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化合物

基本信息

批准号：
7966267
负责人：
Joseph John Barchi
金额：
$ 36.39万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/7966267
关键词：
Alanine Alteplase Amino Acid Substitution Amino Acids Anabolism Antineoplastic Agents Award Baltimore Behavior Binding Biology Biophysics Bladder Bladder Diseases Bladder Neoplasm C-terminal Cells Characteristics Charge Chemicals Chronic Disease Cytoskeleton Disaccharides Disease Down-Regulation Elements Endoplasmic Reticulum Epidermal Growth Factor Epithelial Epithelial Cells Epithelium Excision Extracellular Domain Frizzled Domain Glycobiology Glycopeptides Goals Grant Growth Factor Heparin Binding Hydrophobicity Increased frequency of micturition Indium Individual Interstitial Cystitis Lead Link Location Maintenance Malignant Neoplasms Maps Maryland Minor Molecular Conformation Morphology N-terminal Pain Patients Peptides Permeability Pharmaceutical Chemistry Pharmacy Schools Play Production Property Proteins Publishing Pulmonary Surfactant-Associated Protein A Receptor Signaling Research Role Seminal Series Solutions Stretching Structure Tail Techniques Technology Testing Therapeutic Threonine Tight Junctions Transferase Translating Tumor Cell Line Tumor Tissue Tumor-Associated Carbohydrate Antigens Ulcer Universities Urine Vaccine Design Work alanylalanine analog anticancer activity antiproliferative agents base cancer immunotherapy design drug discovery inhibitor/antagonist interest knock-down methyl group mimetics neoplastic cell novel pharmacophore programs prolylvaline protein aminoacid sequence protein expression protein protein interaction receptor scaffold simulation small molecule stereochemistry sugar trend valyl-valyl-valine water solution

项目摘要

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. This was started in the lab of Dr.Chris Michejda who tragically passed away very suddenly in January of 2007. In 2008 the project was turned over to this section and to date, approximately 60 analogues have been prepared and tested. Analogues of both the peptide and sugar portions of the molecule were modified and specific clues have emerged as to portions of the molecule that are essential for activity. We have determined that most all of the molecule is necessary for full antiproliferative activity. The sugar is essential but the Thomsen Friedenreich disaccharide can be replaced with LacNAc (Gal(beta)1-4GlcNAc) alpha linked to the threonine. The peptide sequence may be truncated at the C-terminal end (removal of alanine) to an 8-mer without detriment to activity; further truncation abolishes function. Other important features for activity are 1) maintenance of charge at the termini; 2) A specific arrangement of methyl groups on the N-terminal amino acid sidechains and the ability to assume some secondary structural element in the C-terminal tail. The AXXXA motif is one that is frequently found within a helical motif and involved in binding to other protein helices (protein-protein interactions). Disruption of proper arrangement of these amino acids is detrimental to activity. A very important discovery was one that showed that specific derivatives with D-amino acid substitutions are inhibitors of APF antiproliferative activity in bladder epithelial cells, and these can now be developed as therapeutic leads for IC/PBS patients. Since we have already published on the peptide portion of the molecule and are now completing a study for a series of 8-mer glycopeptides, we are concentrating on the elements of the sugar portion of the molecule that are necessary for APF to function. We know several things about the sugar requirements already, but are now modifying individual atoms and stereochemistries around the disaccharide to map the binding interactions that are involved with the sugar portion of APF. A significant advance in the research was made by our collaborators when in 2006 they published on the discovery of a cellular receptor for APF in bladder epithelium. Cytoskeletal-associated protein 4 (CKAP4) was identified and characterized as this receptor, and knock down of its function desensitized cells to APF activity. CKAP4 links the cytoskeleton to the endoplasmic reticulum, it binds surfactant protein A and tissue plasminogen activator, but little is known of its actual function or the consequences of its inhibition. We are very interested in the details of the putative interactions of CKAP4 with APF, and a small grant was awarded to us to clone and purify the extracellular domain (474 residues) which has recently been accomplished in the Protein Expression Lab of he Advanced Technology Program here at NCI Frederick. A major effort in our APF work is dedicated to the structural characterization of the preferred conformation of APF and its analogues in solution. Being a small glycopeptide, it is relatively unstructured in water solution, but we have found certain NMR observables that suggest a preferred fold in the C-terminal domain, the portion of the peptide that we have surmised needs to be structured for APF to function. We are exploring the solution biophysics on several fronts: 1) Compare the conformations of active and inactive compounds in both the 9-mer and 8-mer series; 2) Investigate the possible aggregative properties of APF since it has a hydrophilic end (sugar) and a very hydrophobic peptide sequence; 3) Study the binding of analogues to CKAP4 and observe trends with actives and inactive compounds and 4) Develop new force fields for glycopeptide with our collaborators at the School of Pharmacy at the U. of Maryland and perform simulations that may offer other clues to APF behavior in solution. All of these studies will be geared toward defining a pharmacophore for APFs anticancer activity to design novel mimetics that may be used to selectively shut down proliferation in tumor tissue. APF constitutes an interesting an confounding study in biosynthesis and medicinal chemistry. It is a mystery as to how a small glycosylated peptide could be produced and secreted by the bladder epithelium to act like APF. If the peptide is derived from Frizzled 8, how did the transmembrane sequence become glycosylated?? Why is the molecule so sensitive to minor changes in structure and/or hydrophobicity?? We are going to tackle the glycobiology of APF by initially attempting to identify the glycosyl transferases that are involved in its biosynthesis. We would like to use novel chemical biology approaches to dynamically tag the precursors of APF in bladder cells and follow its biosynthesis and ultimate secretion. Unraveling the mysteries of the production and function of APF in the bladder will have dramatic implications that will translate to the anticancer drug discovery arena. Overall, every aspect of this project will advance our basic understanding of novel small molecule biosynthesis function and relevance to a variety of disease states including a host of different cancers.

