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Updated: Jul 31, 2026
Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors
Published on: November 23, 2019
Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia
Chorlada Paiboonrungruang1, Zhaohui Xiong1, David Lamson2
1Coriell Institute for Medical Research, Camden, NJ, 08103, USA; Cancer Research Program, Julius L. Chambers Biomedical Biotechnology Research Institute, North Carolina Central University, Durham, NC, 27707, USA.
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 NRF2high 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 NRF2Mut 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 Nrf2E79Q allele in mouse esophageal epithelium (Sox2CreER;LSL-Nrf2E79Q/+) resulted in an NRF2high phenotype, which included squamous hyperplasia, hyperkeratinization, and hyperactive glycolysis. Treatment with pyrimethamine (30 mg/kg/day, p.o.) suppressed the NRF2high 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 for NRF2high ESCC.
Insights
Researchers identified pyrimethamine as a novel Nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor. This compound effectively targets NRF2-activated esophageal squamous cell carcinoma (ESCC) and shows promise for clinical testing.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Nuclear factor erythroid 2-related factor 2 (NRF2) is a key regulator of cellular stress response.
- NRF2 activation is common in esophageal squamous cell carcinoma (ESCC) and contributes to treatment resistance.
- Targeted NRF2 inhibition is needed for effective ESCC therapy.
Purpose of the Study:
- To identify novel inhibitors of NRF2 for the treatment of NRF2-high ESCC.
- To validate the efficacy and mechanism of action of identified compounds.
Main Methods:
- High-throughput screening of compound libraries to identify NRF2 inhibitors.
- Validation of hit compounds in human ESCC cell lines and a genetically modified mouse model.
- Biochemical assays and RNA sequencing to elucidate the mechanism of action.
Main Results:
- Eleven hit compounds were identified, with pyrimethamine and mitoxantrone showing significant NRF2 inhibition in ESCC cells.
- Pyrimethamine was found to reduce NRF2 half-life by promoting its ubiquitination and degradation.
- In a mouse model, pyrimethamine suppressed NRF2-driven esophageal hyperplasia and associated metabolic changes without toxicity.
Conclusions:
- Pyrimethamine is a validated NRF2 inhibitor with potential for treating NRF2-high ESCC.
- The findings support the rapid clinical evaluation of pyrimethamine for ESCC patients.
