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An efficient assay for identification and quantitative evaluation of potential polysialyltransferase inhibitors.
Analyst ( IF 3.6 ) Pub Date : 2020-05-08 , DOI: 10.1039/d0an00721h Xiaoxiao Guo 1 , Jodie R Malcolm , Marrwa M Ali , Goreti Ribeiro Morais , Steven D Shnyder , Paul M Loadman , Laurence H Patterson , Robert A Falconer
Analyst ( IF 3.6 ) Pub Date : 2020-05-08 , DOI: 10.1039/d0an00721h Xiaoxiao Guo 1 , Jodie R Malcolm , Marrwa M Ali , Goreti Ribeiro Morais , Steven D Shnyder , Paul M Loadman , Laurence H Patterson , Robert A Falconer
Affiliation
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The polysialyltransferases (polySTs) catalyse the polymerisation of polysialic acid, which plays an important role in tumour metastasis. While assays are available to assess polyST enzyme activity, there is no methodology available specifically optimised for identification and quantitative evaluation of potential polyST inhibitors. The development of an HPLC-fluorescence-based enzyme assay described within includes a comprehensive investigation of assay conditions, including evaluation of metal ion composition, enzyme, substrate and acceptor concentrations, temperature, pH, and tolerance to DMSO, followed by validation using known polyST inhibitors. Thorough analysis of each of the assay components provided a set of optimised conditions. Under these optimised conditions, the experimentally observed Ki value for CMP, a competitive polyST inhibitor, was strongly correlated with the predicted Ki value, based on the classical Cheng–Prusoff equation [average fold error (AFE) = 1.043]. These results indicate that this assay can provide medium-throughput analysis for enzyme inhibitors with high accuracy, through determining the corresponding IC50 values with substrate concentration at the KM, without the need to perform extensive kinetic studies for each compound. In conclusion, an in vitro cell-free assay for accurate assessment of polyST inhibition is described. The utility of the assay for routine identification of potential polyST inhibitors is demonstrated, allowing quantitative measurement of inhibition to be achieved, and exemplified through assessment of full competitive inhibition. Given the considerable and growing interest in the polySTs as important anti-metastatic targets in cancer drug discovery, this is a vital tool to enable preclinical identification and evaluation of novel polyST inhibitors.
中文翻译：
一种有效的测定方法，用于鉴定和定量评估潜在的聚唾液酸转移酶抑制剂。
聚唾液酸转移酶（polySTs）催化聚唾液酸的聚合，这在肿瘤转移中起着重要作用。尽管有可用的方法来评估polyST酶的活性，但尚没有专门针对识别和定量评估潜在的polyST抑制剂而专门优化的方法。其中描述的基于HPLC荧光的酶测定方法的开发包括对测定条件的全面研究，包括评估金属离子组成，酶，底物和受体的浓度，温度，pH和对DMSO的耐受性，然后使用已知的polyST进行验证抑制剂。对每个测定成分的彻底分析提供了一组优化条件。在这些优化条件下，实验观察到的K i基于经典的Cheng–Prusoff方程[平均倍数误差（AFE）= 1.043] ，竞争性polyST抑制剂CMP的K值与预测的K i值密切相关。这些结果表明，该测定法可以通过确定相应的IC 50值和底物浓度为K M的情况下，对酶抑制剂的中通量分析进行高精度分析，而无需对每种化合物进行广泛的动力学研究。总之，体外描述了用于准确评估polyST抑制作用的无细胞检测方法。证明了该测定法用于常规鉴定潜在的polyST抑制剂的实用性，可以实现对抑制作用的定量测量，并通过评估完全竞争性抑制作用来举例说明。鉴于人们对polyST作为癌症药物发现中重要的抗转移靶标的兴趣日益浓厚，这是实现临床前鉴定和评估新型polyST抑制剂的重要工具。
更新日期：2020-06-29
中文翻译：
一种有效的测定方法，用于鉴定和定量评估潜在的聚唾液酸转移酶抑制剂。
聚唾液酸转移酶（polySTs）催化聚唾液酸的聚合，这在肿瘤转移中起着重要作用。尽管有可用的方法来评估polyST酶的活性，但尚没有专门针对识别和定量评估潜在的polyST抑制剂而专门优化的方法。其中描述的基于HPLC荧光的酶测定方法的开发包括对测定条件的全面研究，包括评估金属离子组成，酶，底物和受体的浓度，温度，pH和对DMSO的耐受性，然后使用已知的polyST进行验证抑制剂。对每个测定成分的彻底分析提供了一组优化条件。在这些优化条件下，实验观察到的K i基于经典的Cheng–Prusoff方程[平均倍数误差（AFE）= 1.043] ，竞争性polyST抑制剂CMP的K值与预测的K i值密切相关。这些结果表明，该测定法可以通过确定相应的IC 50值和底物浓度为K M的情况下，对酶抑制剂的中通量分析进行高精度分析，而无需对每种化合物进行广泛的动力学研究。总之，体外描述了用于准确评估polyST抑制作用的无细胞检测方法。证明了该测定法用于常规鉴定潜在的polyST抑制剂的实用性，可以实现对抑制作用的定量测量，并通过评估完全竞争性抑制作用来举例说明。鉴于人们对polyST作为癌症药物发现中重要的抗转移靶标的兴趣日益浓厚，这是实现临床前鉴定和评估新型polyST抑制剂的重要工具。
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