Bioorthogonal Profiling of a Cancer Cell Proteome Identifies a Large Set of 3‑Bromopyruvate Targets beyond Glycolysis

3-Bromopyruvate
(3BP) is a potential anticancer agent viewed as
a glycolytic inhibitor that preferentially kills cancer cells through
inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), resulting
in severe energy depletion. We previously identified four cysteine
residues in GAPDH that are alkylated by 3BP, resulting in its inactivation.
However, we also showed that addition of excess pyruvate, the final
product of glycolysis, was unable to rescue cells from 3BP treatment.
This result indicates that GAPDH may not be the only relevant target
and is consistent with the chemical reactivity of 3BP that should
result in the modification of cysteine residues in many different
proteins. To directly test this hypothesis, we first synthesized a
probe of 3BP activity bearing an alkyne functionality, termed AO3BP,
and then demonstrated that this probe could modify a variety of proteins
in living cells. Subsequent competition of AO3BP labeling with pretreatment
by 3BP identified 62 statistically significant proteins of various
functions as targets of 3BP, confirming that 3BP labeling is indeed
widespread. We conclude that 3BP’s cytotoxic impact on cancer
cells does not only result from selective inhibition of glycolysis
but rather from a more widespread effect on cellular proteins that
could be driven by the pharmacokinetics of the 3BP. These pleiotropic
consequences should be considered when thinking about the potential
toxicity of this highly reactive compound.