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，肝素结合的表皮生长因子样生长因子（HB-EGF）水平降低。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，Gal(β)1-3GalNAc(α)-O-Thr是众所周知的Thomsen Friedenreich双糖，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。它们是由一家小公司开发的抗肿瘤药物。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。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。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。我们也刚刚发表了一篇关于APF类似物在新药研究中的抗癌活性的论文。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)两种类似物在11种不同的癌细胞系中的抗癌活性；3)两种抑制剂分子在apf转化细胞上的作用。碳水化合物类似物的合成和另一份手稿中所有这些数据的汇编正在进行中。

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 protein 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 transmebrane domain of Frizzled 8, a Wnt signaling receptor. Whereas the sugar portion, Gal(beta)1-3GalNAc(alpha)-O-Thr is 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 by a small company as antitumor agents. 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. 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. 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. We also just published a paper on the anticancer activity of APF analogues in Investigational New Drugs. 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 3) Exploration of the two inhibitor molecules on APF-transformed cells. Synthesis of the carbohydrate analogues and compilation of all these data for another manuscript is in progress.