Novel synthetic tools for mucin glycobiology

描述（由申请人提供）：细胞表面糖基化异常已成为癌症的新标志。粘液蛋白1（MUC 1）较短糖型的表达增加和密度改变是恶性和癌前上皮细胞中常见的变化。尽管O-连接的N-乙酰半乳糖胺（Tn）、唾液酸帽化的Tn（sTn）的功能作用尚不清楚，但已显示出TF抗原积极参与肿瘤转移，通过与内源性β-半乳糖苷特异性凝集素半乳糖凝集素-3（gal-3）结合促进几种关键的细胞-细胞相互作用。反过来，gal-3促进癌细胞的侵袭和转移。由于TF抗原和gal-3之间的相互作用可能代表了异型癌-内皮细胞粘附和血管内转移性沉积物形成的重要早期步骤，因此在分子水平上彻底了解这一过程对于将其用于生物医学应用至关重要。我们推测，聚糖的介绍和密度模式的O-连接的聚糖有一个重大影响的识别TF抗原的癌症相关MUC 1的半乳糖苷酶-3。我们最近的突破性研究结果表明，天然肽支架的聚糖配体的介绍是高度相关的半乳糖醛酸-3结合，并提供了机会，在半乳糖醛酸-3抑制剂的设计变构位点靶向。在这个建议中，我们将准备一个MUC 1衍生的糖肽位置扫描组合库显示天然样异质性和异常肿瘤相关的O-聚糖表位（目标1）。将使用基于AlphaScreen技术的新型珠基邻近测定法筛选合成的糖肽文库的gal-3结合。已识别的单个糖肽对gal-3和一组半乳糖凝集素（gal-1、gal-4、gal-7和gal-3 CRD结构域）的结合亲和力和选择性将由ITC在更生理相关的环境中使用代表聚糖链天然复杂性的癌细胞系来确定（目标2）。显示出对gal-3的良好效力和选择性的基于MUC 1的糖肽将被探测与肿瘤侵袭和转移相关的肿瘤细胞功能（Aim 3）。这些研究将通过定义MUC 1-gal-3相互作用在癌症进展和转移形成中的作用和特异性，提供对异常MUC 1糖基化的功能意义的见解。我们的长期目标是研究MUC 1-gal-3相互作用相对于参与识别和结合肿瘤相关形式的MUC 1的其他凝集素样受体的特异性，评估MUC 1/gal-3相互作用的其他功能，例如它们在免疫调节中的作用，并将鉴定的结构用于设计和开发基于小分子的选择性和有效的半乳糖，3抑制剂，具有理想的药理学特征，用于临床前评价新的抗癌治疗策略。

DESCRIPTION (provided by applicant): Aberrant cell surface glycosylation has emerged as a new hallmark of cancer. The increased expression and altered density of shorter glycoforms of mucin 1 (MUC1) are commonly observed changes in malignant and premalignant epithelia. Whereas the functional role of O-linked N-acetylgalactosamine (Tn), sialic acid capped Tn (sTn) remains unclear, Thomsen-Friedenreich (TF) antigen has been shown to be actively involved in tumor metastasis, promoting several key cell-cell interactions via association with the endogenous �- galactoside-specific lectin, galectin-3 (gal-3). In turn, gal-3 promotes cancer cell invasion and metastasis. Since the interaction between TF antigen and gal-3 potentially represents an important early step in heterotypic cancer-endothelial adhesion and the formation of intravascular metastatic deposits, a thorough understanding of this process at the molecular level is essential for it to be used for biomedical applications. We hypothesize that glycan presentation and the density patterns of O-linked glycans have a major impact on recognition of TF antigen of cancer-associated MUC1 by gal-3. Our recent breakthrough findings have shown that the presentation of the glycan ligand by the natural peptide scaffold is highly relevant for gal-3 binding and provides opportunities for allosteric site targeting in the design of gal-3 inhibitors. Within this proposal, we will prepare a MUC1-derived glycopeptide positional scanning combinatorial library displaying native-like heterogeneous and aberrant tumor-associated O-glycan epitopes (Aim 1). Synthesized glycopeptide libraries will be screened for gal-3 binding using a novel bead-based proximity assay based on AlphaScreen technology. The binding affinities and selectivity of the identified individual glycopeptides for gal-3 and a panelof galectins (gal-1, gal-4, gal-7, and gal-3 CRD domain) will be determined by ITC and in a more physiologically relevant setting, using cancer cell lines representing the natural complexity of glycan chains (Aim 2). MUC1-based glycopeptides that show good potency and selectivity for gal-3 will be probed for tumor cell functions relevant to tumor invasion and metastasis (Aim 3). These studies will provide insights into the functional significance of the aberrant MUC1 glycosylation by defining the role and specificity of MUC1-gal-3 interactions in cancer progression and metastasis formation. Our long-term goals are to study the specificity of MUC1-gal-3 interactions in relation to other lectin-like receptors involved in recognition and binding o tumor- associated forms of MUC1, assess other functions of MUC1/gal-3 interactions such as their role in immune modulation, and to use identified structures in design and development of small molecule-based selective and potent gal-3 inhibitors with desirable pharmacological profiles for preclinical evaluation of a novel anti-cancer therapeutic strategy.