Nannocystin Reagents to Elucidate the Role of Elongation Factor 1a in Apoptosis

Nannocystin 试剂阐明延伸因子 1a 在细胞凋亡中的作用

• 批准号: 10112421
• 负责人: STEVEN T DIVER
• 金额: $ 7.86万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-09 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10112421
• 关键词: Address; Affect; Alkenes; Alkynes; Amino Acids; Antineoplastic Agents; Apoptosis; Attenuated; Binding; Binding Proteins; Binding Sites; Biology; Catalysis; Cell Death; Cell Nucleus; Cell Proliferation; Cells; Chemicals; Coupling; Data; Detection; Development; Diazomethane; Dissociation; Docking; Drug resistance; Elongation Factor; Goals; Growth; Growth Inhibitors; HCT116 Cells; Hybrids; Immunofluorescence Immunologic; In Vitro; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mitochondria; Modeling; Molecular Probes; Myxococcales; Natural Products; Outcome Study; Pathway interactions; Positioning Attribute; Preparation; Process; Production; Protein Biosynthesis; Proteins; Publishing; Reaction; Reagent; Role; Signal Pathway; Signal Transduction; Site; Stress; Structure; Structure-Activity Relationship; Suppressor-Effector T-Lymphocytes; TP53 gene; Therapeutic; Time; Tumor Suppressor Proteins; Uncertainty; analog; anti-cancer therapeutic; base; cancer cell; cancer therapy; chemical synthesis; chemotherapy; covalent bond; design; diene; flexibility; nanomolar; novel; p53 Signaling Pathway; prevent; recruit; response; small molecule; therapeutically effective; unnatural amino acids

Project Summary Nannocystin reagents will be used to interrogate the role of the protein elongation factor 1A in apoptosis of cancer cells. Nannocystins are small molecule natural products isolated from myxobacteria that potently inhibit cancer cell proliferation and trigger apoptosis at an early time point. Nannocystin is a hybrid molecule consisting of an upper tripeptide domain and a lower polyketide domain. Nannocystin binds the protein eukaryotic elongation factor 1α (EEF1A), however it is presently unclear how binding to this protein causes apoptosis. The related molecule didemnin B also binds EEF1A, inhibiting EEF1A-mediated elongation at micromolar concentrations but triggering apoptosis at nanomolar concentrations. Elongation factors are vital for protein synthesis, but additional cellular roles have recently been found. In particular, EEF1A was found to inhibit p53's activity as a tumor suppressor, and cancer cells under stress increase EEF1A production. It is hypothesized that nannocystin disrupts EEF1A-p53 binding, which releases p53 to activate the apoptosis pathway, leading to cell death. Nannocystin is thought to bind EEF1A at the same site as didemnin B. However, there are no cocrystal structures of EEF1A-nannocystin, and a recent structure-activity relationship study challenged this putative binding model. Details of the binding site are critical for rational development of new anticancer therapeutics related to nannocystin. To investigate apoptosis in cancer cells, we propose studies in which nannocystin and molecular probe reagents will be prepared by total synthesis, an approach that requires efficient and convergent chemical synthesis. We will also employ a tandem reaction between a simple alkene and alkyne to access the unique polyketide domain. This proposal has three specific aims: (1) to use Ru coupling and Co-promoted isomerization in the total synthesis of nannocystin, for which we have made significant progress toward both the tripeptide and polyketide fragments; (2) to synthesize two bifunctional photoaffinity reagents to elucidate the site of binding between nannocystin and EEF1A, incorporating a novel tripeptide to introduce the photoaffinity probe, designed to covalently bond to the protein at the two extreme ends of the binding site; and (3) to determine if the p53 pathway is involved in nannocystin-triggered apoptosis and identify the downstream effectors of p53 activation.

项目摘要 微囊藻毒素试剂将用于询问蛋白延伸因子1A在微囊藻细胞凋亡中的作用。 癌细胞微囊藻毒素是从粘细菌中分离的小分子天然产物， 癌细胞增殖并在早期时间点触发凋亡。微囊藻毒素是一种杂合分子， 由上三肽结构域和下聚酮结构域组成。微囊藻毒素结合蛋白质真核生物 延伸因子1α（EEF 1A），但目前尚不清楚如何结合这种蛋白质引起细胞凋亡。的 相关分子didemnin B也结合EEF 1A，抑制EEF 1A介导的微摩尔伸长 浓度，但在纳摩尔浓度下触发细胞凋亡。延伸因子对蛋白质至关重要 EEF 1A是一种抑制p53合成的蛋白质，但最近发现了另外的细胞作用，特别是EEF 1A被发现抑制p53的合成， EEF 1A作为肿瘤抑制因子的活性，并且癌细胞在应激下增加EEF 1A的产生。它是假设 微囊藻毒素破坏EEF 1A-p53结合，从而释放p53以激活凋亡途径，导致细胞凋亡。 死亡认为微囊藻毒素在与地德霉素B相同的位点结合EEF 1A。然而，没有共晶 EEF 1A-微囊藻毒素的结构，最近的结构-活性关系研究挑战了这一假定的 绑定模型结合位点的细节对于合理开发新的抗癌疗法至关重要 与nannocystin有关为了研究癌细胞的凋亡，我们提出了研究，其中微囊藻毒素和 分子探针试剂将通过全合成制备，这是一种需要高效和收敛的方法， 化学合成我们还将采用简单烯烃和炔烃之间的串联反应来获得 独特的聚酮结构域。该方案有三个具体目标：（1）利用Ru偶联和Co促进 在微囊藻毒素的全合成中的异构化，为此，我们已经朝着两个方向取得了重大进展， 三肽和聚酮片段;（2）合成两种双功能光亲和试剂， 微囊藻毒素和EEF 1A之间的结合，掺入新的三肽以引入光亲和探针， 设计成在结合位点的两个末端共价键合到蛋白质上;和（3）确定 p53通路参与微囊藻毒素诱导的细胞凋亡并鉴定p53的下游效应子 activation.