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: 10348198
• 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/10348198
• 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.

项目概要 Nannocystin 试剂将用于探究蛋白质延伸因子 1A 在细胞凋亡中的作用 癌细胞。 Nannocystins 是从粘细菌中分离出来的小分子天然产物，可有效抑制 癌细胞增殖并在早期时间点引发细胞凋亡。 Nannocystin 是一种混合分子，由 上部三肽结构域和下部聚酮结构域。 Nannocystin 结合真核蛋白质 延伸因子 1α (EEF1A)，但目前尚不清楚与该蛋白的结合如何导致细胞凋亡。这 相关分子 didemnin B 也结合 EEF1A，抑制 EEF1A 介导的微摩尔伸长 但在纳摩尔浓度下会引发细胞凋亡。延伸因子对于蛋白质至关重要 合成，但最近发现了其他细胞作用。特别是，EEF1A 被发现可以抑制 p53 作为肿瘤抑制因子，癌细胞在应激下会增加 EEF1A 的产生。假设 nannocystin 破坏 EEF1A-p53 结合，释放 p53 激活细胞凋亡途径，导致细胞 死亡。 Nannocystin 被认为在与didemnin B 相同的位点结合 EEF1A。然而，没有共晶体 EEF1A-nannocystin 的结构，最近的一项结构-活性关系研究挑战了这一假设 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 与南诺胱氨酸有关。为了研究癌细胞的凋亡，我们提出了以下研究：nannocystin 和 分子探针试剂将通过全合成来制备，这种方法需要高效和收敛 化学合成。我们还将采用简单的烯烃和炔烃之间的串联反应来获得 独特的聚酮结构域。该提案有三个具体目标：（1）使用Ru耦合和共同促进 南诺胱氨酸全合成中的异构化，我们在这方面取得了重大进展 三肽和聚酮片段； (2) 合成两种双功能光亲和试剂以阐明位点 nannocystin 和 EEF1A 之间的结合，结合新型三肽引入光亲和探针， 设计为在结合位点的两个极端与蛋白质共价结合； (3) 确定是否 p53 通路参与南诺胱素触发的细胞凋亡并鉴定 p53 的下游效应子 激活。