针对SMAD4-SMAD3-DNA复合物的多路时间分辨荧光共振能量转移超高通量筛选试验

IF 5.3 2区 生物学 Q2 CELL BIOLOGY
Wukun Ouyang, Qianjin Li, Qiankun Niu, Min Qui, Haian Fu, Yuhong Du, Xiulei Mo
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引用次数: 0

摘要

转化生长因子- β (tgf - β)信号通路在建立免疫抑制肿瘤微环境中起着至关重要的作用,这使得抗tgf - β药物成为癌症免疫治疗的一个重要领域。然而,目前针对上游细胞因子和受体的抗tgf β药物的临床翻译仍然具有挑战性。因此,开发专门针对tgf -β通路下游主调控因子SMAD4的小分子抑制剂,将为抗tgf -β信号传导提供一种具有显著治疗潜力的替代方法。在这项研究中,我们提出了一种基于细胞裂解液的多路时间分辨荧光共振能量转移(TR-FRET)的超高通量筛选(uHTS) 1536孔板格式检测方法。该分析能够同时监测SMAD4和SMAD3之间的蛋白质-蛋白质相互作用,以及smad之间的蛋白质- dna相互作用及其一致的dna结合基序。多重TR-FRET测定具有高灵敏度,允许在单氨基酸分辨率下动态分析SMAD4-SMAD3-DNA复合物。此外,多重uHTS试验在筛选小分子抑制剂方面具有稳健性。通过fda批准的生物活性化合物库的试点筛选,我们确定了藤黄酸和藤黄原酸作为潜在的打击化合物。这些概念验证的发现强调了我们优化的多路复用TR-FRET平台在大规模筛选中发现靶向SMAD4-SMAD3-DNA复合物的小分子抑制剂作为新型抗tgf - β信号剂的效用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A multiplexed time-resolved fluorescence resonance energy transfer ultrahigh-throughput screening assay for targeting the SMAD4-SMAD3-DNA complex.

The transforming growth factor-beta (TGFβ) signaling pathway plays crucial roles in the establishment of an immunosuppressive tumor microenvironment, making anti-TGFβ agents a significant area of interest in cancer immunotherapy. However, the clinical translation of current anti-TGFβ agents that target upstream cytokines and receptors remains challenging. Therefore, the development of small-molecule inhibitors specifically targeting SMAD4, the downstream master regulator of the TGFβ pathway, would offer an alternative approach with significant therapeutic potential for anti-TGFβ signaling. In this study, we present the development of a cell lysate-based multiplexed time-resolved fluorescence resonance energy transfer (TR-FRET) assay in an ultrahigh-throughput screening (uHTS) 1536-well plate format. This assay enables simultaneous monitoring of the protein‒protein interaction between SMAD4 and SMAD3, as well as the protein‒DNA interaction between SMADs and their consensus DNA-binding motif. The multiplexed TR-FRET assay exhibits high sensitivity, allowing the dynamic analysis of the SMAD4-SMAD3-DNA complex at single-amino acid resolution. Moreover, the multiplexed uHTS assay demonstrates robustness for screening small-molecule inhibitors. Through a pilot screening of an FDA-approved bioactive compound library, we identified gambogic acid and gambogenic acid as potential hit compounds. These proof-of-concept findings underscore the utility of our optimized multiplexed TR-FRET platform for large-scale screening to discover small-molecule inhibitors that target the SMAD4-SMAD3-DNA complex as novel anti-TGFβ signaling agents.

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来源期刊
CiteScore
9.60
自引率
1.80%
发文量
1383
期刊介绍: The Journal of Molecular Cell Biology ( JMCB ) is a full open access, peer-reviewed online journal interested in inter-disciplinary studies at the cross-sections between molecular and cell biology as well as other disciplines of life sciences. The broad scope of JMCB reflects the merging of these life science disciplines such as stem cell research, signaling, genetics, epigenetics, genomics, development, immunology, cancer biology, molecular pathogenesis, neuroscience, and systems biology. The journal will publish primary research papers with findings of unusual significance and broad scientific interest. Review articles, letters and commentary on timely issues are also welcome. JMCB features an outstanding Editorial Board, which will serve as scientific advisors to the journal and provide strategic guidance for the development of the journal. By selecting only the best papers for publication, JMCB will provide a first rate publishing forum for scientists all over the world.
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