Cysteine-reactive covalent chloro-N-acetamide ligands induce ferroptosis mediated cell death

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• Covalent inhibitors offer a strategy for targeting difficult proteins like RNF4, a potential vulnerability in acute myeloid leukemia (AML).
• High RNF4 expression correlates with poor AML patient survival, and its depletion increases sensitivity to antileukemic drugs.

Purpose of the Study:

• To develop proteolysis-targeting chimeras (PROTACs) for RNF4 degradation using a known covalent ligand, CCW16.
• To investigate the cellular activity and specificity of CCW16-based PROTACs.

Main Methods:

• Synthesis of CCW16-derived PROTACs incorporating E3 ligase ligands (CRBN, VHL).
• In vitro testing of covalent binding to recombinant RNF4.
• Cellular assays to assess RNF4 degradation, protein binding, and cell viability.
• Analysis of ferroptosis markers and off-target effects.

Main Results:

• CCW16 and its PROTACs covalently bound RNF4 in vitro but showed broad off-target binding to cellular proteins, including peroxiredoxins.
• CCW16-based PROTACs failed to degrade RNF4, instead inducing heme oxygenase-1, a ferroptosis marker.
• These compounds impaired cell viability via an RNF4-independent ferroptotic pathway.
• Another chloro-N-acetamide ligand, EN219, also induced ferroptosis, suggesting this electrophile causes toxicity.

Conclusions:

• The chloro-N-acetamide moiety in CCW16 leads to off-target toxicity and ferroptosis, independent of RNF4 degradation.
• RNF4-targeting PROTACs based on CCW16 are not suitable for AML therapy due to these toxicities.
• Ligands with the chloro-N-acetamide electrophile may cause undesired off-target ferroptotic toxicity.