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伙伴关系（UW-BRP）正在开发新的分子分析工具，以识别和验证生物学终点，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。这项工作的重点是表皮生长因子受体（EGFR），因为它的过度表达和突变与一些最无法治愈的癌症密切相关。然而，这些工具足够通用，可以应用于其他关键信号分子。鉴于许多现有筛选的基础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样品分析的工具的原则，以及异种移植物和自发性肿瘤组织的活检。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。在过去的4年中，UW-BRP定义了基于液晶的分子分析工具的关键分析特征，并证明了EGFR表达和激活（磷酸化）的报告以及小分子对EGFR酪氨酸激酶活性的抑制（在复杂样品中，如膜提取物和细胞裂解物）。本提案寻求继续支持在化学和生物工程、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，通过整合以下领域的进展，进一步开发这种广泛适用的生物分析方法：a）识别和优化识别细胞裂解物中关键EGFR突变体表位的均匀固定的单链抗体，B）整合基于液晶的分析方法和样品制备，用于使用小样品（少数细胞）定量野生型和突变型EGFR的表达、活化和激酶抑制，和c）研究参与与致癌相关的过程的关键细胞信号传导蛋白。具体而言，将UW-BRP分子分析工具与研究中的常规方法进行比较，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EGFR的水平和活性，B）检验EGFR的野生型和致癌形式将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将表现出拮抗细胞培养物中EGFR表达和/或活性的能力。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。公共卫生相关性：威斯康星州生物工程研究伙伴关系的大学是专注于开发一个综合的生物工程系统的方法，将提供新的分子工具来分析的蛋白质调控途径的基础细胞转化成癌状态的基础。这些工具有可能加速生物学终点的识别和验证，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。

DESCRIPTION (provided by applicant): The University of Wisconsin Bioengineering Research Partnership (UW-BRP) is developing new molecular analysis tools 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 is focused on epidermal growth factor receptor (EGFR), given that its overexpression and mutation has been closely-associated with some of the most incurable cancers. However, the tools are sufficiently versatile to be applicable to other key signaling molecules. Whereas the basis of many existing screens 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. Over the past 4 years, the UW-BRP has defined key analytic characteristics of liquid crystal-based tools for molecular analysis, and demonstrated reporting of EGFR expression and activation (phosphorylation) as well as inhibition of EGFR tyrosine kinase activity by small molecules (in complex samples such as membrane extracts and cell lysates). The present proposal seeks continued support for the multi-disciplinary team of researchers with diverse expertise in chemical and biological engineering, chemistry and biochemistry, and the biomolecular and biomedical sciences to develop further this broadly-applicable bioanalytical approach via integration of advances in the following areas: a) identification and optimization of uniformly immobilized, single chain antibodies that recognize epitopes of key EGFR mutants in cell lysates, b) integration of liquid crystal-based analytic methodologies and sample preparation for quantification of expression, activation and kinase inhibition of wild-type and mutant EGFRs using small samples (a few cells), and c) the investigation of key cell signaling proteins that participate in processes associated with carcinogenesis. Specifically, the UW-BRP molecular analysis tools will be compared to conventional methods in a study that will a) rapidly and sensitively assess the levels and activity of wild-type and mutant human EGFRs in biological samples, b) test the hypothesis that wild-type and oncogenic forms of the EGFR will exhibit differential inhibitor specificity, and c) assess if agents that potently inhibit EGF-mediated events in vitro will also exhibit a capacity to antagonize EGFR expression and/or activity in cell culture. 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. PUBLIC HEALTH RELEVANCE: The University of Wisconsin Bioengineering Research Partnership is focused on the development of an integrated bioengineering systems approach that will provide the foundations for new molecular tools to analyze the protein regulatory pathways that underlie cellular transformation into cancerous states. These tools have the potential to accelerate the identification and validation of biological endpoints whereby the activity of novel anti-cancer agents can be more accurately and rapidly evaluated for their molecular mechanisms and clinical relevance.