Sammons Rae M, Ghosh Soma, Yapindi Lacin, Cho Eun Jeong, Johnson Faye M, Dalby Kevin N
Targeted Therapeutic Drug Discovery & Development Program, The University of Texas at Austin, Austin, TX, United States.
Thoracic, Head and Neck Medical Oncology, The University of Texas, MD Anderson Cancer Center, Houston, TX, United States.
SLAS Discov. 2025 Jun;33:100233. doi: 10.1016/j.slasd.2025.100233. Epub 2025 Apr 12.
TRIP13, a promising target for cancer therapy, has been identified as a key regulator of the mitotic checkpoint. Overexpression of TRIP13 is associated with poor clinical outcomes in various cancers. Inhibition of TRIP13 has the potential to address therapeutic challenges in cancer, particularly in therapy-resistant and Rb-deficient cancers. Despite the potential therapeutic benefits of TRIP13 inhibition, the development of TRIP13 inhibitors has been hindered by the lack of a robust high-throughput screening (HTS) assay. We developed a luminescence-based biochemical assay for TRIP13 activity to address this challenge using the ADP-Glo detection system. This assay offers high sensitivity, low background signal, and ease of automation, making it ideal for HTS applications. A pilot screen of kinase-focused inhibitors library and a large-scale screen of 4000 additional compounds demonstrated the assay's robust performance with a z'-factor exceeding 0.85 and a signal-to-background (S/B) ratio near 6. From the 50 initial hits, rigorous validation identified anlotinib as the most potent TRIP13 inhibitor with an IC of 5 μM. A cellular thermal shift assay (CETSA) confirmed the direct binding of anlotinib to TRIP13, validating the potential of our biochemical assay for identifying novel TRIP13 inhibitors. Our study provides a valuable tool for discovering novel TRIP13 inhibitors and advances our understanding of the therapeutic potential of targeting TRIP13 in cancer.
TRIP13是一种很有前景的癌症治疗靶点，已被确定为有丝分裂检查点的关键调节因子。TRIP13的过表达与多种癌症的不良临床结果相关。抑制TRIP13有可能应对癌症治疗中的挑战，特别是在耐药性和Rb缺陷型癌症中。尽管抑制TRIP13具有潜在的治疗益处，但由于缺乏强大的高通量筛选（HTS）检测方法，TRIP13抑制剂的开发受到了阻碍。我们开发了一种基于发光的TRIP13活性生化检测方法，使用ADP-Glo检测系统来应对这一挑战。该检测方法具有高灵敏度、低背景信号且易于自动化的特点，使其成为HTS应用的理想选择。对激酶聚焦抑制剂库的初步筛选以及对另外4000种化合物的大规模筛选表明，该检测方法性能强大，z'因子超过0.85，信号与背景（S/B）比接近6。从最初的50个命中化合物中，经过严格验证确定安罗替尼是最有效的TRIP13抑制剂，IC50为5 μM。细胞热位移分析（CETSA）证实了安罗替尼与TRIP13的直接结合，验证了我们的生化检测方法在鉴定新型TRIP13抑制剂方面的潜力。我们的研究为发现新型TRIP13抑制剂提供了一个有价值的工具，并推进了我们对靶向TRIP13在癌症治疗中的潜力的理解。
