Reactivity-Based Probe of the Iron(II)-Dependent Interactome Identifies New Cellular Modulators of Ferroptosis

Ferroptosis is an iron-dependent
form of cell death resulting from
loss or inhibition of cellular machinery that protects from the accumulation
of lipid hydroperoxides. Ferroptosis likely serves a tumor suppressing
function in normal cellular homeostasis, but certain cancers exploit
and become highly dependent on specific nodes of the pathway, presumably
to survive under conditions of increased oxidative stress and elevated
labile ferrous iron levels. Here we introduce Ferroptosis Inducing
Peroxide for Chemoproteomics-1 (FIPC-1), a reactivity-based probe
that couples Fenton-type reaction with ferrous iron to subsequent
protein labeling via concomitant carbon-centered radical generation.
We show that FIPC-1 induces ferroptosis in susceptible cell types
and labels cellular proteins in an iron-dependent fashion. Use of
FIPC-1 in a quantitative chemoproteomics workflow reproducibly enriched
protein targets in the thioredoxin, oxidoreductase, and protein disulfide
isomerase (PDI) families, among others. In further interrogating the
saturable targets of FIPC-1, we identified the PDI family member P4HB
and the functionally uncharacterized protein NT5DC2, a member of the
haloacid dehalogenase (HAD) superfamily, as previously unrecognized
modulators of ferroptosis. Knockdown of these target genes sensitized
cells to known ferroptosis inducers, while PACMA31, a previously reported
inhibitor of P4HB, directly induced ferroptosis and was highly synergistic
with erastin. Overall, this study introduces a new reactivity-based
probe of the ferrous iron-dependent interactome and uncovers new targets
for the therapeutic modulation of ferroptosis.