High throughput glutathione and Nrf2 assays to assess chemical and biological reactivity of cysteine-reactive compounds
High throughput glutathione and Nrf2 assays to assess chemical and biological reactivity of cysteine-reactive compounds
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DOI：
10.1039/c3tx50027f
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发表时间：
2013-01-01
影响因子：
2.1
通讯作者：
Leach, Karen L.
中科院分区：
文献类型：
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作者：
Naven, Russell T.;Kantesaria, Sheila;Leach, Karen L.
通过共价结合抑制其靶标的化合物作为潜在的治疗剂具有许多独特的优势。它们可以实现高配体效率，从而实现高效力，这可以转化为减少的药物剂量。此外，共价结合可以导致化合物的作用持续时间较长，从而减少药物给药的频率。尽管有这些优点，但由于其固有的反应性和与细胞蛋白质非特异性结合的潜力，此类化合物仍存在一些安全问题。本研究的主要目的是通过测量对各种含半胱氨酸的细胞成分的反应性来确定丙烯酰胺和其他半胱氨酸反应基团的硫醇反应性。将化合物与谷胱甘肽、牛血清蛋白 (BSA) 或人肝微粒体 (HLM) 一起孵育，并使用埃尔曼试剂对硫醇水平的降低进行定量。此外，化合物在报告基因测定中诱导Nrf2活性的能力被用作化合物反应性的功能读数。使用已知的硫醇反应性化合物和 Nrf2 诱导剂（例如萝卜硫素 (SFP) 和 1,2-二硫醇-3-硫酮 (D3T)）对测定进行了验证。我们的结果表明，除非被吸电子 N-取代基激活，否则丙烯酰胺具有低反应性。类似的丙炔酰胺和乙烯基砜在谷胱甘肽活性方面比丙烯酰胺更具反应性，但在 Nrf2 测定中反应性较低。大的药物样分子，包括针对 BTK 和 EGFR 的不可逆激酶抑制剂，其反应性远低于其低分子量对应物，这表明亲电子试剂的存在不足以预测硫醇反应性。这些研究证明了在评估共价抑制剂的硫醇反应性时，除了谷胱甘肽之外，多种硫醇来源的效用。
Compounds that inhibit their target through covalent binding offer a number of unique advantages as potential therapeutic agents. They can achieve high ligand efficiency, and thus high potency, which can translate into a reduced drug dosage. In addition, covalent binding can result in a long duration of action of the compound and thus less frequent drug dosing. Despite these advantages, there are several safety concerns about this class of compounds because of their inherent reactivity and potential for nonspecific binding to cellular proteins. The primary aim of this study was to establish the thiol reactivity of acrylamides and other cysteine-reactive groups by measuring reactivity against various cysteine-containing cellular components. Compounds were incubated with glutathione, bovine serum albumen (BSA) or human liver microsomes (HLM), and the reduction in thiol levels was quantitated using Ellman's reagent. In addition, the ability of compounds to induce Nrf2 activity in a reporter gene assay was used as a functional readout of compound reactivity. The assays were validated using known thiol-reactive compounds and Nrf2 inducers such as sulforaphane (SFP) and 1,2-dithiole-3-thione (D3T). Our results demonstrate that acrylamides possess low reactivity unless activated by electron-withdrawing N-substituents. Analogous propynamides and vinyl sulphones were more reactive than acrylamides with respect to glutathione activity but were less reactive in the Nrf2 assay. Large drug-like molecules, including irreversible kinase inhibitors targeting BTK and EGFR, were far less reactive than their lower molecular weight counterparts, suggesting that the presence of an electrophile is not sufficient to predict thiol reactivity. These studies demonstrate the utility of multiple thiol sources in addition to glutathione when assessing the thiol reactivity of covalent inhibitors.