A Glycopeptide from Interstitial Cystitis Patients as a Novel Anticancer Lead

IC/PBS是一种慢性膀胱疾病，其特征在于膀胱上皮层变薄和溃疡，引起剧烈疼痛、尿频和尿急。位于巴尔的摩的马里兰州大学的精液研究表明，一种特定的因子是IC/PBS中发生的许多特征性病理变化的原因。该因子被称为APF，在亚纳摩尔浓度下对膀胱上皮细胞具有抗增殖活性。APF引起细胞旁通透性增加，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抗膀胱肿瘤细胞增殖剂，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Frizzled 8的第6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Neu 5Ac（alpha）2-3Gal（beta）1-3GalNAc（alpha）-O-Thr是众所周知的Alpensen Friedenreich二糖的唾液酸化形式，其是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，开始合成APF的脱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），以确定天然糖肽的结构-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完全活性的最低要求。我们今年发表了我们确定的两种抑制剂，以及用这些药物治疗时IC/PBS样膀胱细胞的正常化。它们被开发为IC/PBS的治疗剂。我们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电子等排体取代，以绘制糖的重要相互作用。已经制备了其中的几种，并且已经将两种掺入肽中。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，在标准条件下聚合非常迅速。因此，我们无法开发和测定所有我们的类似物。这是正在修订和修改的蛋白质片段将被探索。我们与马里兰州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模拟方法，我们已经确定了特定的图案在各种类似物，是重要的支配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的特定受体结合有关。主要成绩如下：1）通过NMR和建模分析了所有8-mer类似物以及4种最重要的9-mer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）与我们的合作者一起在11种不同的癌细胞系中研究了其中两种类似物的抗癌活性，并在Investigational New Drugs上发表了全文;和3）APF转化细胞上的两种抑制剂分子的探索;和4）碳水化合物类似物的合成和另一手稿的所有这些数据的汇编。此外，由于在过去一年中就这一主题进行了几次讲座，我们为APF获得了几项新的合作。第一个是与纽约汉密尔顿学院的Myriam Cotton教授合作，在存在膜模拟结构的情况下通过固态NMR研究APF类似物。Cotten博士现在正在用APF膜模拟物进行固态核磁共振研究，以观察它在膜环境中的行为。与加拿大安大略金斯顿皇后学院的Inka Brockhausen的合作表明，APF可以作为各种糖基转移酶的受体底物，以制备其他糖相关类似物。作为底物的效率并不高，但可能足以确保一些新的类似物。我们已经开始与NCI Frederick高级技术计划的蛋白质组学实验室合作，对IC细胞的糖蛋白质组进行细胞范围的糖蛋白质组学评估。这将告诉我们携带TF抗原的蛋白质的起源，并可能引导我们找到APF分子的起源！

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. In addition, as a result of several lectures given on this subject in the past year, we have secured several new collaborations for APF. The first is with Professor Myriam Cotton of Hamilton College in New York, on the study of APF analogues by solid state NMR in the presence of membrane mimcking constructs. Dr Cotten is now performing solid state NMR studies with APF membrane mimics to see how it behaves while sequestered in membrane environment. The collaboration with Inka Brockhausen of Queens college in Kingston, Ontario, Canada, has shown that APF can be an acceptor substrate for various glycosyltransferases to prepare other sugar-related analogues. The efficiency as a substrate is not that high, but probably good enough to secure some new analogues. We have started a collaboration with the Proteomics lab at the Advanced Technology Program at NCI Frederick to perform a cell-wide glycoproteomics evaluation of the glycoproteome of IC cells. This will tell us the origin of proteins that bear the TF antigen and may lead us to the origin of the APF molecule!