Cell-free assay technologies for the identification of active compounds

用于鉴定活性化合物的无细胞测定技术

• 批准号: 8763550
• 负责人: Barry Okeefe
• 金额: $ 81.5万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763550
• 关键词: ATF2 gene; Affinity; Age; Amides; Amino Acids; Arginine; Autoimmune Diseases; B-Lymphocytes; Basic Science; Biochemical; Biological Assay; Biological Factors; Buffers; CCR; Cell Adhesion; Cell Line; Cell model; Cells; Chemicals; Cleaved cell; Collaborations; Data; Development; Disease; Egtazic Acid; Ensure; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Escherichia coli; Etiology; Family; Family member; Fibronectins; GADD45; Glutathione S-Transferase; Goals; Human; Immune system; Inflammation; Inflammatory; Inflammatory Response; Kinetics; Length; Libraries; Ligands; Lupus; MAPK14 gene; Malignant Neoplasms; Measurement; Mediating; Mediator of activation protein; Mitogen-Activated Protein Kinases; Molecular Sieve Chromatography; Monitor; N-terminal; NCI Center for Cancer Research; NF-kappa B; Pathway interactions; Peptide Hydrolases; Peptides; Phosphorylation; Phosphotransferases; Population; Production; Protease Inhibitor; Protein Isoforms; Protein translocation; Proteins; RNA Interference; Reaction; Reagent; Receptor Activation; Recombinants; Research; Research Personnel; Rheumatoid Arthritis; Scaffolding Protein; Series; Signal Transduction; Signaling Molecule; Specificity; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; TEV protease; Technology; Tertiary Protein Structure; Testing; Threonine; Titrations; Tyrosine; Ursidae Family; Work; caspase-8; combat; cost; cytokine; expression vector; feeding; high throughput screening; inhibitor/antagonist; large cell Diffuse non-Hodgkin' s lymphoma; leucylarginine; macromolecule; member; mucosa-associated lymphoid tissue lymphoma; novel; novel therapeutics; pre-clinical; response; screening; small molecule; sodium citrate; tyrosyl-DNA phosphodiesterase

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.