IC/PBS 是一种慢性膀胱疾病，其特征是膀胱上皮层变薄和溃疡，导致剧烈疼痛、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造成的。这种称为 APF 的因子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针对膀胱肿瘤细胞的抗增殖剂，并且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 个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 Gal(β)1-3GalNAc(α)-O-Thr 是众所周知的 Thomsen Friedenreich 二糖，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，开始合成 APF 去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），以定义天然糖肽的结构-活性特征。该项目由 Chris Michejda 博士的实验室开始，他于 2007 年 1 月不幸去世。2008 年该项目移交给该部门，迄今为止，已制备并测试了大约 60 个类似物。该分子的肽和糖部分的类似物都被修饰，并且已经出现了关于该分子对于活性至关重要的部分的具体线索。我们已经确定该分子的大部分对于完整的抗增殖活性是必需的。糖是必需的，但 Thomsen Friedenreich 二糖可以用与苏氨酸连接的 LacNAc (Gal(beta)1-4GlcNAc) α 代替。肽序列可以在 C 末端截短（去除丙氨酸）为 8 聚体，而不会损害活性；进一步截断会废除功能。活动的其他重要特征是 1) 终点站的充电维持； 2) N 端氨基酸侧链上甲基的特定排列以及在 C 端尾部呈现某些二级结构元件的能力。 AXXXA 基序是一种经常在螺旋基序中发现的基序，并参与与其他蛋白质螺旋的结合（蛋白质-蛋白质相互作用）。破坏这些氨基酸的正确排列不利于活性。一项非常重要的发现表明，具有 D-氨基酸取代的特定衍生物是膀胱上皮细胞中 APF 抗增殖活性的抑制剂，现在可以将它们开发为 IC/PBS 患者的治疗先导药物。由于我们已经发表了关于该分子的肽部分的文章，并且现在正在完成一系列 8 聚体糖肽的研究，因此我们将重点放在 APF 发挥功能所必需的分子糖部分的元素上。我们已经了解了有关糖需求的一些信息，但现在正在修改二糖周围的单个原子和立体化学，以绘制与 APF 糖部分相关的结合相互作用。我们的合作者在这项研究中取得了重大进展，他们于 2006 年发表了在膀胱上皮细胞中发现 APF 细胞受体的文章。细胞骨架相关蛋白 4 (CKAP4) 被鉴定和表征为这种受体，敲低其功能会使细胞对 APF 活性不敏感。 CKAP4将细胞骨架连接到内质网，它结合表面活性蛋白A和组织纤溶酶原激活剂，但对其实际功能或其抑制的后果知之甚少。我们对 CKAP4 与 APF 的假定相互作用的细节非常感兴趣，并获得了一笔小额资助来克隆和纯化细胞外结构域（474 个残基），这一工作最近在高级技术计划的蛋白质表达实验室中完成。这里是 NCI 弗雷德里克。我们 APF 工作的主要工作是致力于 APF 及其类似物在溶液中的首选构象的结构表征。作为一种小糖肽，它在水溶液中相对非结构化，但我们发现某些 NMR 观察结果表明 C 端结构域存在首选折叠，我们推测该肽部分需要结构化才能使 APF 发挥作用。我们正在几个方面探索生物物理学的解决方案：1）比较9聚体和8聚体系列中活性和非活性化合物的构象； 2）研究APF可能的聚集特性，因为它具有亲水末端（糖）和非常疏水的肽序列； 3) 研究类似物与 CKAP4 的结合，观察活性化合物和非活性化合物的趋势，4) 与我们在马里兰大学药学院的合作者一起开发糖肽的新力场，并进行模拟，这可能为 APF 提供其他线索溶液中的行为。所有这些研究都将致力于定义 APF 抗癌活性的药效团，以设计可用于选择性地阻止肿瘤组织增殖的新型模拟物。 APF 是生物合成和药物化学领域一项有趣且令人困惑的研究。膀胱上皮细胞如何产生和分泌一种小糖基化肽以发挥 APF 的作用仍然是一个谜。如果肽源自Frizzled 8，那么跨膜序列是如何糖基化的？为什么分子对结构和/或疏水性的微小变化如此敏感？我们将通过最初尝试鉴定参与 APF 生物合成的糖基转移酶来解决 APF 的糖生物学问题。我们希望使用新的化学生物学方法来动态标记膀胱细胞中 APF 的前体，并跟踪其生物合成和最终分泌。解开膀胱中 APF 的产生和功能之谜将对抗癌药物发现领域产生巨大影响。总体而言，该项目的各个方面都将增进我们对新型小分子生物合成功能及其与包括许多不同癌症在内的各种疾病状态的相关性的基本理解。