孔雀石绿测定法用于发现热休克蛋白 90 抑制剂
Summary
孔雀石绿测定方案是一种简单且经济高效的方法，用于发现热休克蛋白 90 （Hsp90） 抑制因子以及其他针对 ATP 依赖性酶的抑制剂化合物。
Abstract
热休克蛋白90（Hsp90）是一种有前途的抗癌靶点，因为它对多种致癌蛋白具有伴侣作用。Hsp90的活性取决于其将三磷酸腺苷（ATP）水解为二磷酸腺苷（ADP）和游离磷酸盐的能力。Hsp90的ATP酶活性与其伴侣功能有关;ATP与Hsp90的N端结构域结合，破坏其结合被认为是抑制Hsp90功能最成功的策略。ATP酶活性可以通过比色孔雀石绿测定法测量，该测定法确定ATP水解形成的游离磷酸盐的量。这里描述了使用孔雀石绿磷酸测定试剂盒测定酵母Hsp90的ATP酶活性的程序。此外，还提供了通过服用格尔达霉素作为真实抑制剂来发现Hsp90抑制剂的详细说明。最后，讨论了该测定方案通过针对酵母Hsp90的抑制剂分子的高通量筛选（HTS）的应用。
Introduction
热休克蛋白90（Hsp90）是一种分子伴侣，可维持负责癌症发展和进展的蛋白质的稳定性。此外，负责抗肿瘤药物耐药性的蛋白质也是Hsp901的客户。Hsp90在所有癌细胞类型（>90%的细胞蛋白）中普遍过度表达，而正常细胞可能占总蛋白的2%以下。此外，癌细胞的Hsp90与共伴侣存在于复合物中，而在正常细胞中，它主要以游离，未复合的状态存在2，3。近年来，几种Hsp90抑制剂在体外和体内研究中已被证明具有senolyticality作用，它们显着改善了小鼠的寿命4，5，6。上述所有发现都证实了这样一个事实，即Hsp90抑制剂可能对多种癌症类型有效，不良反应更少，产生耐药性的机会也更少。Hsp90的伴侣功能是通过在Hsp90的N端结构域结合ATP并将其水解成ADP和游离磷酸盐7来实现的。发现与Hsp90的ATP结合口袋竞争性结合的小分子成功地抑制了蛋白质的伴侣作用。迄今为止，这仍然是Hsp90抑制的最佳策略，这种抑制剂已达到临床试验的事实支持了这一策略8。其中之一Pimitespib于2022年6月在日本被批准用于治疗胃肠道间质瘤（GIST）9。这是自1994年建立伴侣的成药性以来第一个批准的Hsp90抑制剂10。
孔雀石绿测定是一种简单、灵敏、快速且廉价的无机磷酸盐检测程序，适用于针对其所需靶标11 对化合物进行自动化和高通量筛选 （HTS）。该测定已成功用于在小型实验室规模设置以及HTS12，13，14，15，16，17中筛选Hsp90抑制剂。该测定使用比色法测定由于Hsp90的ATP酶活性而形成的游离无机磷酸盐。这种定量的基础是在游离磷酸盐和钼之间形成磷钼酸盐复合物，随后与孔雀石绿反应产生绿色（图1）。这种快速的颜色形成是在分光光度计或读板器上测量的，在600-660nm18，19之间。
在本协议中，描述了用酵母Hsp90进行孔雀石绿测定以及随后鉴定针对伴侣的抑制剂的程序。首先建立Hsp90成药性的天然产物分子geldanamycin （GA）被视为真正的抑制剂10。由于有大量分子用于测试，HTS已成为当前药物发现计划的一个组成部分。由于迫切需要重新利用药物治疗Covid-19感染，该技术在过去2年中变得更加重要20，21。因此，提出了采用孔雀石绿测定法对酵母Hsp90蛋白的分子HTS的详细概述。
Protocol
1. 实验室规模的孔雀石绿色测定 测定缓冲液的制备按照 表1中所示的组合物和制备制备测定缓冲液。 磷酸盐标准品的制备使用孔雀石绿色测定磷酸盐测定试剂盒（储存在4°C）中提供的1mM磷酸盐标准品。 将 40 μL 1 mM 磷酸盐标准品移入 960 μL 超纯水中，以获得 40 μM 磷酸盐溶液（预混溶液）。按照制造商的说明，将预混料溶液与?…
Representative Results
测定结果根据游离磷酸根离子浓度引起的吸光度来解释。由于酵母Hsp90在620nm处水解ATP引起的游离磷酸盐的吸光度被认为是100%ATP酶活性或零百分比蛋白质抑制。蛋白质的抑制导致ATP水解停止（游离磷酸盐减少）。这反映在620nm处的吸光度降低。 实验室规模的孔雀石绿色测定结果磷酸盐标准品的标准图如图 3所示。Hsp90的活性是根据其将ATP水…
Discussion
Hsp90是发现新型抗癌药物分子的重要靶标。自1994年建立成药性以来，已有10、18种分子进入临床试验。目前，有7种分子处于临床试验的不同阶段，或单独使用，或组合22。所有这些小分子都是N端ATP结合抑制剂。抑制伴侣的其他手段（C末端抑制剂，中域抑制剂）的进展不如N末端抑制剂快。因此，N端ATP结合抑制剂化合物仍然有望发展为临床上可销售的分子。此外?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了韩国研究奖学金（KRF）计划的支持，该计划是韩国国家研究基金会（NRF）的博士后研究员，由科学与信息通信技术部（NRF-2019H1D3A1A01102952）资助。作者感谢KIST校内赠款和海洋和渔业部第2MRB130号赠款为该项目提供财政援助。
Materials
| 1M Magnesium chloride solution in water | Sigma-Aldrich | 63069-100ml | |
| 1M Potassium chloride solution in water | Sigma-Aldrich | 60142-100ml | |
| 96-well plate | SPL Life Sciences | Not applicable | |
| Adenosine 5′-triphosphate disodium salt hydrate | Sigma-Aldrich | A7699-5G | |
| Biomek FX laboratory automation workstation | Beckman Coulter | Not applicable | |
| Compounds 3-96 | Not applicable | Not applicable | Histidine tagged yeast Hsp90 was obtained from Dr. Chrisostomos Prodromou, School of Life Sciences, University of Sussex, United Kingdom, and protein was expressed in KIST Gangneung Institute of Natural Products. Details cannot be disclosed due to patent infringement issues. |
| Dimethyl sulfoxide | Sigma-Aldrich | D8418 | |
| Geldanamycin, 99% (HPLC), powder | AK Scientific, Inc. | V2064 | |
| Invitroge UltraPure DNase/RNase-Free Distilled Water | ThermoFisher Scientific | 10977015 | |
| Malachite Green Phosphate Assay Assay kit | Sigma-Aldrich | MAK307-1KT | |
| Multi-Detection Microplate Reader Synergy HT | Biotek Instruments, Inc. | Not applicable | |
| Synergy HT multi-plate reader | Biotek Instruments, Inc. | Not applicable | |
| Trizma hydrochloride buffer solution, pH7.4 | Sigma-Aldrich | 93313-1L | |
| Yeast Hsp90 | Not applicable | Not applicable | School of Life Sciences, University of Sussex, United Kingdom and protein was expressed in KIST Gangneung Institute of Natural Products. Primary Accession number: P02829 |
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Cite This Article
Gupta, S. D., Song, D., Lee, S., Lee, J. W., Park, J., Prodromou, C., Pan, C. Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors. J. Vis. Exp. (191), e64693, doi:10.3791/64693 (2023).