Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia

Chorlada Paiboonrungruang; Zhaohui Xiong; David Lamson; Yahui Li; Brittany Bowman; Julius Chembo; Caizhi Huang; Jianying Li; Eric W Livingston; Jon E Frank; Vivian Chen; Yong Li; Bernard Weissman; Hong Yuan; Kevin P Williams; M Ben Major; Xiaoxin Chen

doi:10.1016/j.redox.2023.102901

Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia

Redox Biol. 2023 Nov:67:102901. doi: 10.1016/j.redox.2023.102901. Epub 2023 Sep 22.

Authors

Chorlada Paiboonrungruang¹, Zhaohui Xiong¹, David Lamson², Yahui Li¹, Brittany Bowman³, Julius Chembo³, Caizhi Huang⁴, Jianying Li⁵, Eric W Livingston⁶, Jon E Frank⁶, Vivian Chen⁴, Yong Li⁷, Bernard Weissman⁸, Hong Yuan⁹, Kevin P Williams¹⁰, M Ben Major¹¹, Xiaoxin Chen¹²

Affiliations

¹ Coriell 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.
² Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC, 27707, USA.
³ Department of Cell Biology and Physiology, Department of Otolaryngology, Washington University in St. Louis, St. Louis, MO, 63110, USA.
⁴ Cancer Research Program, Julius L. Chambers Biomedical Biotechnology Research Institute, North Carolina Central University, Durham, NC, 27707, USA.
⁵ Euclados Bioinformatics Solutions, Cary, NC, 27519, USA.
⁶ Biomedical Research Imaging Center, University of North Carolina, Chapel Hill, NC, 277599, USA.
⁷ Department of Thoracic Surgery, National Cancer Center, Cancer Hospital of Chinese Academy of Medical Sciences, Beijing, 100021, China.
⁸ Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 277599, USA.
⁹ Biomedical Research Imaging Center, University of North Carolina, Chapel Hill, NC, 277599, USA; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 277599, USA; Department of Radiology, University of North Carolina, Chapel Hill, NC, 277599, USA.
¹⁰ Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC, 27707, USA. Electronic address: kpwilliams@nccu.edu.
¹¹ Department of Cell Biology and Physiology, Department of Otolaryngology, Washington University in St. Louis, St. Louis, MO, 63110, USA. Electronic address: bmajor@wustl.edu.
¹² Coriell 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; Surgical Research Lab, Department of Surgery, Cooper University Health Care, Camden, NJ, 08103, USA; MD Anderson Cancer Center at Cooper, Camden, NJ, 08103, USA; Cooper Medical School of Rowan University, Camden, NJ, 08103, USA. Electronic address: lchen@coriell.org.

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 (30 mg/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 for NRF2^high ESCC.

Keywords: Esophageal squamous cell carcinoma; KEAP1; Mitoxantrone; NRF2; Pyrimethamine.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Line, Tumor
Cell Proliferation
Esophageal Neoplasms* / genetics
Esophageal Squamous Cell Carcinoma* / genetics
Esophageal Squamous Cell Carcinoma* / pathology
Esophageal Squamous Cell Carcinoma* / therapy
Humans
Hyperplasia
Mice
NF-E2-Related Factor 2 / genetics
NF-E2-Related Factor 2 / metabolism
Pyrimethamine / pharmacology
Pyrimethamine / therapeutic use

Substances

NF-E2-Related Factor 2
Pyrimethamine

Abstract

Publication types

MeSH terms

Substances

Grants and funding