Small Molecule Screen Identifies Pyrimethamine as an Inhibitor of NRF2-driven Esophageal Hyperplasia
Bibliographic record
Abstract
Abstract Objective NRF2 is a master transcription factor that regulates the stress response. NRF2 is frequently mutated and activated in human esophageal squamous cell carcinoma (ESCC), which drives resistance to chemotherapy and radiation therapy. Therefore, a great need exists for NRF2 inhibitors for targeted therapy of NRF2 high ESCC. Design We performed high-throughput screening of two compound libraries from which hit compounds were further validated in human ESCC cells and a genetically modified mouse model. The mechanism of action of one compound was explored by biochemical assays. Results Using high-throughput screening of two small molecule compound libraries, we identified 11 hit compounds as potential NRF2 inhibitors with minimal cytotoxicity at specified concentrations. We then validated two of these compounds, pyrimethamine and mitoxantrone, by demonstrating their dose- and time-dependent inhibitory effects on the expression of NRF2 and its target genes in two NRF2 Mut human ESCC cells (KYSE70 and KYSE180). RNAseq and qPCR confirmed the suppression of global NRF2 signaling by these two compounds. Mechanistically, pyrimethamine reduced NRF2 half-life by promoting NRF2 ubiquitination and degradation in KYSE70 and KYSE180 cells. Expression of an Nrf2 E79Q allele in mouse esophageal epithelium ( Sox2CreER;LSL-Nrf2 E79Q/+ ) resulted in an NRF2 high phenotype, which included squamous hyperplasia, hyperkeratinization, and hyperactive glycolysis. Treatment with pyrimethamine (30mg/kg/day, p . o .) suppressed the NRF2 high esophageal phenotype with no observed toxicity. Conclusion We have identified and validated pyrimethamine as an NRF2 inhibitor that may be rapidly tested in the clinic as a radiation and chemotherapy sensitizer for NRF2 high ESCC. Summary What is already known on this topic – summarise the state of scientific knowledge on this subject before you did your study and why this study needed to be done Mutational activation of the NRF2 transcription factor drives ESCC progression and therapeutic resistance. Targeted therapies to block NRF2 have not yet been realized, despite great needs. What this study adds – summarise what we now know as a result of this study that we did not know before A screen of >35,000 small molecules identified eleven potential NRF2 inhibitors. Pyrimethamine and mitoxantrone were validated to inhibit the expression of NRF2 and NQO1 in human ESCC cells in both dose- and time-dependent manners. Pyrimethamine enhanced NRF2 protein ubiquitination and degradation, resulting a decreased half-life. A genetically modified mouse model was established to express the Nrf2 E79Q mutant allele in the mouse esophageal epithelium upon tamoxifen induction. Pyrimethamine suppressed the NRF2 high esophageal phenotype induced by the mutant allele. How this study might affect research, practice or policy – summarise the implications of this study As an FDA-approved drug, Pyrimethamine has the potential for immediate translation to a clinical trial on NRF2 high ESCC in humans. Further exploration of its mechanisms of action may lead to more potent NRF2 inhibitors for future use.
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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) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.001 | 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) | 0.002 | 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".