该项目与 Drs. 合作开发了针对粘膜相关淋巴组织淋巴瘤易位蛋白 1 (MALT1) 和非规范 p38 目标的高通量筛选。路易斯·斯塔特和乔纳森·阿什韦尔 (CCR)。此外，我们与 Yves Pommier 博士 (CCR) 合作，对酪氨酰 DNA 磷酸二酯酶 1 (Tdp1) 抑制剂进行了动力学表征。 MALT1 是一种 824 个氨基酸的多结构域蛋白，具有能够裂解含精氨酸底物的蛋白水解副半胱天冬酶结构域。 MALT1 已被证明可以裂解参与 NF-kappa B 激活和 Jun N 末端激酶激活的多个信号分子。此外，MALT1 已被证明可以调节 T 细胞对纤连蛋白的粘附并激活 caspase-8。特定 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 蛋白酶活性会导致活化 B 细胞样弥漫性大 B 细胞淋巴瘤细胞系的存活率降低，这为分子靶向 MALT1 疗法提供了原理验证。我们与 Staudt 实验室合作，开发并验证了 MALT1 蛋白酶活性抑制剂的 HTS 测定。 PCMBS承担的第一个任务是产生活性MALT1。创建了编码谷胱甘肽-S-转移酶 (GST) 标记的全长人 MALT1 同工型 A 的表达载体。 GST-MALT1 酶使用 BL21(DE3) 大肠杆菌表达。使用亲和力、TEV 蛋白酶裂解/GST 重吸附和尺寸排阻色谱的组合将 MALT1 纯化至接近均质。通过裂解 Bcl-10 衍生的荧光肽底物乙酰基-亮氨酸-精氨酸-丝氨酸-精氨酸-4-甲基-香豆酰基-7-酰胺 (Ac-LRSR-MCA) 来监测纯化的重组 MALT1 的蛋白酶活性。 MALT1 测定的优化缓冲液为 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，通过用 MALT1 不可逆抑制剂 Z-VRPR-FMK 滴定来测定活性 MALT1 的浓度。获得的活性酶浓度为497 nM，占总蛋白的31%。然后对 MALT1 的线性和稳态动力学参数进行了表征。 MALT1 反应的 Km 测定为 103 uM。为了鼓励竞争性和非竞争性抑制剂的发现，底物浓度设置为 100 uM 以近似 MALT1 的 Km。 100 nM MALT1 和 100 uM Ac-LRSR-MCA 的组合在 130 分钟内产生线性测定响应，并在 60 分钟时产生可接受的信号与背景比率 (4.9)。向蛋白酶反应中添加 0.1% SDS 可终止 MALT1 蛋白酶活性。 MALT1 抑制化合物的 HTS 正在进行中，并将包括 MTL 合成化合物和天然产物提取物库的测试。我们与 Ashwell 实验室合作的目标是开发我们独特库的高通量筛选，以找到能够特异性抑制替代激活的 p38 而不抑制经典激活的 p38 激酶的小分子。 Ashwell 实验室提供了 p38 的替代激活形式，我们已经建立了一种酶测定法，能够灵敏地检测该酶的抑制剂。为了确保这些抑制剂仅针对替代途径的特异性，我们使用经典激活的 p38 激酶建立了二次测定。在优化高通量生化测定时，确定最合适的筛选底物浓度可能是最重要的参数。因此，我们将在接近计算的 Km（300 nM）的底物（ATF2-GST，[S]）浓度下进行测定。在此浓度下，60 分钟后底物消耗低于 25%，并且分数活性应与化合物的预期 Ki 直接相关。在此浓度下，我们相信我们可以以可接受的基质成本合理地收集 500,000 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核心介质。自身免疫性疾病已经非常普遍，并且预计随着全球人口老龄化而增加。因此，了解这些疾病的病因并开发新的疗法来对抗它们至关重要。为此，我们与 Jonathan Ashwell 博士 (LICB) 合作，他发现了一种新的机制，可以通过这种机制不当激活免疫系统。 T 细胞激活的一种机制是通过 T 细胞受体 (TCR) 的参与，启动细胞内激酶信号级联反应，最终导致促炎细胞因子的产生并诱导炎症反应。将 TCR 激活信号转导为炎症反应的典型激酶类型是丝裂原激活蛋白激酶 (MAPK) 家族，其中 p38 激酶是其中的杰出成员。在 Ashwell 实验室最近的工作之前，人们了解到 p38 激活是依赖于一系列上游 MAPK 家族成员 (MKK3/4/6) 的磷酸中继的信号级联的结果。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 实验室现已证明，TCR 参与可以通过 TCR 近端激酶 ZAP70 的激活介导 p38 激活，无需经典信号级联，从而导致 p38 在酪氨酸 323 (Y323) 处发生新型磷酸化。与经典激活的 p38 不同，Y323 的磷酸化使 p38 具有自身磷酸化其自身激活环的能力，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 细胞中被抑制性支架蛋白 GADD45-α 所对抗。在缺乏 GADD45-α 介导的抑制作用的情况下，替代激活的 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 实验室合作的中心目标是利用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库，以找到能够特异性抑制替代激活的 p38（磷酸化位点）的小分子。 Y323），但不会抑制经典激活的 p38 激酶（Y323 未磷酸化）。为了实现这一目标，Ashwell 实验室提供了 p38 的替代激活形式，并且我们建立了一种酶测定法，能够灵敏地检测该酶的抑制剂。为了确保这些抑制剂仅针对替代途径的特异性，我们使用经典激活的 p38 激酶（在 T180 和 Y182 处磷酸化）建立了二次测定。出于本次筛选的目的，只有那些特异性抑制替代激活途径的抑制剂才会被视为命中。此外，Ashwell 实验室将测试这些命中药物在几种 T 细胞模型中仅特异性抑制替代途径的能力。我们相信，我们在初步筛选中具有必要的灵敏度，在二次测定中具有相应的特异性，可以探测该途径抑制剂的化合物库的完整化学多样性。