P1‐028: Methylene blue inhibits caspases by oxidation of the catalytic cysteine
Bibliographic record
Abstract
Methylene blue is a well-tolerated treatment for many conditions in humans and is currently being tested in phase 3 clinical trials for Alzheimer Disease as a Tau aggregation inhibitor. Methylene blue oxidizes cysteine residues on Tau protein to inhibit its oligomerization. The Caspase-6 protease has been implicated in axonal degeneration and cognitive impairment in AD. Whether methylene blue can also inhibit caspases by oxidation of the catalytic cysteine is unknown. Inhibition of recombinant active Caspase-1, Caspase-3, and Caspase-6 by methylene blue and its derivatives, azure A and azure B, was determined by fluorogenic assay for caspase activity. Mass spectrometry was performed on recombinant active Caspase-6 treated with methylene blue or azure B to assess the oxidation state of the catalytic cysteine. The toxicity of methylene blue and its derivatives was monitored by phase contrast microscopy and MTT assay in human colon carcinoma cells and human primary neurons. The inhibitory activity of methylene blue on Caspase-6 was measured in Caspase-6 transfected human colon carcinoma cells and serum-deprived human primary neurons by fluorogenic assay and western blot. A mouse model of liver toxicity was used to test the ability of methylene blue to inhibit Caspase-3 activity in vivo. Methylene blue, azure A, and azure B efficiently inhibited recombinant active Caspase-6, Caspase-1, and Caspase-3. Methylene blue competitively inhibited recombinant Caspase-6, suggesting an inhibitory mechanism through the active site of the enzyme. Methylene blue and azure B were non-toxic and significantly inhibited Caspase-6 activity in human colon carcinoma cells and primary human neurons cultures. Furthermore, methylene blue inhibited Casp3 activity in an acute mouse model of liver toxicity. Mass spectrometry confirmed methylene blue and azure B-mediated sulfenation of the catalytic Cys163 of Casp6. This study reveals sulfenation of caspase-6 by methylene blue and its derivatives as a novel inhibitory post-translational modification regulating caspase activity. Therefore, methylene blue or its derivatives could (1) have a positive effect against AD by inhibiting brain Casp6 activity, (2) be used as a drug to prevent caspase activation in other conditions, and (3) predispose chronically treated individuals to cancer via the inhibition of caspases.
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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 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.006 | 0.002 |
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".