A chemoproteomic strategy for identifying protein covalent binding targets of clozapine: An approach for advancing clozapine toxicity research
Bibliographic record
Abstract
Schizophrenia affects a significant proportion of individuals, wherein a subset of patients are described as treatment-resistant. Clozapine 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 clozapine’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-clozapine adducts will contribute to our understanding of toxicity pathways. We have developed a novel clickable clozapine (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 clozapine-bound protein targets that will be used to advance our understanding of the toxicity of clozapine. • Clozpaine was modified to produce “Click-CLOZ” for click chemistry protein ID • Click-CLOZ protein binding was conducted in human neutrophils and HL-60 cells • Click-CLOZ demonstrates similar cytotoxicity and metabolic behaviour as CLOZ • MPO catalyzed Click-CLOZ protein adduct formation and was a target itself • Neutrophil extracellular trap proteins are susceptible to CLOZ adduction
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.002 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".