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: 9556504
• 负责人: Joel Schneider
• 金额: $ 47.24万
• 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/9556504
• 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; Epithelium; 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; Molecular Models; 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; anticancer activity; antiproliferative agents; base; biophysical techniques; cancer cell; cancer immunotherapy; carbohydrate analog; cell transformation; design; glycosyltransferase; hydroxyl group; information model; inhibitor/antagonist; micturition urgency; molecular modeling; nanomolar; neoplastic cell; novel; novel anticancer drug; receptor; scaffold; sugar

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.

IC/PBS 是一种慢性膀胱疾病，其特征是膀胱上皮层变薄和溃疡，导致剧烈疼痛、尿频和尿急。巴尔的摩马里兰大学的开创性工作表明，IC/PBS 中发生的许多特征性病理变化是由一个特定因素造成的。这种称为 APF 的因子在亚纳摩尔浓度下对膀胱上皮细胞具有抗增殖活性。 APF 导致细胞旁通透性增加、参与紧密连接形成的几种蛋白质的下调，并降低肝素结合表皮生长因子样生长因子 (HB-EGF) 的水平。此外，APF 在同样低的浓度下也是一种有效的针对膀胱肿瘤细胞的抗增殖剂，并且随后被证明可以抑制其他肿瘤细胞系的增殖。 APF 的肽部分与 Wnt 信号受体 Frizzled 8 的第 6 个跨膜结构域中的一段氨基酸具有 100% 的序列同一性。而糖部分 Neu5Ac(α)2-3Gal(β)1-3GalNAc(α)-O-Thr 是众所周知的 Thomsen Friedenreich 双糖的唾液酸化形式，Thomsen Friedenreich 双糖是一种肿瘤相关糖抗原，用于疫苗设计和癌症的免疫治疗。 2006 年，开始合成 APF 去唾液酸衍生物的一系列类似物（as-APF，与天然唾液酸化化合物等效），以定义天然糖肽的结构-活性特征。在上一份年度报告中，我们概述了我们对该分子所做的广泛的结构活性研究，并报告了该分子作为抗增殖剂的全部活性的最低要求。我们今年发表了关于我们确定的两种抑制剂以及用这些药物治疗时 IC/PBS 样膀胱细胞正常化的文章。它们正在被开发为 IC/PBS 的治疗剂。我们正在继续进行 SAR 工作，制备碳水化合物类似物，其中特定的羟基被去除或被氟等电子等排物取代，以绘制糖的重要相互作用。其中几种已被制备，其中两种已被掺入肽中。我们对 CKAP 蛋白的研究陷入停滞，因为我们制备的构建体不稳定并且在标准条件下聚集得非常快。因此，我们无法开发和分析所有类似物。目前正在对此进行修订，并将探索修饰的蛋白质片段。我们与马里兰大学的合作者在结构方面取得了很大进展。通过核磁共振和分子建模方法，我们已经确定了各种类似物中的特定基序，这些基序对于指示这些结构的构象偏差非常重要。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，从而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。我们现在还与国家牙科和颅面研究所的合作者合作，以确定参与 APF 生物合成的特定糖基转移酶，并探索糖部分是否与癌细胞上特定受体的结合有关。主要成就是： 1) 通过 NMR 和建模分析了所有 8 聚体类似物以及 4 个最重要的 9 聚体类似物的数据，定义了分子与其自身的重要相互作用，现在扩展到蛋白质结合； 2) 与我们的合作者一起研究了两种类似物在 11 种不同癌细胞系中的抗癌活性，并在《研究性新药》上发表了一篇完整论文； 3）两种抑制剂分子对APF转化细胞作用的探索； 4）碳水化合物类似物的合成以及另一份手稿的所有这些数据的汇编。我们现在已经制备了几种具有合成挑战性的 APF 氟化碳水化合物类似物，并发现其中一种几乎与天然材料一样活跃。与马里兰大学的 Alex Mackerell 博士的富有成效的合作已经产生了 APF 的结构模型，我们现在可以在其中进行药效团搜索并尝试设计一种与天然产物一样活跃的非肽 APF 类似物。我们还可以尝试设计选择性靶向癌细胞的类似物。