A Glycopeptide from Interstitial Cystitis Patients as a Novel Anticancer Lead

来自间质性膀胱炎患者的糖肽作为新型抗癌先导药物

• 批准号: 10702513
• 负责人: Joseph John Barchi
• 金额: $ 30.28万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702513
• 关键词: Amino Acids; Anabolism; Annual Reports; Baltimore; Binding; Binding Proteins; Biological Assay; Bladder; Bladder Diseases; Bladder Neoplasm; Cancer cell line; Cells; Characteristics; Chemicals; Chronic Disease; Collaborations; DTR gene; Data; Data Analyses; Disaccharides; Down-Regulation; Drug Design; Epidermal Growth Factor; Epithelial; Epithelial Cells; Fluorine; Frizzled Domain; Glycobiology; Glycopeptides; Goals; Growth Factor; Heparin Binding; Increased frequency of micturition; Interstitial Cystitis; Investigational Drugs; Journals; Lead; Location; Manuscripts; Maps; Maryland; Methods; Modeling; Molecular Conformation; Morphology; National Institute of Dental and Craniofacial Research; Natural Products; Pain; Paper; Pathologic; Patients; Peptides; Permeability; Pharmaceutical Preparations; Play; Protein Fragment; Proteins; Publishing; Receptor Signaling; Reporting; Role; Seminal; Series; Stretching; Structural Models; Structure; Therapeutic Agents; Thinness; Tight Junctions; Translating; Transmembrane Domain; Tumor Cell Line; Tumor-Associated Carbohydrate Antigens; Ulcer; Universities; Urine; Vaccine Design; WNT Signaling Pathway; Work; alanylproline; analog; anti-cancer; anticancer activity; antiproliferative agents; base; biophysical techniques; cancer cell; cancer immunotherapy; carbohydrate analog; cell transformation; design; glycosyltransferase; hydroxyl group; information model; inhibitor; micturition urgency; molecular modeling; nanomolar; neoplastic cell; novel; novel anticancer drug; receptor; scaffold; sugar

Interstitial cystitis/painful bladder disease (IC/PBS) is a chronic disease of the bladder characterized by thinning and ulceration of the bladder epithelial layer causing severe pain, urinary frequency and urgency. Seminal work at the University of Maryland, Baltimore, showed that a specific factor was responsible for many of the characteristic pathological changes that occur in IC/PBS. This factor, called APF, was shown to have antiproliferative activity toward bladder epithelial cells at sub-nanomolar concentrations. APF caused an increase in paracellular permeability, the down regulation of several proteins involved in tight junctions formation and reduced the levels of heparin-binding epidermal growth factor-like growth factor (HB-EGF). In addition, APF was also a potent antiproliferative agent against bladder tumor cells at equally low concentrations and has subsequently been shown to inhibit proliferation of other tumor cell lines. The peptide portion of APF has 100% sequence identity to a stretch of amino acids in the 6th trans-membrane domain of Frizzled 8, a Wnt signaling receptor. Whereas the sugar portion, Neu5Ac(alpha)2-3Gal(beta)1-3GalNAc(alpha)-O-Thr is the sialylated form of the well-known Thomsen Friedenreich disaccharide, a tumor associated carbohydrate antigen used in vaccine design and in the immunotherapy of cancer. In 2006, synthesis began on a series of analogues of the asialo derivative of APF (as-APF, equipotent to the natural sialylated compound) to define the structure-activity profile of the natural glycopeptide. In the last annual report we outlined the extensive structure-activity studies we had done with this molecule, and reported in the minimal requirements for full activity of the molecule as an antiproliferative agent. We published this year on the two inhibitors we identified and the normalization of IC/PBS-like bladder cells when treated with these drugs. They are being developed as therapeutic agents for IC/PBS. We are continuing with the SAR work by preparing carbohydrate analogues where specific hydroxyl groups are removed or replaced with isosteres like fluorine to map the important interactions of the sugar. Several of these have been prepared and two have been incorporated into the peptide. Our work with the CKAP protein was stalled since the construct we prepared as unstable and aggregated very rapidly under standard conditions. Thus we were not able to develop and assay for all our analogues. This is being revised and modified protein fragments will be explored. We have made a lot of progress on the structural front with our collaborators at the University of Maryland. By NMR and molecular modeling methods, we have identified specific motifs in various analogues that are important for dictating the conformational bias of those structures. These data have helped in elucidation the manner in which the sugar portion of the glycopeptides interacts with the peptide portion: this could be highly relevant to its interactions with specific cellular receptors and thus aid in actual drug design of particular analogues that may have selective anticancer activity. A manuscript on this work was published in the Journal of Chemical Information and Modeling. We are also working with collaborators now at the National Institute of Dental and Craniofacial Research to determine the specific glycosyltransferases that are involved in the biosynthesis of APF and to explore whether or not the sugar portion is relevant to binding with specific receptors on cancer cells. The major accomplishments were: 1) Analysis of data on all 8-mer analogues as well as 4 of the most important 9-mer analogues by NMR and modeling, defined the important interactions of the molecule with itself and now expanding to protein binding; 2) Anticancer activity of two of the analogues in 11 different cancer cell lines with our collaborators and publishing a full paper in Investigational New Drugs; and 3) Exploration of the two inhibitor molecules on APF-transformed cells; and 4) Synthesis of the carbohydrate analogues and compilation of all these data for another manuscript. We have now prepared several synthetically challenging fluorinated carbohydrate analogues of APF and found that one of them is almost as active as the natural material. A fruitful collaboration with Dr. Alex Mackerell of the University of Maryland has yielded a structural model of APF where we can now perform pharmacaphore searches and try to design a non-peptidic APF anlogue that is as active as the natural product. We can also attempt to design analogues that will selectively target cancer cells.

