位点的磷酸化使p38具有自磷酸化自身激活环的能力。已经证明这种现象是t细胞特异性的，并且通常在t细胞中由抑制性支架蛋白GADD45 α对抗。在缺乏GADD45 α介导的抑制的情况下，可选择性激活的p38启动促炎级联反应。我们与Ashwell实验室合作的目标是开发我们独特文库的高通量筛选，以找到能够特异性抑制可选活化p38而不抑制经典活化p38激酶的小分子。Ashwell实验室已经提供了p38的可选活化形式，我们已经建立了一种酶分析方法，能够灵敏地检测这种酶的抑制剂。为了确保这些抑制剂仅对替代途径具有特异性，我们使用经典活化的p38激酶建立了二级分析。确定筛选最合适的底物浓度可能是优化高通量生化测定时最重要的参数。因此，我们将在接近计算Km， 300 nM的底物（ATF2-GST, [S]）浓度下进行测定。在此浓度下，60分钟后底物损耗小于25%，分数活性应与化合物的预期Ki直接相关。在这个浓度下，我们相信我们可以合理地以可接受的基板成本收集50万个数据点。

This project resulted in the development high throughput screens for the targets mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) and Polo-like kinase 1 (Plk1) in collaboration with Drs. Louis Staudt and Kyung Lee (CCR). In addition we kinetically characterized inhibitors of tyrosyl-DNA phosphodiesterase-1 (Tdp1) in collaboration with Dr. Yves Pommier (CCR). Plk1 belongs to the polo-like kinase family of Ser/Thr protein kinases, and similarly to the cyclin dependent kinases, plays an important role in cellular proliferation. It is overexpressed in 80% of human cancers. In contrast, the two most closely related kinases, Plk2 and Plk3, appear to play a role in checkpoint-mediated cell cycle arrest to ensure genetic stability. Specific inhibition of Plk1, but not Plk2 or Plk3, is critical for anti-Plk cancer therapy. The Plk proteins have two functionally crucial target sites, a kinase domain that is closely related to several other protein kinases and the unique polo-box domain (PBD), which has been shown to confer specificity. As such, compounds that target the kinase domain may be less specific for Plk1, whereas those that target the PBD domain may be more specific. The goal of this project is to identify inhibitors of Plk1. In collaboration with Dr. Kyung Lee (LM) we have designed a competitive binding assay for natural product extracts that inhibit binding between a pT78 peptide and the PBD of Plk1. An ELISA assay was modified to 384-well plates and screened against MTL pure compound libraries as well as the MTL pre-fractionated natural product extract library. The assay begins with a coating step, where a biotinylated pT78 peptide (a 9-mer PBIP sequence) is added to the bottom of a streptavidin-coated black 384-well plate. The assay progresses to a binding step where full-length GST-Plk1 is added (alone and in the presence of test samples) to the surface-bound pT78 peptide. The level of bound Plk1 is measured by ELISA using a Plk1-specific primary antibody visualized by colorimetric detection. A total of 97,504 pre-fractionated natural product extract samples were screened resulting in 1573 initial hits. These were filtered to prioritize 440 extracts with a Z score >4 for confirmation assays (0.45% initial hit rate). The confirmation assays resulted in 185 natural product extract fractions, which were then prioritized according to NPCS criteria and tested in dose response assays. Dose response results identified 72 fractions (0.07% final hit rate) for further bioassay-guided fractionation. Out of the 72 lead fraction samples, 24 hits had IC50 <20 mg/ml and 48 hits had IC50 values between 20-50 mg/ml. MALT1 is an 824 amino acid, multi-domain protein that possesses a proteolytic paracaspase domain capable of cleaving arginine-containing substrates. MALT1 has been demonstrated to cleave multiple signaling molecules involved in NF-kappa B activation and Jun N-terminal kinase activation. In addition, MALT1 has been demonstrated to modulate T cell adhesion to fibronectin and activate caspase-8. The discovery of specific MALT1 small-molecule protease inhibitors would impact numerous aspects of cancer and immunological research. Recent research has shown that inhibition of MALT1 protease activity by RNAi and peptide inhibitors resulted in decreased survival in activated B cell-like diffuse large B-cell lymphoma cell lines, offering proof-of-principle for molecularly-targeted MALT1 therapies. In collaboration