A Glycopeptide from Interstitial Cystitis Patients as a Novel Anticancer Lead

IC/PBS是一种慢性膀胱疾病，其特征是膀胱上皮变薄和溃疡，引起剧烈疼痛、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引起的。这种被称为APF的因子被证明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下调，并降低肝素结合的表皮生长因子样生长因子（HB-EGF）的水平。此外，在同样低的浓度下，APF也是一种有效的膀胱肿瘤细胞的抗增殖剂，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frizzle8的第6跨膜结构域的一段氨基酸序列100%相同。而糖部分Neu5Ac(α)2-3Gal(β)1-3GalNAc(α)-O-Thr是Thomsen Friedenreich双糖的唾液化形式，Thomsen Friedenreich双糖是一种用于疫苗设计和癌症免疫治疗的肿瘤相关碳水化合物抗原。2006年，人们开始合成一系列APF的asialo衍生物的类似物（as-APF，与天然唾液化化合物等效），以确定天然糖肽的结构-活性谱。在上一份年度报告中，我们概述了我们对这种分子所做的广泛的结构-活性研究，并报告了作为抗增殖剂的分子充分活性的最低要求。我们今年发表了关于我们发现的两种抑制剂以及用这些药物治疗后IC/ pbs样膀胱细胞的正常化的文章。它们正被开发为IC/PBS的治疗剂。我们正在通过制备碳水化合物类似物来继续SAR工作，其中特定的羟基被去除或被氟等同分异构体取代，以绘制糖的重要相互作用图。其中几种已制备，两种已纳入肽中。由于我们制备的构建物不稳定，在标准条件下聚集速度非常快，因此我们对CKAP蛋白的研究陷入了停滞。因此，我们无法开发和分析我们所有的类似物。这是正在修订和修改的蛋白质片段将探索。我们和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倚很重要。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：这可能与它与特定细胞受体的相互作用高度相关，因此有助于实际药物设计可能具有选择性抗癌活性的特定类似物。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的特定受体结合有关。主要成果有：1)通过核磁共振和建模分析了所有8-聚类似物以及4个最重要的9-聚类似物的数据，定义了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研究了两种类似物的抗癌活性，并在《新药研究》上发表了一篇完整的论文；3)两种抑制剂分子在apf转化细胞中的作用探索；4)碳水化合物类似物的合成，并将这些数据汇编为另一篇论文。我们现在已经制备了几种具有合成挑战性的APF的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具有活性。与马里兰大学的Alex Mackerell博士卓有成效的合作已经产生了APF的结构模型，我们现在可以进行药效团搜索，并尝试设计一种与天然产物一样活跃的非肽类APF类似物。我们也可以尝试设计类似物，选择性地靶向癌细胞。

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.