The intracellular concentration of diadenosine tetraphospate (Ap₄A) increases upon exposure to stress conditions. Despite being discovered over 50 years ago, the cellular functions of Ap₄A are still enigmatic. If and how the varied Ap₄A is a signal and involved in the signaling pathways leading to an appropriate cellular response remain to be discovered. Because the turnover of Ap₄A by Ap₄A cleaving enzymes is rapid, small molecule inhibitors for these enzymes would provide tools for the more detailed study of the role of Ap₄A. Here, we describe the development of a high-throughput screening assay based on a fluorogenic Ap₄A substrate for the identification and optimization of small molecule inhibitors for Ap₄A cleaving enzymes. As proof-of-concept we screened a library of over 42 000 compounds toward their inhibitory activity against the Ap₄A phosphorylase (Rv2613c) of Mycobacterium tuberculosis (Mtb). A sulfanylacrylonitril derivative with an IC₅₀ of 260 ± 50 nM in vitro was identified. Multiple derivatives were synthesized to further optimize their properties with respect to their in vitro IC₅₀ values and their cytotoxicity against human cells (HeLa). In addition, we selected two hits to study their antimycobacterial activity against virulent Mtb to show that they might be candidates for further development of antimycobacterial agents against multidrug-resistant Mtb.

中文翻译：

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结核分枝杆菌二磷酸腺苷四磷酸磷酸化酶Rv2613c的抑制剂

暴露于应激条件下，胞苷四磷酸腺苷（Ap ₄ A）浓度增加。尽管在50多年前就被发现，但Ap ₄ A的细胞功能仍然是令人迷惑的。变化的Ap ₄ A是否以及如何成为信号并参与导致适当细胞应答的信号传导途径尚待发现。由于Ap ₄ A裂解酶对Ap ₄ A的周转速度很快，因此这些酶的小分子抑制剂将为更详细研究Ap ₄ A的作用提供工具。在这里，我们描述了高通量筛选的发展基于荧光Ap _4的检测用于鉴定和优化Ap ₄ A裂解酶的小分子抑制剂的底物。作为概念验证，我们针对其对结核分枝杆菌（Mtb）Ap ₄ A磷酸化酶（Rv2613c）的抑制活性筛选了超过42 000种化合物的文库。鉴定了在体外IC ₅₀为260±50 nM的亚磺酰基丙烯腈衍生物。合成了多种衍生物，以进一步优化其体外IC ₅₀值及其对人细胞的细胞毒性（HeLa）的性能。此外，我们选择了两个命中来研究它们对强毒的抗分枝杆菌活性Mtb表明它们可能是进一步开发抗多药耐药性Mtb的抗分枝杆菌药物的候选药物。