A chemoproteomic strategy for identifying protein covalent binding targets of clozapine: An approach for advancing clozapine toxicity research

Schizophrenia affects a significant proportion of individuals, wherein a subset of patients is described as treatment-resistant. Clozapine (CLOZ) is an atypical antipsychotic, which is reserved for these patients and is superior in its anti-suicidal activity. However, it carries numerous serious warnings and is well-known for its risk of drug-induced agranulocytosis. The mechanism of toxicity is unclear and could be due to CLOZ's protein covalent binding and off-target effects through its reactive metabolites produced from neutrophil myeloperoxidase (MPO) activity. We hypothesize that identifying and analyzing the protein-CLOZ adducts will contribute to our understanding of toxicity pathways. We have developed a novel clickable CLOZ (Click-CLOZ) derivative and have designed click chemistry protocols for protein identification. The HL-60 (human promyelocytic leukemia) cell line and isolated human neutrophils express MPO significantly and were used to identify the protein covalent targets of Click-CLOZ. In HL-60 cells, LC/MS analysis revealed many Click-CLOZ-bound proteins (compared to the vehicle control). Some captured proteins were known for their roles in DNA replication, immune responses and oxidative stress, such as cathepsin G, MPO, ribophorin I and P1-MCM3. In neutrophils, Click-CLOZ-bound proteins included MPO, S100, and DEFA1B, which are also associated with neutrophil-mediated oxidative stress and immune responses. In conclusion, the application of click chemistry proteomics has facilitated a novel approach to identify multiple CLOZ-bound protein targets that will be used to advance our understanding of the toxicity of CLOZ.