Screening for Small Molecule Inhibitors of Eukaryotic Translation Initiation

真核翻译起始小分子抑制剂的筛选

• 批准号: 7303688
• 负责人: JERRY PELLETIER
• 金额: $ 2.34万
• 依托单位: MCGILL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7303688
• 关键词: Adopted; Amino Acids; Apoptotic; Bacteriophages; Binding; Binding Sites; Biological; Biological Assay; Cancer Model; Cell Cycle Progression; Cell Death; Cell physiology; Cells; Chemistry; Complex; Cues; Enzyme-Linked Immunosorbent Assay; Eukaryotic Initiation Factor-4F; Fluorescence Resonance Energy Transfer; Follow-Up Studies; Genotype; Human; Hypoxia; In Vitro; Insulin; Lead; Lesion; Link; Lymphoma; Malignant - descriptor; Malignant Neoplasms; Mediating; Messenger RNA; Mitotic Cell Cycle; Monitor; Mus; NIH 3T3 Cells; Peptide Initiation Factors; Phase; Process; Property; Proteins; RNA Cap-Binding Proteins; RNA Helicase; Rate; Recombinants; Regulation; Relative (related person); Ribosomes; Screening procedure; Signal Pathway; Signal Transduction; Sirolimus; Staphylococcus aureus; Starvation; Structure; Testing; Thinking; Time; Translation Initiation; Translation Process; Translations; Tumor Suppressor Genes; base; cytotoxicity; design; high throughput screening; in vivo; inhibitor/antagonist; mRNA capping; programs; protein protein interaction; response; scaffold; small molecule; translation factor

DESCRIPTION (provided by applicant): Translation is an essential cellular process whose deregulation is associated with alterations in cell growth, cell cycle progression, and cell death responses. The initiation phase of translation is a key target for regulation when cells are exposed to various environmental cues (e.g. - insulin, amino acid starvation, mitogenic stimulation, hypoxia, etc). As well, this process is deregulated in many human cancers. Over-expression of certain translation factors can lead to malignant transformation and many of the components of the translational apparatus are over-expressed in human cancers. Several tumor suppressor genes directly influence the translation process and recently, chemoresistance in vivo has been linked to deregulated translation initiation. In a transformed setting, where translation can be inhibited by a small molecule modulator (e.g. rapamycin), decreased translation rates are associated with reversal of chemoresistance, possibly by inhibition of (a) pro-survival or resetting of (a) pro-apoptotic program(s). These results validate translation initiation, and in particular eIF4F, a heterotrimeric complex involved in the ribosome/mRNA recruitment phase, as a potential chemotherapeutic target. The Specific Aims of the current application are to implement a High Throughput Screen (HTS) at the MLSCN in order to identify small molecules that alter eIF4F complex integrity, by disrupting the association between two of its subunits, eIF4E and eIF4G. Following the initial identification of compounds that show activity in the HTS assay, false-positives will be identified and eliminated using a counterscreen designed to identify non-specific effects of compounds on the TR-FRET assay or on protein-protein interaction (for example, denaturants or chemically reactive compounds). The activity of compounds that are not eliminated in the counterscreen will then be confirmed in a secondary assay that monitors the interaction between eIF4E and eIF4G, but utilizes a different readout. Follow-up studies with optimized compounds will be performed to characterize their biological properties in vitro and in vivo. Should any of the optimized hits show activity in vivo, relative cytotoxicities will be determined in lymphomas of defined genotypes generated from a mechanism-based mouse cancer model.

描述（由申请人提供）：翻译是一个必不可少的细胞过程，其放松管制与细胞生长，细胞周期进展和细胞死亡反应的改变有关。当细胞暴露于各种环境线索时（例如 - 胰岛素，氨基酸饥饿，有丝分裂刺激，缺氧等），翻译的起始阶段是调节的关键目标。同样，在许多人类癌症中，此过程受到管制。某些翻译因子的过度表达会导致恶性转化，而翻译设备的许多组成部分在人类癌症中被过表达。几种肿瘤抑制基因直接影响翻译过程，最近，体内化学抗性已与失控的翻译起始有关。在转化的环境中，小分子调节剂（例如雷帕霉素）可以抑制翻译，翻译速率降低与化学抗性的逆转有关，可能是通过抑制（a）亲寿命或（a）proptotic prototic计划的抑制。这些结果验证了核糖体/mRNA募集阶段涉及的异三聚体复合物，尤其是EIF4F的翻译起始，作为潜在的化学治疗靶标。当前应用程序的具体目的是通过破坏其两个亚基EIF4E和EIF4G之间的关联来确定改变EIF4F复合完整性的小分子，以识别改变EIF4F复杂完整性的小分子。在最初鉴定出显示HTS测定中活性的化合物后，将使用旨在识别化合物对TR-FRET分析或蛋白质蛋白质相互作用的非特异性作用的圆屏鉴定和消除假阳性（例如，变性或化学反应性化合物）。然后将在次级测定中确认未消除的化合物的活性，该测定法可以监测EIF4E和EIF4G之间的相互作用，但使用了不同的读数。将进行具有优化化合物的随访研究，以表征其体外和体内的生物学特性。如果任何优化的命中均显示在体内活性，则将在基于机制的小鼠癌模型产生的定义基因型的淋巴瘤中确定相对细胞毒性。