Biomolecular Analysis using Liquid Crystals

描述（由申请人提供）：威斯康星州大学生物工程研究合作伙伴关系（UW-BRP）将专注于开发新的分子分析工具，这些工具具有用于识别和验证生物学终点的潜力，从而可以更准确和快速地评估新型抗癌药物的分子机制和临床相关性。这项工作最初将集中在表皮生长因子受体（EGFR）的分析上，因为它的过度表达和突变与一些最难治愈的癌症有很好的关联。然而，可以预期的是，待开发的原理将是足够通用的，以便在未来应用于其他关键信号分子。鉴于现有的高通量筛选的基础在很大程度上限制了其应用于受体或其他信号分子的富集制剂的体外分子分析，UW-BRP寻求建立也可应用于培养细胞和异种移植物和自发性肿瘤组织活检样品分析的工具的原则。这种能力将最终实现一种跨越分子、细胞和组织水平的基本方法，并将用于基础研究以及动物和人体临床试验。在本提案中，一个在化学和生物工程、化学和生物化学以及生物分子和生物医学科学方面具有不同专长的多学科研究人员团队提议开发一种广泛适用的生物分析方法，该方法将以下领域的进展整合在一起：a）表面的纳米制造，B）开发用于将蛋白质和肽共价和定向固定在表面上的合成和生物化学策略，c）液晶作为表面捕获蛋白质存在的高灵敏度报告物的实施，以及d）参与与致癌相关的过程的关键细胞信号传导蛋白的研究。具体地，将在研究中将液晶用于报告公认的抗癌靶标（即EGF受体）的行为的分析特性与常规分析方法进行比较，所述研究将a）快速且灵敏地评估生物样品中野生型和突变型人EGF受体的水平和活性，B）检验EGF受体的野生型和致癌形式表现出不同的抑制剂特异性的假设，和c）评估在体外有效抑制EGF介导的事件的试剂是否也表现出拮抗EGF受体表达的能力和/或或在细胞培养中的活性。这些研究将使用EGF受体系统作为原型，预计该技术将很容易适用于广泛的其他分子靶点。从长远来看，这些新工具应该有助于评估抗癌药物的分子机制和后果，从而促进从基础生物学到临床疗效评估的研究。

DESCRIPTION (provided by applicant): The University of Wisconsin Bioengineering Research Partnership (UW-BRP) will focus on the development of new molecular analysis tools that possess the potential to be used to identify and validate biological endpoints whereby the activity of novel anti-cancer agents can be more accurately and rapidly evaluated as to their molecular mechanism(s) and clinical relevance. The work will initially be focused on the analysis of the epidermal growth factor receptor (EGFR), given that its over expression and mutation has been well-associated with some of the most incurable cancers. However, it is anticipated that the principles to be developed will be sufficiently versatile to be applied to other key signaling molecules in the future. Whereas the basis of existing high throughput screens largely restricts their application to in vitro molecular analyses of enriched preparations of receptors or other signaling molecules, the UW-BRP seeks to establish principles for tools that can also be applied to the analysis of samples from cultured cells and from biopsies of xenographs and spontaneous tumor tissues. This capability will ultimately enable a fundamental approach that will span the molecular, cellular and tissue levels and will be used in both basic research and in animal and human clinical trials. In the present proposal, a multidisciplinary team of researchers with diverse expertise in chemical and biological engineering, chemistry and biochemistry, and the biomolecular and biomedical sciences proposes to develop a broadly-applicable bioanalytical approach that integrates advances in the following areas: a) the nano-fabrication of surfaces, b) the development of synthetic and biochemical strategies for the covalent and oriented immobilization of proteins and peptides on surfaces, c) the implementation of liquid crystals as highly sensitive reporters of the presence of proteins captured on surfaces, and d) the investigation of key cell signaling proteins that participate in processes associated with carcinogenesis. Specifically, the analytical characteristics of liquid crystals for reporting the behavior of the well-recognized anti-cancer target, i.e. the EGF receptor, will be compared to conventional analytical methods in a study that will a) rapidly and sensitively assess the levels and activity of wild-type and mutant human EGF receptor in biological samples, b) test the hypothesis that wild-type and oncogenic forms of the EGF receptor exhibit differential inhibitor specificity, and c) assess if agents that potently inhibit EGF-mediated events in vitro will also exhibit a capacity to antagonize EGF receptor expression and/or activity in cell culture. These studies will use the EGF receptor system as a prototype and it is anticipated that the technology will be readily adaptable to a wide range of other molecular targets. In the long term, these new tools should be useful for the assessment of the molecular mechanisms and consequences of anti-cancer agents, thereby facilitating their research from basic biology through to clinical assessment of efficacy.