Establishing MALDI-TOF as Versatile Drug Discovery Readout to Dissect the PTP1B Enzymatic Reaction
Establishing MALDI-TOF as Versatile Drug Discovery Readout to Dissect the PTP1B Enzymatic Reaction
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DOI：
10.1177/2472555218759267
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发表时间：
2018-07-01
期刊：
影响因子：
3.1
通讯作者：
Buettner, Frank H.
中科院分区：
文献类型：
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作者：
Winter, Martin;Bretschneider, Tom;Buettner, Frank H.
无标记质谱 (MS) 检测是药物发现领域的一项新兴技术。与传统的基于标签的读数相比，酶反应的无偏见解读具有明显的优势，这些读数受到化合物干扰和定制信号介质的复杂生成。基质辅助激光解吸/电离飞行时间 (MALDI-TOF) MS 以及相关液体处理仪器的重大发展引发了药物发现界的广泛努力，将全面的 MS 读数集成到高通量筛选 (HTS) 产品组合中。提供与传统基于标签的读数相当的速度、灵敏度和准确性，结合基于 MS 的技术的优点（例如多个生理成分的无标签并行测量），强调了 MALDI-TOF 对于 HTS 方法的优势。在这里，我们描述了用于鉴定蛋白酪氨酸磷酸酶 1B (PTP1B) 抑制剂的检测方法的开发。在这一珍贵的药物靶点的背景下，MALDI-TOF被集成到HTS环境中，并与成熟的AlphaScreen技术进行交叉比较。我们展示了稳健且准确的 IC50 测定，与 AlphaScreen 生成的数据高度一致。此外，还开发了一种定制的 MALDI-TOF 测定法来监测 PTP1B 活性半胱氨酸的化合物依赖性、不可逆修饰。总体而言，所提供的数据证明了将 MALDI-TOF 整合到药物发现活动中的前景广阔。
Label-free, mass spectrometric (MS) detection is an emerging technology in the field of drug discovery. Unbiased deciphering of enzymatic reactions is a proficient advantage over conventional label-based readouts suffering from compound interference and intricate generation of tailored signal mediators. Significant evolvements of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, as well as associated liquid handling instrumentation, triggered extensive efforts in the drug discovery community to integrate the comprehensive MS readout into the high-throughput screening (HTS) portfolio. Providing speed, sensitivity, and accuracy comparable to those of conventional, label-based readouts, combined with merits of MS-based technologies, such as label-free parallelized measurement of multiple physiological components, emphasizes the advantages of MALDI-TOF for HTS approaches. Here we describe the assay development for the identification of protein tyrosine phosphatase 1B (PTP1B) inhibitors. In the context of this precious drug target, MALDI-TOF was integrated into the HTS environment and cross-compared with the well-established AlphaScreen technology. We demonstrate robust and accurate IC50 determination with high accordance to data generated by AlphaScreen. Additionally, a tailored MALDI-TOF assay was developed to monitor compound-dependent, irreversible modification of the active cysteine of PTP1B. Overall, the presented data proves the promising perspective for the integration of MALDI-TOF into drug discovery campaigns.