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

项目摘要 纳米囊藻毒素试剂将被用来询问蛋白延伸因子1A在细胞凋亡中的作用 癌细胞。纳米囊藻毒素是从粘细菌中分离出来的小分子天然产物，它能有效地抑制 癌细胞增殖并在早期触发细胞凋亡。纳米囊藻毒素是一种杂化分子，由 上三肽结构域和下多酮结构域的结合。纳米囊藻毒素与真核细胞结合蛋白 延长因子1α(EEF1a)，但目前尚不清楚如何与该蛋白结合导致细胞凋亡。这个 相关分子白粉菌素B也与EEF1a结合，抑制EEF1a介导的微摩尔伸长 但在纳摩尔浓度下会触发细胞凋亡。伸长因子对蛋白质至关重要 合成，但最近发现了更多的细胞作用。特别是，EEF1a被发现对P53‘S有抑制作用 作为肿瘤抑制因子的活性，以及应激状态下的癌细胞增加EEF1a的产生。据推测， 纳米囊藻毒素破坏EEF1A-P53结合，从而释放P53激活凋亡途径，导致细胞 死亡。Nannocystin被认为与Didemnin B在同一位置与EEF1a结合，但没有共晶体 EEF1a-纳米囊藻毒素的结构，以及最近的构效关系研究对这一假设提出了挑战 绑定模型。结合部位的详细信息对于合理开发新的抗癌治疗药物至关重要 与超微囊藻毒素有关。为了研究癌细胞的凋亡，我们建议进行研究，在这些研究中，纳米囊藻毒素和 分子探针试剂将通过全合成来制备，这是一种要求高效和收敛的方法 化学合成。我们还将使用简单的烯烃和炔烃之间的串联反应来访问 唯一的聚酮结构域。这一建议有三个具体目的：(1)利用Ru耦合和共同推动 在纳米囊藻毒素的全合成中的异构化，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结合部位 纳米藻毒素与EEF1a的结合，加入一种新的三肽以引入光亲和探针， 被设计成在结合部位的两端与蛋白质共价结合；以及(3)确定 P53通路参与微囊藻毒素诱导的细胞凋亡及其下游效应因子的确定 激活。

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.