Department of Cellular and Molecular Biology, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
Laboratory for Structural Bioinformatics, Center for Biosystems Dynamics Research, RIKEN, 1-7-22 Suehiro, Tsurumi, Yokohama, Kanagawa 230-0045, Japan.
Int J Mol Sci. 2020 Nov 30;21(23):9150. doi: 10.3390/ijms21239150.
Nuclear factor-κB (NF-κB) is an important transcription factor involved in various biological functions, including tumorigenesis. Hence, NF-κB has attracted attention as a target factor for cancer treatment, leading to the development of several inhibitors. However, existing NF-κB inhibitors do not discriminate between its subunits, namely, RelA, RelB, cRel, p50, and p52. Conventional methods used to evaluate interactions between transcription factors and DNA, such as electrophoretic mobility shift assay and luciferase assays, are unsuitable for high-throughput screening (HTS) and cannot distinguish NF-κB subunits. We developed a HTS method named DNA strand exchange fluorescence resonance energy transfer (DSE-FRET). This assay is suitable for HTS and can discriminate a NF-κB subunit. Using DSE-FRET, we searched for RelA-specific inhibitors and verified RelA inhibition for 32,955 compounds. The compound A55 (2-(3-carbamoyl-6-hydroxy-4-methyl-2-oxopyridin-1(2H)-yl) acetic acid) selectively inhibited RelA-DNA binding. We propose that A55 is a seed compound for RelA-specific inhibition and could be used in clinical applications.
核因子-κB（NF-κB）是一种参与多种生物学功能的重要转录因子，包括肿瘤发生。因此，NF-κB 作为癌症治疗的靶因子引起了关注，导致了几种抑制剂的开发。然而，现有的 NF-κB 抑制剂不能区分其亚基，即 RelA、RelB、cRel、p50 和 p52。用于评估转录因子与 DNA 之间相互作用的传统方法，如电泳迁移率变动分析和荧光素酶测定，不适合高通量筛选（HTS），并且不能区分 NF-κB 亚基。我们开发了一种称为 DNA 链交换荧光共振能量转移（DSE-FRET）的 HTS 方法。该测定法适用于 HTS，并且可以区分 NF-κB 亚基。使用 DSE-FRET，我们搜索了 RelA 特异性抑制剂，并验证了 32,955 种化合物对 RelA 的抑制作用。化合物 A55（2-(3-氨甲酰基-6-羟基-4-甲基-2-氧代-1,2-二氢吡啶-1(2H)-基)乙酸）特异性抑制 RelA-DNA 结合。我们提出 A55 是 RelA 特异性抑制的起始化合物，可用于临床应用。
