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

• 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
• 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

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。这是在chris Michejda博士的实验室里开始的他在2007年1月突然不幸去世。2008年，该项目移交给该部门，到目前为止，已经准备和测试了大约60个类似物。分子的肽和糖部分的类似物都被修改了，并且已经出现了关于分子中对活性至关重要的部分的特定线索。我们已经确定，大多数分子都是完全抗增殖活性所必需的。糖是必需的，但是Thomsen Friedenreich双糖可以用连接苏氨酸的LacNAc (Gal(β)1-4GlcNAc) α代替。肽序列可以在c端截断（去除丙氨酸）至8聚体而不损害活性；进一步的截断消除了函数。活动的其他重要特征是1)在终点站保持电荷；2) n端氨基酸侧链上甲基的特殊排列，以及在c端尾部承担某些二级结构元件的能力。AXXXA基序是一个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）。打乱这些氨基酸的正确排列对活性是有害的。一个非常重要的发现是，具有d -氨基酸取代的特异性衍生物是膀胱上皮细胞中APF抗增殖活性的抑制剂，这些衍生物现在可以作为IC/PBS患者的治疗先导。由于我们已经发表了分子的肽部分，并且现在正在完成一系列8-mer糖肽的研究，因此我们将重点放在分子的糖部分的元素上，这些元素是APF发挥作用所必需的。我们已经知道了一些关于糖的需求，但现在正在修改双糖周围的单个原子和立体化学，以绘制与APF糖部分有关的结合相互作用。2006年，我们的合作者在膀胱上皮中发现了一种APF细胞受体，这一研究取得了重大进展。细胞骨架相关蛋白4 （CKAP4）被鉴定为这种受体，其功能的下调使细胞对APF活性脱敏。CKAP4将细胞骨架连接到内质网，它结合表面活性剂蛋白A和组织纤溶酶原激活物，但对其实际功能或其抑制的后果知之甚少。我们对CKAP4与APF相互作用的细节非常感兴趣，我们获得了一笔小额资助，用于克隆和纯化细胞外结构域（474个残基），这项工作最近在NCI Frederick的先进技术项目的蛋白质表达实验室完成。我们在APF工作中的主要工作是致力于对溶液中APF及其类似物的首选构象进行结构表征。作为一个小的糖肽，它在水溶液中是相对非结构化的，但我们发现某些核磁共振观察结果表明，c端结构域有一个优先折叠，我们推测肽的这一部分需要被结构化才能使APF发挥作用。我们正在从几个方面探索溶液生物物理学：1)比较9-聚和8-聚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，观察活性和非活性化合物的变化趋势；4)与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，可能为溶液中APF的行为提供其他线索。所有这些研究都将致力于确定APFs抗癌活性的药效团，以设计新的模拟物，可用于选择性地关闭肿瘤组织中的增殖。APF在生物合成和药物化学领域是一项有趣而又令人困惑的研究。膀胱上皮如何产生和分泌一种小的糖基化肽来发挥APF的作用，这是一个谜。如果肽来源于frizzled8，那么跨膜序列是如何被糖基化的？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识别参与其生物合成的糖基转移酶来解决APF的糖生物学问题。我们希望利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。揭示膀胱中APF的产生和功能的奥秘将具有戏剧性的意义，这将转化为抗癌药物发现领域。总的来说，这个项目的每个方面都将促进我们对新型小分子生物合成功能及其与多种疾病状态（包括许多不同的癌症）的相关性的基本理解。