Development of enzymatic tools for rapid measurement of Advanced Glycation End Product-protein adducts

项目摘要 晚期糖基化终产物（AGE）的积累是哺乳动物衰老的标志。AGEs起着致病作用 通过干扰细胞信号通路和在细胞表面形成加合物， 大分子，有助于其在许多组织/器官中的失活和病理生理学。 Nε-羧甲基赖氨酸（CML）是赖氨酸的美拉德反应产物，是最具特征的AGE， 临床上用于糖尿病肾病的预测性筛查，并作为糖尿病视网膜病变的生物标志物 和黄斑水肿。尽管CML作为年龄相关疾病的生物标志物和促成因素的重要性， 目前还没有用于快速、定量检测患者样品中CML的技术。 因此，改进测量CML的方法对于推进该研究具有非常重要的意义， 治疗衰老疾病。 Revel制药公司提出开发旨在切割CML的酶，从而使试剂能够用于 基于酶的检测衰老、糖尿病和糖生物学中最重要的AGEs之一。发展 一种简便的酶法检测临床样本中的CML将是重要的一步 在诊断医学上的进步。 Revel发现了一种CML氧化酶，能够将CML氧化为赖氨酸。新发现的CML 氧化酶提供了开发用于监测CML修饰的诊断用途的酶的机会。的 该I期SBIR的目标是开发用于快速测量CML的酶测定法。拟议 将开发一种类似于用于评估患者血糖的酶促HbA 1c检测的检测方法 水平和诊断糖尿病。在第二阶段，我们将开发一个在患者样本中检测CML的工作流程。

PROJECT SUMMARY Advanced glycation end product (AGE) accumulation is a hallmark of mammalian aging. AGEs play causative roles in various age-related diseases by interfering with cell signaling pathways and forming adducts on cellular macromolecules, contributing to their inactivation and pathophysiology in many tissues/organs. Nε-carboxymethyl-lysine (CML), a maillard reaction product of lysine, is the best characterized AGE and is used clinically for predictive screening of diabetic kidney disease and as a biomarker for diabetic retinopathy and macular oedema. Despite the importance of CML as a biomarker of and contributor to age related disease, there are currently no techniques available for rapid, quantitative detection of CML in patient samples. Therefore, improved methods for measuring CML would be highly significant for advancing the study and treatment of diseases of aging. Revel Pharmaceuticals proposes to develop enzymes designed to cleave CML, thus enabling reagents for enzyme-based detection of one of the most important AGEs in aging, diabetes and glycobiology. Development of a convenient enzymatic method for measurement of CML in clinical samples would be an important step forward in diagnostic medicine. Revel has discovered a CML oxidase enzyme, capable of oxidizing CML to lysine. The newly discovered CML oxidase provides an opportunity to develop enzymes for diagnostic use in monitoring CML-modifications. The goal of this Phase I SBIR is to develop an enzymatic assay for rapid measurement of CML. The proposed assay will be developed analogous to the enzymatic HbA1c test which is used to assess patient blood sugar levels and diagnose diabetes. In Phase II, we will develop a workflow for CML detection in patient samples.