间质性膀胱炎/疼痛性膀胱疾病（IC/PBS）是一种慢性疾病， 以膀胱上皮层变薄和溃疡为特征的膀胱， 剧烈疼痛、尿频和尿急。在马里兰州大学从事种子工作， 巴尔的摩的研究表明，一个特定的因素是造成许多特征的原因。 在IC/PBS中发生的病理变化。这个因素，称为APF，被证明具有 在亚纳摩尔浓度下对膀胱上皮细胞的抗增殖活性。 APF引起细胞旁通透性增加，几种蛋白质表达下调， 参与紧密连接的形成，并降低肝素结合表皮细胞的水平。 生长因子样生长因子（HB-EGF）此外，APF也是一种有效的 以同样低的浓度使用抗膀胱肿瘤细胞增殖剂， 随后显示抑制其它肿瘤细胞系的增殖。所述肽 APF的一部分与第6个氨基酸序列中的一段氨基酸具有100%的序列同一性。 Frizzled 8是一种Wnt信号受体。而糖的部分， Neu5Ac（alpha）2 - 3Gal（beta）1 - 3GalNAc（alpha）-O-Thr是众所周知的Neu5Ac（alpha）2 - 3Gal（beta）1 - 3GalNAc（alpha）-O-Thr的唾液酸化形式。 一种用于疫苗的肿瘤相关糖抗原--福氏二糖 设计和癌症的免疫治疗。2006年，开始合成一系列类似物， APF的去唾液酸衍生物（as-APF，等效于天然唾液酸化化合物）， 定义天然糖肽的结构-活性特征。上一份年报所 我们概述了我们对这种分子所做的广泛的结构-活性研究， 在作为抗增殖药物的分子的完全活性的最低要求中报道了 剂我们今年发表了关于我们发现的两种抑制剂以及 IC/PBS样膀胱细胞，当用这些药物治疗时。他们正在开发， 用于IC/PBS的治疗剂。我们正在通过制备碳水化合物继续SAR工作 类似物，其中特定的羟基基团被去除或被电子等排体如氟取代 来绘制糖的重要相互作用。其中一些已经准备好， 已经被整合到肽中。我们对CKAP蛋白的研究在2000年被搁置， 我们制备的构建体是不稳定的，并且在标准条件下非常迅速地聚集。因此 我们不能开发和分析所有我们的类似物。目前正在修订， 将探索修饰的蛋白质片段。我们在结构上取得了很大的进展， 与我们在马里兰州大学的合作者一起。通过核磁共振和分子模拟 方法，我们已经确定了各种类似物中的特定基序， 决定了这些结构的构象偏差。这些数据有助于阐明 糖肽的糖部分与肽部分相互作用的方式： 这可能与其与特定细胞受体的相互作用高度相关，因此 有助于可能具有选择性抗癌活性的特定类似物的实际药物设计。 这项工作的手稿发表在《化学信息与建模杂志》上。 我们现在也在与国家牙科研究所的合作者合作， 颅面研究，以确定具体的糖基转移酶，参与了 APF的生物合成，并探索糖部分是否与结合有关 与癌细胞上的特定受体结合。主要成果有：1）数据分析 通过NMR和建模对所有8-mer类似物以及4种最重要的9-mer类似物进行了分析， 定义了分子与自身的重要相互作用，现在扩展到蛋白质 2）两种类似物在11种不同的癌细胞系中的抗癌活性， 我们的合作者，并在研究性新药上发表全文;以及3）探索 两种抑制剂分子在APF转化细胞上的作用;和4）碳水化合物的合成 类似物和汇编所有这些数据的另一个手稿。我们已经准备好了 几种合成具有挑战性的APF氟化碳水化合物类似物，并发现其中一种 其中的活性几乎与天然材料一样。与Alex博士的合作 马里兰州大学的Mackerell提出了一个APF的结构模型， 进行药效学搜索，并尝试设计一种非肽类APF类似物， 作为天然产物。我们还可以尝试设计类似物， 癌细胞