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 7 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 5 (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.