with the Staudt lab, we have developed and validated a HTS assay for inhibitors of MALT1 protease activity. The first task the PCMBS undertook was the production of active MALT1. An expression vector encoding glutathione-S-transferase (GST)-tagged, full-length human MALT1 isoform A was created. GST-MALT1 enzyme was expressed using BL21(DE3) E. coli. MALT1 was purified to near homogeneity using a combination of affinity, TEV protease cleavage/GST-readsorption, and size-exclusion chromatography. The protease activity of purified, recombinant MALT1 was monitored by cleavage of the Bcl-10 derived fluorescent peptide substrate acetyl-Leu-Arg-Ser-Arg-4-methyl-coumaryl-7-amide (Ac-LRSR-MCA). The optimized buffer for the MALT1 assay was determined to be 50 mM Tris-HCl, 1 mM DTT, 0.05% CHAPS, 0.1 mM EGTA, 0.8 M sodium citrate, pH = 7.5. Initially, the concentration of active MALT1 was determined by titration with the MALT1 irreversible inhibitor Z-VRPR-FMK. An active enzyme concentration of 497 nM was obtained, which represented 31% of the total protein. The linearity and steady-state kinetic parameters of MALT1 were then characterized. The Km of the MALT1 reaction was determined to be 103 uM. To encourage the discovery of both competitive and uncompetitive inhibitors, the substrate concentration was set to 100 uM to approximate the Km of MALT1. A combination of 100 nM MALT1 and 100 uM Ac-LRSR-MCA yielded a linear assay response through 130 minutes, and an acceptable ratio (4.9) of signal to background at 60 minutes. The addition of 0.1% SDS to the protease reaction stopped MALT1 protease activity. HTS for MALT1 inhibitory compounds is ongoing, and will include testing of MTL synthetic compound and natural product extract libraries. A major class of immune cells that mediate the immune response is the T-cell. One mechanism of T-cell activation is through the engagement of the T-cell receptor (TCR), which initiates an intracellular kinase signaling cascade ultimately resulting in the production of pro-inflammatory cytokines and the induction of an inflammatory response. The Ashwell lab has now demonstrated that TCR engagement can mediate p38 activation without the classical signaling cascade through the activation of a TCR proximal kinase, ZAP70, which results in the novel phosphorylation of p38 at tyrosine 323 (Y323). Unlike classically activated p38, phosphorylation at Y323 imbues p38 with the ability to autophosphorylate its own activation loop. It has been demonstrated that this phenomenon is T-cell specific and is normally opposed in T-cells by an inhibitory scaffolding protein, GADD45 alpha. In the absence of GADD45 alpha mediated inhibition the alternatively activated p38 initiates a pro-inflammatory cascade.The goal of our collaboration with the Ashwell lab is to develop a high-throughput screen of our unique libraries to find small molecules that will specifically inhibit the alternatively activated p38 without inhibiting classically activated p38 kinase. The Ashwell lab has provided the alternatively activated form of p38 and we have established an enzymatic assay capable of sensitively detecting inhibitors of this enzyme. To ensure the specificity of these inhibitors for only the alternative pathway we have established a secondary assay using the classically activated p38 kinase.Determination of the most appropriate substrate concentration for screening is likely the most important parameter when optimizing a high-throughput biochemical assay. We will therefore carry out the assay at a substrate (ATF2-GST, [S]) concentration near the calculated Km, 300 nM. At this concentration substrate depletion is less than 25% after 60 minutes and the fractional activity should directly correlate to the anticipated Ki of the compound. At this concentration, we believe that we can reasonably collect 500,000 data points at an acceptable substrate cost.