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是膀胱的慢性疾病，其特征是膀胱上皮层变薄和溃疡，导致严重的疼痛，尿频和紧迫性。巴尔的摩马里兰大学的开创性工作表明，一个特定因素是导致IC/PBS中发生的许多特征性病理变化。该因子称为APF，在亚纳米尔浓度下对膀胱上皮细胞具有抗增殖活性。 APF导致细胞细胞渗透性的增加，几种涉及紧密连接形成的蛋白质的下降调节，并降低了肝素结合表皮表皮生长因子样生长因子（HB-EGF）的水平。此外，APF也是针对膀胱肿瘤细胞的有效抗增殖剂，浓度同样低，随后被证明可以抑制其他肿瘤细胞系的增殖。 APF的肽部分具有100％的序列身份，与毛躁8（Wnt信号受体）的第6个跨脑域中的氨基酸相同。而糖的部分，GAL（beta）1-3galnac（alpha）-o-thR是众所周知的Thomsen Friedenreich二糖，这是一种用于疫苗设计和癌症免疫疗法的肿瘤相关碳水化合物抗原。 2006年，合成始于APF的ASIALO衍生物的一系列类似物（AS-APF，对天然溶解化合物的等式），以定义天然糖肽的结构活性谱。这是从Chris Michejda博士的实验室开始的，他于2007年1月突然惨败地去世了。2008年，该项目已移交给本节，迄今为止，已经准备好了60个类似物。对分子的肽和糖部分的类似物进行了修改，并且已经出现了针对活性必不可少的部分的特定线索。我们已经确定大多数分子对于完全抗增生活性都是必需的。糖是必不可少的，但是汤姆森·弗里德内里希二糖可以用lacNAC（gal（beta）1-4Glcnac）αα链接的alpha代替。肽序列可以在C末端（将丙氨酸去除）截断为8-Mer，而不会损害活性。进一步的截断废除了功能。 活动的其他重要特征是1）在终点站维护电荷； 2）甲基在N末端氨基酸侧链上的特定排列，以及在C末端尾部假设某些二级结构元件的能力。 AXXXA基序是在螺旋基序中经常发现的，并与其他蛋白螺旋（蛋白质 - 蛋白质相互作用）结合。这些氨基酸适当排列的破坏对活性有害。一个非常重要的发现是表明具有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，现在可以作为IC/PBS患者的治疗铅开发这些衍生物。由于我们已经在分子的肽部分发表了，现在正在完成一系列8-Mer糖肽的研究，因此我们集中于APF功能所需的分子糖部分的元素。我们已经了解了有关糖需求的几件事，但是现在正在修改二糖周围的单个原子和立体体，以绘制与APF的糖部分有关的结合相互作用。当我们的合作者在2006年发表有关在膀胱上皮中发现APF的细胞受体时，我们的合作者取得了重大进展。将细胞骨架相关的蛋白4（CKAP4）鉴定为该受体，并将其功能脱敏细胞降低至APF活性。 CKAP4将细胞骨架与内质网连接起来，结合表面活性剂A和组织纤溶酶原激活剂，但对其实际功能或抑制作用的后果知之甚少。我们对CKAP4与APF的推定相互作用的详细信息非常感兴趣，并授予我们一笔少量赠款，以克隆并净化细胞外域（474个残基）（474个残基），该结构域（474个残基）最近在NCI Frederick的HE先进技术计划的蛋白质表达实验室中完成了。我们的APF工作中的一项重大努力致力于APF及其在解决方案中的类似物中首选构象的结构表征。作为一个小糖肽，它在水溶液中相对非结构化，但是我们发现了某些NMR可观察物，这些NMR可观察到C-末端结构域中的首选折叠，我们刺激的肽的一部分需要为APF构造，以使APF起作用。我们正在几个方面探索溶液生物物理学：1）比较9-Mer和8-Mer系列中活性和非活性化合物的构象； 2）研究APF的可能聚集特性，因为它具有亲水性端（糖）和非常疏水的肽序列； 3）研究类似物与CKAP4的结合，并通过活性和非活性化合物观察趋势，以及4）与我们在马里兰州大学药学院的合作者一起开发新的力场，用于糖肽，并进行模拟，并可能为解决方案中的APF行为提供其他线索。所有这些研究都将旨在定义APFS抗癌活性的药效团，以设计新型的模拟物，可用于选择性地关闭肿瘤组织中的增殖。 APF构成了一项有趣的生物合成和药物化学研究。对于如何由膀胱上皮产生和分泌的小糖基化肽如何像APF一样，这是一个谜。 如果肽是从卷曲8衍生的，则跨膜序列如何变为糖基化？为什么该分子对结构和/或疏水性的微小变化如此敏感？我们将通过最初试图鉴定与其生物合成有关的糖基转移酶来解决APF的糖生物学。我们想使用新型的化学生物学方法在膀胱细胞中动态标记APF的前体，并遵循其生物合成和最终分泌。揭开APF在膀胱中生产和功能的奥秘将具有巨大的含义，这将转化为抗癌药物发现领域。总体而言，该项目的每个方面都将提高我们对新型小分子生物合成功能的基本理解以及与包括许多不同癌症在内的多种疾病状态的相关性。