A high-throughput fluorimetric assay for 2-hydroxyglutarate identifies Zaprinast as a glutaminase inhibitor
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Abstract
UNLABELLED: Recently identified isocitrate dehydrogenase (IDH) mutations lead to the production of 2-hydroxyglutarate (2HG), an oncometabolite aberrantly elevated in selected cancers. We developed a facile and inexpensive fluorimetric microplate assay for the quantitation of 2HG and performed an unbiased small-molecule screen in live cells to identify compounds capable of perturbing 2HG production. Zaprinast, a phosphodiesterase 5 inhibitor, was identified as an efficacious modulator of 2HG production and confirmed to lower 2HG levels in vivo. The mechanism of action was not due to cGMP stabilization, but rather, profiling of metabolites upstream of mutant IDH1 pointed to targeted inhibition of the enzyme glutaminase (GLS). Zaprinast treatment reversed histone hypermethylation and soft-agar growth of IDH1-mutant cells, and treatment of glutamine-addicted pancreatic cancer cells reduced growth and sensitized cells to oxidative damage. Thus, Zaprinast is efficacious against glutamine metabolism and further establishes the therapeutic linkages between GLS and 2HG-mediated oncogenesis. SIGNIFICANCE: Gain-of-function IDH mutations are common events in glioma, acute myelogenous leukemia, and other cancer types, which lead to the accumulation of the oncometabolite 2HG. We show that the drug Zaprinast is capable of reducing cellular 2HG levels by inhibiting the upstream enzyme GLS, thus identifying a new strategy to target 2HG production in selected IDH-mutant cancers.
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Zaprinast can alter aspartate and glutamate metabolism via the MPC and glutaminase.
“Moreover, recent work shows the phosphodiesterase inhibitor Zaprinast can alter aspartate and glutamate metabolism via the MPC (Du et al., 2013) and glutaminase (Elhammali et al., 2014).”
From Regulation of Substrate Utilization by the Mitochondrial Pyruvate Carrier (2014) · cites this paper as Elhammali et al., 2014
The lack of evidence of glutaminolysis through malic enzyme in Du et al. is potentially due to off-target effects on mitochondrial glutaminase.
“While Du et al. similarly observed an increase in aspartate levels, no evidence of glutaminolysis through malic enzyme was observed (Du et al., 2013), potentially due to off-target effects on mitochondrial glutaminase (Elhammali et al., 2014).”
From Regulation of Substrate Utilization by the Mitochondrial Pyruvate Carrier (2014) · cites this paper as Elhammali et al., 2014
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References 34 · Cited by 77
- Authors
- Elhammali, A., Ippolito, J. E., Collins, L., Crowley, J., Marasa, J., & Piwnica-Worms, D.
- Affiliations
- Authors' Affiliations: 1BRIGHT Institute, Molecular Imaging Center, Mallinckrodt Institute of Radiology;
- Washington University in St. Louis (US)
- The University of Texas MD Anderson Cancer Center (US)
- Corresponding author
- Piwnica-Worms, David
- Venue
- Cancer Discovery
- Published
- 2014-07-01
- Volume
- 4
- Issue
- 7
- Pages
- 828-839
- ISSN
- 2159-8274 (ISSN-L)
- 2159-8290
- PMC
- PMC4197823
- OpenAlex
- W2147845373
- Cited by
- 77
- References
- 34
- Citations by year
- 2026: 2 · 2025: 2 · 2024: 4 · 2023: 8 · 2022: 7 · 2021: 10 · 2020: 5 · 2019: 4 · 2018: 7 · 2017: 7 · 2016: 5 · 2015: 12 · 2014: 4
- FWCI
- 2.99
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Cancer Discovery (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- Alvin J. Siteman Cancer Center
- National Institutes of Health (5p60dk020579-35, 5p41rr000954-34)
- National Cancer Institute (P50CA094056, P30CA091842)
- National Institute of Diabetes and Digestive and Kidney Diseases (P60-DK-020579, P30DK056341, P30DK020579)
- National Center for Research Resources (P41-RR000954)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
- Health Sciences › Medicine › Genetics › Glioma Diagnosis and Treatment
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Amino Acid Enzymes and Metabolism
- Keywords
- 2-hydroxyglutarate, IDH mutation, glutaminase inhibition, zaprinast, phosphodiesterase-5 inhibitors, oncometabolites, glutamine metabolism, pancreatic cancer, fluorometric assay, small molecule screening, oxidative damage
- MeSH terms
- Animals, Enzyme Inhibitors, Fibrosarcoma, Glutaminase, Glutarates, Histones, Humans, Isocitrate Dehydrogenase, Methylation, Mice, Nude, Neoplasms, Experimental, Purinones, Cell Line, Tumor, Mice, High-Throughput Screening Assays, HEK293 Cells
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