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 machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.001 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.001 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 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 source (direct Gemma or distilled Codex), 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".