BellBrook Labs, R&D, Madison, WI, USA.
Dane County Youth Apprenticeship Program in Biotechnology, Verona Area High School, Verona, WI, USA.
SLAS Discov. 2020 Mar;25(3):320-326. doi: 10.1177/2472555219893632. Epub 2019 Dec 22.
Production of adenosine in the extracellular tumor microenvironment elicits strong immunosuppression and is associated with tumor progression. Thus, targeting adenosine-generating ectonucleotidases is a potential strategy to stimulate and prolong antitumor immunity. Because the reaction products of ectonucleotidases differ by a single phosphate group, selective detection in an assay format that is compatible with high-throughput screening (HTS) has been elusive. We report the development of biochemical assays capable of measuring the activity of ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1; also known as CD39) and ecto-5'-nucleotidase (CD73). Both assays leverage the Transcreener HTS Assay platform, which facilitates selective immunodetection of nucleotides with homogenous fluorescent readouts, fluorescence polarization or time-resolved fluorescence energy transfer. The Transcreener AMP2 Assay was used to measure CD39 activity, allowing detection of adenosine monophosphate (AMP) production (Z' > 0.6) with subnanomolar amounts of CD39, allowing IC determination for tool compounds, consistent with previously reported values. To detect the production of adenosine by CD73, the Transcreener ADP2 Assay was coupled with adenosine kinase (AK); conversion of adenosine to AMP and adenosine diphosphate (ADP) by AK allows detection with ADP2 antibody. The Transcreener AMP2 Assay was used to screen a 1280 Library of Pharmacologically Active Compounds (LOPAC) library and a 1600-compound subset of a ChemBridge diversity library for CD39 inhibitors, allowing the identification of nine and eight candidate compounds from each library, respectively. The Transcreener ADP2 Assay was used to screen 1600 compounds from the ChemBridge diversity library for CD73 inhibitors and identified 14 potential candidates. HTS-compatible assays for ectonucleotidase activity may allow identification of purinergic signaling pathway inhibitors important for tumor-specific immune responses during tumor pathogenesis.
细胞外肿瘤微环境中腺苷的产生会引发强烈的免疫抑制，并与肿瘤进展相关。因此，靶向产生腺苷的细胞外核苷酸酶是刺激和延长抗肿瘤免疫的一种潜在策略。由于细胞外核苷酸酶的反应产物仅相差一个磷酸基团，因此在与高通量筛选 (HTS) 兼容的测定形式中进行选择性检测一直难以实现。我们报告了能够测量外核苷酸三磷酸二磷酸水解酶-1（ENTPD1；也称为 CD39）和外 5'-核苷酸酶（CD73）活性的生化测定方法的开发。这两种测定方法都利用了 Transcreener HTS 测定平台，该平台可通过均相荧光读数、荧光偏振或时间分辨荧光能量转移选择性地检测核苷酸。使用 Transcreener AMP2 测定法来测量 CD39 活性，允许以亚纳摩尔数量的 CD39 检测 AMP 的产生（Z' > 0.6），从而可以确定工具化合物的 IC 值，这与之前报道的值一致。为了检测 CD73 产生的腺苷，将 Transcreener ADP2 测定法与腺苷激酶（AK）偶联；AK 将腺苷转化为 AMP 和腺苷二磷酸（ADP），可通过 ADP2 抗体进行检测。使用 Transcreener AMP2 测定法筛选了 Pharmacologically Active Compounds 文库（LOPAC）中的 1280 种化合物库和 ChemBridge 多样性文库中的 1600 种化合物亚库，分别从每个文库中鉴定出 9 种和 8 种候选化合物。使用 Transcreener ADP2 测定法筛选了 ChemBridge 多样性文库中的 1600 种化合物，鉴定出 14 种潜在候选药物。用于细胞外核苷酸酶活性的 HTS 兼容测定法可能有助于确定在肿瘤发病过程中对肿瘤特异性免疫反应重要的嘌呤能信号通路抑制剂。
