Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, Florida 33458, United States.
Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.
J Am Chem Soc. 2021 Dec 8;143(48):20332-20342. doi: 10.1021/jacs.1c09724. Epub 2021 Nov 24.
Chemoproteomic profiling of cysteines has emerged as a powerful method for screening the proteome-wide targets of cysteine-reactive fragments, drugs, and natural products. Herein, we report the development and an in-depth evaluation of a tetrafluoroalkyl benziodoxole (TFBX) as a cysteine-selective chemoproteomic probe. We show that this probe features numerous key improvements compared to the traditionally used cysteine-reactive probes, including a superior target occupancy, faster labeling kinetics, and broader proteomic coverage, thus enabling profiling of cysteines directly in live cells. In addition, the fluorine "signature" of probe constitutes an additional advantage resulting in a more confident adduct-amino acid site assignment in mass-spectrometry-based identification workflows. We demonstrate the utility of our new probe for proteome-wide target profiling by identifying the cellular targets of (-)-myrocin G, an antiproliferative fungal natural product with a to-date unknown mechanism of action. We show that this natural product and a simplified analogue target the X-ray repair cross-complementing protein (XRCC5), an ATP-dependent DNA helicase that primes DNA repair machinery for nonhomologous end joining (NHEJ) upon DNA double-strand breaks, making them the first reported inhibitors of this biomedically highly important protein. We further demonstrate that myrocins disrupt the interaction of XRCC5 with DNA leading to sensitization of cancer cells to the chemotherapeutic agent etoposide as well as UV-light-induced DNA damage. Altogether, our next-generation cysteine-reactive probe enables broader and deeper profiling of the cysteinome, rendering it a highly attractive tool for elucidation of targets of electrophilic small molecules.
半胱氨酸的化学蛋白质组学分析方法已成为筛选半胱氨酸反应性片段、药物和天然产物的蛋白质组靶标的强大方法。在此，我们报告了四氟苯并恶唑（TFBX）作为半胱氨酸选择性化学蛋白质组学探针的开发和深入评估。我们表明，与传统使用的半胱氨酸反应性探针相比，该探针具有许多关键改进，包括更高的靶标占有率、更快的标记动力学和更广泛的蛋白质组覆盖范围，从而能够直接在活细胞中对半胱氨酸进行分析。此外，探针的氟"特征"构成了另一个优势，在基于质谱的鉴定工作流程中，对半加合物-氨基酸位点的分配更有信心。我们通过鉴定（-）-槐霉素 G 的细胞靶标，展示了我们新探针在蛋白质组范围内进行靶标分析的实用性，槐霉素 G 是一种具有未知作用机制的抗增殖真菌天然产物。我们表明，这种天然产物和简化的类似物靶向 X 射线修复交叉互补蛋白 5（XRCC5），一种 ATP 依赖性 DNA 解旋酶，可在 DNA 双链断裂时为非同源末端连接（NHEJ）启动 DNA 修复机制，使其成为该生物医学上高度重要的蛋白质的首批报道抑制剂。我们进一步证明，槐霉素会破坏 XRCC5 与 DNA 的相互作用，导致癌细胞对化疗药物依托泊苷以及紫外线诱导的 DNA 损伤更加敏感。总之，我们的下一代半胱氨酸反应性探针能够更广泛和更深入地对半胱氨酸组进行分析，使其成为阐明亲电小分子靶标的极具吸引力的工具。
