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/疼痛膀胱疾病（IC/PBS）是膀胱的慢性疾病，其特征是膀胱上皮层的变薄和溃疡，导致严重的疼痛，尿频和紧急情况。巴尔的摩马里兰大学的开创性工作表明，一个特定因素是导致IC/PBS中发生的许多特征性病理变化。该因子称为APF，在亚纳米尔浓度下对膀胱上皮细胞具有抗增殖活性。 APF导致细胞细胞通透性的增加，几种参与紧密连接形成的蛋白质的下降调节，并降低了肝素结合表皮表皮生长因子样生长因子（HB-EGF）的水平。此外，APF也是针对膀胱肿瘤细胞的有效抗增殖剂，浓度同样低，随后被证明可以抑制其他肿瘤细胞系的增殖。 APF的肽部分具有100％的序列身份，与毛躁8（Wnt信号受体）的第6跨膜结构域中的氨基酸延伸。而糖的部分，neu5ac（alpha）2-3gal（beta）1-3galnac（alpha）-o-thR是众所周知的Thomsen Friedenreich二糖，肿瘤相关的碳水化合物抗原抗原抗原和癌症的免疫疗法。 2006年，合成始于APF的ASIALO衍生物的一系列类似物（AS-APF，对天然溶解化合物的等式），以定义天然糖肽的结构活性谱。在上次年度报告中，我们概述了我们对该分子进行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。我们今年发表了有关两种抑制剂的发表，当用这些药物处理时，我们确定了IC/PBS样细胞的标准化。它们正在作为IC/PBS的治疗剂开发。我们通过制备碳水化合物类似物来继续进行SAR工作，在该类似物中，在碳水化合物类似物中，将特定的羟基被除去或用氟等当等渗剂代替，以绘制糖的重要相互作用。其中几个已经制备，两种已掺入肽中。自从我们准备的构造中，我们与CKAP蛋白的工作停滞不前，因为我们在标准条件下制定了不稳定和聚集的速度。因此，我们无法为所有类似物开发和分析。将进行修订，并将探索修改的蛋白质片段。我们在马里兰州大学的合作者中在结构方面取得了很多进步。通过NMR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，这些基序对于决定这些结构的构象偏置很重要。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，从而有助于对可能具有选择性抗癌活性的特定类似物的实际药物设计。 有关这项工作的手稿发表在《化学信息与建模杂志》上。 我们还与美国国家牙科和颅面研究所的合作者合作，以确定与APF生物合成有关的特定糖基转移酶，并探讨该糖部分是否与与癌细胞上特定受体结合的糖分是否相关。主要成就是：1）通过NMR和建模对所有8-mer类似物以及4个最重要的9-Mer类似物的数据分析，定义了该分子与自身的重要相互作用，现在扩展到蛋白质结合； 2）与我们的合作者在11种不同的癌细胞系中的两个类似物的抗癌活性，并发表了研究新药的完整论文； 3）在APF转化细胞上探索两个抑制剂分子； 4）碳水化合物类似物的合成以及所有这些数据的汇编以及其他手稿的汇编。现在，我们已经准备了APF的几种含氟化碳水化合物类似物的合成碳水化合物类似物，发现其中一个几乎与天然物质一样活跃。与马里兰州大学的Alex Mackerell博士进行了富有成果的合作，它产生了APF的结构模型，我们现在可以进行药物搜索，并尝试设计一种与天然产品一样活跃的非肽APF ANLOGUE。我们还可以尝试设计将选择性靶向癌细胞的类似物。