基于2-氧戊二酸类似物的生物分子工具,用于探索非血红素铁酶的结构-活性关系。

IF 2.6 4区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
ChemBioChem Pub Date : 2025-06-22 DOI:10.1002/cbic.202500177
Peter Windsor, Sourav Chatterjee, Anoop Rama Damodaran, Ambika Bhagi-Damodaran
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引用次数: 0

摘要

2-氧戊二酸(2OG)依赖性非血红素铁(NHFe)酶是一类利用2OG和氧催化未活化的C(sp3)-H键羟基化的酶。由于这些酶在调节各种细胞过程中的作用,它们具有重要的生物学意义和治疗意义。在此,我们合理地设计了两类2OG类似物,并将它们作为工具来研究依赖于2OG的NHFe酶,脯氨酸羟化酶结构域2 (PHD2)的活性位点。通过结合稳态动力学的活性分析,我们发现了一类新的芳基共轭2OG类似物,它们表现出12倍的不同抑制作用,并与2OG竞争PHD2活性位点。免疫印迹研究表明,这些类似物具有生物活性,可以靶向细胞内的PHD2。此外,计算模型研究表明,与2OG相比,类似物以“翻转”的构象结合在活性位点上,功能基团的位置是它们不同抑制能力的原因。我们的诱变研究进一步验证了这种独特的结合模式,并提出了几种对抑制至关重要的相互作用。总的来说,这些研究提供了一个2OG类似物的工具包,可以建立结构-活性关系,并确定相互作用,这可能对PHD2抑制剂的设计有用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
2-oxoglutarate analogue-based biomolecular tools for exploring structure-activity relationships in non-heme iron enzymes.

2-oxoglutarate (2OG)-dependent non-heme iron (NHFe) enzymes constitute a family of enzymes that use 2OG and oxygen to catalyze the hydroxylation of unactivated C(sp3)-H bonds. These enzymes are of biological importance and therapeutic interest due to their role in regulating various cellular processes. Herein, we have rationally designed two classes of 2OG analogues and have used them as tools to investigate the active site of a 2OG-dependent NHFe enzyme, prolyl hydroxylase domain 2 (PHD2). Using an activity assay in conjunction with steady-state kinetics, we identify a new class of aryl-conjugated 2OG analogues that exhibit 12-fold varied inhibition and compete with 2OG for the PHD2 active site. Immunoblot studies suggest that these analogues are biologically active and could target PHD2 intracellularly. Furthermore, computational modelling studies reveal that the analogues bind to the active site in a "flipped" conformation relative to 2OG, and functional group placement is responsible for their different inhibition capabilities. Our mutagenesis studies further validate this unique binding mode and suggest several interactions that are crucial for inhibition. Overall, these studies provide a toolkit of 2OG analogues to establish structure-activity relationships and identify interactions that could be useful for PHD2 inhibitor design.

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来源期刊
ChemBioChem
ChemBioChem 生物-生化与分子生物学
CiteScore
6.10
自引率
3.10%
发文量
407
审稿时长
1 months
期刊介绍: ChemBioChem (Impact Factor 2018: 2.641) publishes important breakthroughs across all areas at the interface of chemistry and biology, including the fields of chemical biology, bioorganic chemistry, bioinorganic chemistry, synthetic biology, biocatalysis, bionanotechnology, and biomaterials. It is published on behalf of Chemistry Europe, an association of 16 European chemical societies, and supported by the Asian Chemical Editorial Society (ACES).
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