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）和非典型p38。路易斯·施塔特和乔纳森·阿什维尔（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。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。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。免疫系统的特发性激活是类风湿性关节炎和系统性红斑狼疮等自身免疫性疾病的中心介质。自身免疫性疾病已经非常普遍，并且预计会随着全球人口老龄化而增加。因此，了解这些疾病的病因和开发新的治疗方法是至关重要的。为此，我们正在与Jonathan Ashwell博士（LICB）合作，他已经确定了一种新的机制，通过这种机制，免疫系统可以被不适当地激活。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。将TCR激活信号转化为炎症反应的激酶的典型类别是丝裂原活化蛋白激酶（MAPK）家族，其中p38激酶是最重要的成员。在Ashwell实验室最近的工作之前，人们了解到p38的激活是通过一系列上游MAPK家族成员（MKK3/4/6）依赖于磷接力的信号级联的结果。这最终导致p38通过苏氨酸180 （T180）和酪氨酸182 （Y182）激活环的双重磷酸化而完全激活。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回路的能力，从而在前馈信号级联中激活自身。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，可选激活的p38启动促炎级联反应，随后发生t细胞依赖性炎症。我们与Ashwell实验室合作的核心目标是发挥我们在开发高通量筛选方面的专业知识，以及我们独特的纯化合物和天然产物提取物文库，以找到能够特异性抑制可选活化p38（在Y323位点磷酸化）但不会抑制经典活化p38激酶（在Y323位点未磷酸化）的小分子。为了实现这一目标，Ashwell实验室提供了p38的可选活化形式，我们建立了一种能够灵敏检测该酶抑制剂的酶分析方法。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（在T180和Y182位点磷酸化）建立了二级检测。只有那些特异性抑制替代激活途径的抑制剂才会被认为是命中的。此外，Ashwell实验室将在几种t细胞模型中测试这些靶向物特异性抑制替代途径的能力。我们相信我们在初级筛选中具有必要的敏感性，在二级分析中具有相应的特异性，可以探索该途径抑制剂化合物库的全部化学多样性。

This project resulted in the development high throughput screens for the targets mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) and non-cannonical p38 in collaboration with Drs. Louis Staudt and Jonathan Ashwell (CCR). In addition we kinetically characterized inhibitors of tyrosyl-DNA phosphodiesterase-1 (Tdp1) in collaboration with Dr. Yves Pommier (CCR). 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. 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. Idiopathic activation of the immune system is a central mediator of autoimmune disorders like rheumatoid arthritis and systemic lupus erythematous. Autoimmune disorders are already highly prevalent and are predicted to increase as the global population ages. Therefore, understanding the etiology of these diseases and developing novel therapeutics to combat them is critically important. To this end we are collaborating with Dr. Jonathan Ashwell (LICB), who has identified a novel mechanism by which the immune system can become inappropriately activated. 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 canonical class of kinases that transduces the TCR activation signal into an inflammatory response is that of the Mitogen Activated Protein Kinase (MAPK) family, of which the p38 kinase is the pre-eminent member. Prior to the recent work of the Ashwell lab it was understood that p38 activation was the result of a signaling cascade dependent on a phosphorelay through a series of upstream MAPK family members (MKK3/4/6). This ultimately results in the complete activation of p38 through the dual phosphorylation of its activation loop at threonine 180 (T180) and tyrosine 182 (Y182). 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 the classically activated p38, phosphorylation at Y323 imbues p38 with the ability to autophosphorylate its own activation loop, thus activating itself in a feed forward signaling cascade. 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 and subsequent T-cell dependent inflammation ensues. The central goal of our collaboration with the Ashwell lab is to bring to bear our expertise in developing high-throughput screens and our unique libraries of both pure compounds and natural product extracts to find small molecules that will specifically inhibit the alternatively activated p38 (phosphorylated at Y323) but will not inhibit the classically activated p38 kinase (unphosphorylated at Y323). To accomplish this goal, 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 (phosphorylated at T180 and Y182). Only those inhibitors that specifically inhibit the alternatively activated pathway will be considered hits for the purposes of this screen. Additionally, the Ashwell lab will test these hits for their ability to specifically inhibit only the alternative pathway in several T-cell models. We believe we have the requisite sensitivity in our primary screen and the commensurate specificity in our secondary assays to probe the full chemical diversity of our compound libraries for inhibitors of this pathway.