Yu Xin, Lin Hanfeng, Li Feng, Wang Jin, Lu Dong
Department of Biochemistry and Molecular Pharmacology, Baylor College of Medicine, Houston, Texas 77030, United States.
Center for Drug Discovery, Baylor College of Medicine, Houston, Texas 77030, United States.
ACS Med Chem Lett. 2024 May 10;15(6):906-916. doi: 10.1021/acsmedchemlett.4c00104. eCollection 2024 Jun 13.
RIPK1 inhibitors have emerged as promising candidates for treating diverse diseases, including inflammatory diseases, autoimmune disorders, Alzheimer's disease, and cancer. However, the previously reported binding assays have limited sensitivity and stability, impeding high-throughput screening and robust characterization of the RIPK1 inhibitors. To address this challenge, we introduced two probes, T2-BDP-FL and T3-BDP-FL, derived from distinct RIPK1 inhibitors with different binding modes to establish time-resolved fluorescence resonance energy transfer (TR-FRET) displacement assays. Employing our TR-FRET displacement assays, we quantified the biochemical binding affinities of a series of RIPK1 inhibitors with diverse structural and binding modes for human RIPK1. Consistent results were obtained with these two probes in the TR-FRET displacement assay. Furthermore, we developed a RIPK1 fluorescent probe, T2-BDP589, for the NanoBRET assay. This assay enabled the characterization of RIPK1 target engagement by various RIPK1 inhibitors for both human and mouse RIPK1 in live cells. Our developed fluorescent probe displacement assays offer a sensitive and high-throughput approach to identify RIPK1 inhibitors based on both biochemical and cellular activities.
RIPK1抑制剂已成为治疗多种疾病的有前景的候选药物，这些疾病包括炎症性疾病、自身免疫性疾病、阿尔茨海默病和癌症。然而，先前报道的结合测定法灵敏度和稳定性有限，阻碍了RIPK1抑制剂的高通量筛选和全面表征。为应对这一挑战，我们引入了两种探针，T2-BDP-FL和T3-BDP-FL，它们源自具有不同结合模式的不同RIPK1抑制剂，以建立时间分辨荧光共振能量转移（TR-FRET）置换测定法。利用我们的TR-FRET置换测定法，我们定量了一系列具有不同结构和结合模式的RIPK1抑制剂与人RIPK1的生化结合亲和力。在TR-FRET置换测定中，这两种探针获得了一致的结果。此外，我们开发了一种用于纳米BRET测定的RIPK1荧光探针T2-BDP589。该测定法能够表征各种RIPK1抑制剂在活细胞中对人和小鼠RIPK1的RIPK1靶点结合情况。我们开发的荧光探针置换测定法提供了一种基于生化和细胞活性来鉴定RIPK1抑制剂的灵敏且高通量的方法。
