A Glycopeptide from Interstitial Cystitis Patients as a Novel Anticancer Lead

间质性膀胱炎/痛性膀胱病(IC/PBS)是一种以膀胱上皮层变薄和溃烂为特征的慢性膀胱疾病，引起剧烈的疼痛、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都是由一个特定的因素造成的。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质下调，并降低肝素结合的表皮生长因子样生长因子(HB-EGF)水平。此外，APF在同样低浓度下对膀胱肿瘤细胞也是一种有效的抗增殖剂，随后被证明对其他肿瘤细胞系的增殖具有抑制作用。APF的多肽部分与Wnt信号受体Frizzled8第6跨膜区的一段氨基酸序列具有100%的同源性。而糖部分，Neu5Ac(alpha)2-3Gal(beta)1-3GalNAc(alpha)-O-Thr是众所周知的汤姆森弗里登瑞克双糖的唾液酸化形式，这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，开始合成APF的asialo衍生物的一系列类似物(AS-APF，等同于天然唾液酸化化合物)，以确定天然糖肽的构效关系。在去年的年度报告中，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。我们今年发表了我们确定的两种抑制剂，以及用这些药物治疗IC/PBS样膀胱细胞时的正常化。它们正被开发为IC/PBS的治疗剂。我们正在继续进行SAR工作，准备碳水化合物类似物，其中特定的羟基被移除或被氟等同位素体取代，以绘制糖的重要相互作用图。其中几个已经被制备出来，其中两个已经被结合到多肽中。我们对CKAP蛋白的研究停滞不前，因为我们准备的构建体不稳定，在标准条件下非常迅速地聚集。因此，我们无法开发和测试我们所有的类似物。这一点正在修改中，并将探索修改后的蛋白质片段。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模拟的方法，我们已经在各种类似物中确定了特定的基序，这些基序对于决定这些结构的构象偏差是重要的。这些数据有助于阐明糖肽的糖部分与多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进行实际药物设计。关于这项工作的手稿发表在《化学信息与建模》杂志上。我们还在与美国国立牙科和颅面研究所的合作者合作，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。取得的主要成就是：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，确定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在11个不同的癌细胞系中研究了其中两个类似物的抗癌活性，并在《研究新药》上发表了一篇全文论文；以及3)探索了两个抑制物分子对APF转化细胞的作用；以及4)碳水化合物类似物的合成，并将所有这些数据汇编成另一份手稿。我们现在已经合成了几种具有挑战性的APF的含氟碳水化合物类似物，发现其中一种几乎与天然物质一样具有活性。与马里兰大学的Alex MacKerell博士卓有成效的合作产生了APF的结构模型，我们现在可以在其中进行药效受体搜索，并试图设计一种与天然产品一样活跃的非肽APF序号。我们还可以尝试设计有选择性地针对癌细胞的类似物。

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.