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High-Throughput Platform for Real-Time Monitoring of ATP-Generating Enzymes in Living Cells Based on a Lanthanide Probe.
ACS Sensors ( IF 8.2 ) Pub Date : 2020-07-02 , DOI: 10.1021/acssensors.0c00897 Moustafa T Gabr 1 , Anand Balupuri 2 , Nam Sook Kang 2
ACS Sensors ( IF 8.2 ) Pub Date : 2020-07-02 , DOI: 10.1021/acssensors.0c00897 Moustafa T Gabr 1 , Anand Balupuri 2 , Nam Sook Kang 2
Affiliation
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Remarkable variation between cell-free and cellular measurements of enzyme activity triggered the unmet need to develop tools for monitoring enzyme activity in living cells. Such tools will advance our understanding of the biological functions of enzymes and their potential impact on drug discovery. We report in this study a universal assay for monitoring ATP-generating enzymes in living cells using a self-assembled Tb3+ complex probe. Modulation of the rheological properties of cell culture media enabled shifting the lifetime of the Tb3+ complex in the presence of ATP from micro-to-millisecond range. Based on the response of the Tb3+ complex to ATP, cellular assays for 5 ATP-generating enzymes were developed. Remarkably, assessment of the activity of these enzymes in living cells is made possible for the first time. The pyruvate kinase M2 (PKM2) assay has been optimized for high-throughput screening (HTS) and further implemented in the identification of novel scaffolds as PKM2 inhibitors.
中文翻译：
基于镧系元素探针的实时监测活细胞中ATP生成酶的高通量平台。
无细胞和细胞对酶活性的测量之间的显着差异触发了对开发用于监测活细胞中酶活性的工具的未满足需求。这些工具将增进我们对酶的生物学功能及其对药物发现的潜在影响的理解。我们在这项研究中报告了一种使用自组装Tb 3+复杂探针监测活细胞中ATP生成酶的通用方法。调节细胞培养基的流变特性可以改变ATP存在下Tb 3+复合物的寿命，从微秒到毫秒。根据Tb 3+的响应与ATP形成复合体后，开发了5种产生ATP的酶的细胞分析方法。值得注意的是，首次可以评估活细胞中这些酶的活性。丙酮酸激酶M2（PKM2）分析已针对高通量筛选（HTS）进行了优化，并进一步用于鉴定新型支架作为PKM2抑制剂。
更新日期：2020-07-24
中文翻译：
基于镧系元素探针的实时监测活细胞中ATP生成酶的高通量平台。
无细胞和细胞对酶活性的测量之间的显着差异触发了对开发用于监测活细胞中酶活性的工具的未满足需求。这些工具将增进我们对酶的生物学功能及其对药物发现的潜在影响的理解。我们在这项研究中报告了一种使用自组装Tb 3+复杂探针监测活细胞中ATP生成酶的通用方法。调节细胞培养基的流变特性可以改变ATP存在下Tb 3+复合物的寿命，从微秒到毫秒。根据Tb 3+的响应与ATP形成复合体后，开发了5种产生ATP的酶的细胞分析方法。值得注意的是，首次可以评估活细胞中这些酶的活性。丙酮酸激酶M2（PKM2）分析已针对高通量筛选（HTS）进行了优化，并进一步用于鉴定新型支架作为PKM2抑制剂。
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