Chemical Resource Development Research Unit, RIKEN CSRS, Wako, Saitama, 351-0198, Japan.
Department of RIKEN Molecular and Chemical Somatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, 113-8510, Japan.
Oncol Res. 2023 Jul 21;31(5):645-654. doi: 10.32604/or.2023.030240. eCollection 2023.
β-transducin repeat-containing protein (β-TrCP) is an F-box protein subunit of the E3 Skp1-Cullin-F box (SCF) type ubiquitin-ligase complex, and provides the substrate specificity for the ligase. To find potent ligands of β-TrCP useful for the proteolysis targeting chimera (PROTAC) system using β-TrCP in the future, we developed a high-throughput screening system for small molecule β-TrCP ligands. We screened the chemical library utilizing the system and obtained several hit compounds. The effects of the hit compounds on ubiquitination activity of SCF and on downstream signaling pathways were examined. Hit compounds NPD5943, NPL62020-01, and NPL42040-01 inhibited the TNFα-induced degradation of IκBα and its phosphorylated form. Hence, they inhibited the activation of the transcription activity of NF-κB, indicating the effective inhibition of β-TrCP by the hit compounds in cells. Next, we performed an analysis of the hit compounds to determine the important moieties of the hit compounds. Carboxyl groups of NPL62020-01 and NPL42040-01 and hydroxyl groups of NPD5943 created hydrogen bonds with β-TrCP similar to those created by intrinsic target phosphopeptides of β-TrCP. Our findings enhance our knowledge of useful small molecule ligands of β-TrCP and the importance of residues that can be ligands of β-TrCP.
β-联重复蛋白（β-TrCP）是 E3 Skp1-Cullin-F 盒（SCF）型泛素连接酶复合物的 F -box 蛋白亚基，为连接酶提供底物特异性。为了找到未来使用β-TrCP 的蛋白水解靶向嵌合体（PROTAC）系统的有效β-TrCP 配体，我们开发了一种用于小分子β-TrCP 配体的高通量筛选系统。我们利用该系统筛选了化学文库，获得了几种命中化合物。研究了这些命中化合物对 SCF 泛素化活性和下游信号通路的影响。命中化合物 NPD5943、NPL62020-01 和 NPL42040-01 抑制了 TNFα 诱导的 IκBα及其磷酸化形式的降解。因此，它们抑制了 NF-κB 的转录活性的激活，表明命中化合物在细胞中有效抑制了β-TrCP。接下来，我们对命中化合物进行了分析，以确定命中化合物的重要部分。NPL62020-01 和 NPL42040-01 的羧基和 NPD5943 的羟基与β-TrCP 形成氢键，类似于β-TrCP 的内在靶标磷酸肽形成的氢键。我们的研究结果增强了我们对有用的β-TrCP 小分子配体的认识，以及β-TrCP 的配体残基的重要性。
