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Published on: February 9, 2021
Pyrimethamine and a potent analog inhibit NRF2 by suppressing one-carbon metabolism
Julius Chembo1, Brittany M Bowman1, Kyle Lapak1
1Department of Cell Biology and Physiology, Washington University in St Louis, St Louis, Missouri, USA.
Abstract:
Nuclear factor erythroid 2-related factor 2 (NFE2L2/NRF2) is a critical mediator of the cellular oxidative stress response. Aberrant activation of NRF2 is common in lung and upper aerodigestive cancers, where it promotes tumor initiation and progression and confers resistance to chemotherapy, radiation therapy, and immune checkpoint inhibitors. As such, NRF2 therapeutic inhibitors are actively being sought. We previously reported that the antiparasitic drug pyrimethamine (PYR) inhibits NRF2 in cell lines and in a NRF2-inducible genetically engineered mouse model. Here we design, synthesize, and define structure-activity relationships across a series of 25 PYR-based derivatives to reveal WCDD115 as a 22-fold more potent inhibitor of NRF2 (57 nM versus 1.2 μM). PYR is known to inhibit plasmodial and human dihydrofolate reductase (DHFR). We found that WCDD115 inhibits hDHFR with 31-fold greater potency than PYR (144 nM versus 4.49 μM). Metabolomics showed strong similarities between PYR, WCDD115 and methotrexate. Genetic, pharmacological and metabolic epistasis studies reveal that DHFR inactivation is required for NRF2 suppression by WCDD115 and PYR. Global and targeted proteomics revealed overlapping profiles for WCDD115, PYR and methotrexate, including suppression of NRF2 oxidative stress response and activation of TP53 and the DNA damage response. Therefore, PYR and a novel potent derivative WCDD115 are effective, indirect inhibitors of NRF2 and its antioxidant functions. These data underscore the importance of one-carbon metabolism for the NRF2 signaling pathway and support a new therapeutic strategy to suppress NRF2-driven cancer biology.
Insights
Pyrimethamine derivatives, like WCDD115, effectively inhibit Nuclear factor erythroid 2-related factor 2 (NRF2) by targeting dihydrofolate reductase (DHFR). This offers a new strategy against NRF2-driven cancers.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Nuclear factor erythroid 2-related factor 2 (NRF2) is crucial for oxidative stress response and is aberrantly activated in cancers, promoting tumor growth and treatment resistance.
- Therapeutic inhibitors targeting NRF2 are urgently needed for cancer treatment.
Purpose of the Study:
- To design and synthesize novel Pyrimethamine (PYR)-based derivatives as potent inhibitors of NRF2.
- To elucidate the mechanism of NRF2 inhibition by PYR and its derivatives, focusing on structure-activity relationships and target engagement.
Main Methods:
- Synthesis and structure-activity relationship (SAR) studies of 25 PYR derivatives.
- In vitro assays to determine NRF2 and human dihydrofolate reductase (hDHFR) inhibitory potencies.
- Metabolomics, genetic, pharmacological, and metabolic epistasis studies.
- Global and targeted proteomics analysis.
Main Results:
- WCDD115, a novel PYR derivative, demonstrated 22-fold greater NRF2 inhibition potency (57nM) compared to PYR (1.2μM).
- WCDD115 exhibited 31-fold greater hDHFR inhibition potency (144nM) than PYR (4.49μM).
- DHFR inactivation was identified as essential for NRF2 suppression by PYR and WCDD115, with observed similarities to methotrexate.
- Proteomics revealed overlapping effects including NRF2 oxidative stress response suppression and activation of TP53 and DNA damage response pathways.
Conclusions:
- Pyrimethamine and its derivative WCDD115 are effective indirect inhibitors of NRF2's antioxidant functions.
- DHFR inhibition is a key mechanism for suppressing NRF2 signaling.
- These findings highlight the importance of one-carbon metabolism in NRF2 signaling and suggest a novel therapeutic strategy for NRF2-driven cancers.
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