CHEMICAL SYNTHESIS & TARGET IDENTIFICATION OF CERATOSPONGAMIDE

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。炎症和自身免疫性疾病，如哮喘和类风湿关节炎，其特征是免疫细胞的积累，导致组织损伤，以响应一系列复杂的分泌信号分子。两种细胞因子被认为是炎症的关键介质是IL-1b和TNF-a。这些细胞因子主要通过改变基因表达模式，对靶组织发挥不同但重叠的作用。IL-1b和TNF-a通过结合不同的细胞表面受体来控制基因表达，从而激活多个蛋白激酶级联反应。我们提出一种药理学方法来研究这些复杂的信号通路。我们的想法源于最近发现的一种海洋天然产物，角藻脂酰胺，能有效抑制IL-1b诱导的基因表达，IC50为32 nM。我们试图阐明其作用机制。角鼻虫酰胺是一种大环七肽，构象受半胱氨酸和苏氨酸侧链环化到肽主链上形成的两个附加环（噻唑和恶唑啉）的约束。我们将合成角朊酰胺及其衍生物，以鉴定其细胞靶标。放射性标记和固定化衍生物亲和纯化将使用现代合成方法合成。在UCSF设施获得的质谱测量将是必不可少的，有两个原因：(1)表征合成中间体和(2)鉴定角朊酰胺的蛋白质目标。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Inflammatory and autoimmune diseases, such as asthma and rheumatoid arthritis, are characterized by the accumulation of immune cells that cause tissue damage in response to a complex array of secreted signaling molecules. Two cytokines thought to be critical mediators of inflammation are IL-1b and TNF-a. These cytokines exert distinct yet overlapping effects on target tissues, primarily by altering patterns of gene expression. IL-1b and TNF-a control gene expression by binding to distinct cell surface receptors, whereupon multiple protein kinase cascades are activated. We propose a pharmacological approach to study these complex signaling pathways. Our idea derives from the recent discovery that a marine natural product, ceratospongamide, potently inhibits gene expression induced by IL-1b with an IC50 of 32 nM. We seek to elucidate its mechanism of action. Ceratospongamide is a macrocyclic heptapeptide that is conformationally constrained by two additional rings (thiazole and oxazoline) formed by the cyclization of cysteine and threonine side chains onto the peptide backbone. We will synthesize ceratospongamide and derivatives that will enable the identification of its cellular targets. both radiolabeled and immobilized derivatives for affinity purification will be synthesized using modern synthetic methodology. Mass spectrometry measurements obtained at the UCSF Facility will be essential for two reasons: (1) characterizing synthetic intermediates and (2) identifying ceratospongamide's protein target.