Chemoproteomic Profiling by Cysteine Fluoroalkylation Reveals Myrocin G as an Inhibitor of the Nonhomologous End Joining DNA Repair Pathway

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.