UNS:Direct measurement of DUB activity in intact single cells using a droplet microfluidic array

UNS：使用液滴微流体阵列直接测量完整单细胞中的 DUB 活性

• 批准号: 1509713
• 负责人: Adam Melvin
• 金额: $ 31.36万
• 依托单位: Louisiana State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-15 至 2019-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1509713&HistoricalAwards=false
• 关键词: UNS; Direct; measurement; DUB; activity

1509713 Melvin, AdamOne of the greatest challenges facing myeloma patients is the resistance to the drugs currently used to treat the disease. This interdisciplinary project involves the development of a microfluidic device and man-made peptide-based biosensors to specifically identify distinct sub-populations of cancer cells found in a tumor, including those that are resistant to particular chemotherapy drugs. Ultimately, the goal of this proposal is to develop a new diagnostic tool that can lead to a personalized treatment protocol for high risk myeloma patients, dramatically increasing the prognosis of individuals battling cancer.Molecularly-targeted therapeutics and personalized medicine have dramatically increased the prognosis of patients suffering from cancer. In the case of multiple myeloma, there has been great success using drugs that specifically target enzymes associated with the ubiquitin proteasome system (UPS). Deubiquitinating enzymes (DUBs) are one such class of enzyme due to their ability to promote drug resistance in multiple myeloma patients. In this proposal, an interdisciplinary approach will be applied to develop a new method to directly measure DUB activity in intact single cells across a heterogeneous population such as tumor biopsy. A long-lived, cell permeable, DUB-specific fluorescent reporter will be developed to directly measure DUB activity. One hallmark of this peptide-based reporter is the inclusion of a â-hairpin ?protectide?, serving to both confer stability onto the DUB-specific substrate, and to act as a potent cell penetrating peptide (CPP). This novel reporting scheme will be incorporated into a microfluidic droplet array that will be developed to facilitate high-throughput screening (HTS) of a population of myeloma cells. The microfluidic droplet array designed in this proposal will allow for on-chip encapsulation followed by real-time quantification of DUB activity in intact single cells. The biochemical assay will serve as a first step in developing a new technique allowing to 1) determine if patients would benefit from a DUB-targeted therapy, 2) identify an ideal dose of drug to maximize efficacy while minimizing side effects, and 3) analyze a heterogeneous sample to identify distinct subpopulations of drug-resistant cells, which cannot be performed using bulk measurement.This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Experimental Program to Stimulate Competitive Research (EPSCoR).

骨髓瘤患者面临的最大挑战之一是对目前用于治疗该疾病的药物的耐药性。这个跨学科项目涉及微流体设备和人造肽基生物传感器的开发，以特异性地识别肿瘤中发现的不同癌细胞亚群，包括对特定化疗药物具有抗性的癌细胞亚群。最终，该提案的目标是开发一种新的诊断工具，可以为高危骨髓瘤患者提供个性化的治疗方案，大大提高癌症患者的预后。分子靶向治疗和个性化药物大大提高了癌症患者的预后。在多发性骨髓瘤的情况下，使用特异性靶向与泛素蛋白酶体系统（UPS）相关的酶的药物已经取得了巨大的成功。去泛素化酶（DUB）是一类这样的酶，因为它们能够促进多发性骨髓瘤患者的耐药性。 在这项提案中，将采用跨学科的方法来开发一种新的方法来直接测量跨异质群体（如肿瘤活检）的完整单细胞中的DUB活性。将开发一种寿命长、可渗透细胞的DUB特异性荧光报告基因，以直接测量DUB活性。这种基于肽的报告基因的一个特点是包含了一个发夹？protectide？，用于赋予DUB特异性底物稳定性，并作为有效的细胞穿透肽（CPP）。这种新的报告方案将被纳入微流体微滴阵列，该微流体微滴阵列将被开发用于促进骨髓瘤细胞群体的高通量筛选（HTS）。 在该提议中设计的微流体液滴阵列将允许芯片上封装，然后实时定量完整单细胞中的DUB活性。生化测定将作为开发新技术的第一步，该技术允许1）确定患者是否将受益于DUB靶向治疗，2）确定理想的药物剂量以最大化疗效同时最小化副作用，以及3）分析异质样品以鉴定不同的耐药细胞亚群，该奖项由CBET部门的生物技术和生物化学工程项目颁发，由刺激竞争性研究实验项目（EPSCoR）共同资助。