title: A high-throughput fluorimetric assay for 2-hydroxyglutarate identifies Zaprinast as a glutaminase inhibitor.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Authors' Affiliations:BRIGHT Institute, Molecular Imaging Center, Mallinckrodt Institute of Radiology;
authors.forename: Adnan
authors.initials: A
authors.lastname: Elhammali
authors.affiliation: Authors' Affiliations:BRIGHT Institute, Molecular Imaging Center, Mallinckrodt Institute of Radiology;
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Ippolito
authors.affiliation: Authors' Affiliations:BRIGHT Institute, Molecular Imaging Center, Mallinckrodt Institute of Radiology;
authors.forename: Lynne
authors.initials: L
authors.lastname: Collins
authors.affiliation: Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri; and.
authors.forename: Jan
authors.initials: J
authors.lastname: Crowley
authors.affiliation: Authors' Affiliations:BRIGHT Institute, Molecular Imaging Center, Mallinckrodt Institute of Radiology;
authors.forename: Jayne
authors.initials: J
authors.lastname: Marasa
authors.affiliation: Authors' Affiliations:BRIGHT Institute, Molecular Imaging Center, Mallinckrodt Institute of Radiology; Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, Houston, Texas dpiwnica-worms@mdanderson.org.
authors.forename: David
authors.initials: D
authors.lastname: Piwnica-Worms
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005354:Fibrosarcoma / Q000188:drug therapy* / Q000201:enzymology; D005972:Glutaminase; D005977:Glutarates / Q000378:metabolism; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006657:Histones; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D008745:Methylation; D051379:Mice; D008819:Mice, Nude; D009374:Neoplasms, Experimental; D011688:Purinones / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D005977:Glutarates; D006657:Histones; D011688:Purinones; C019417:alpha-hydroxyglutarate; D007521:Isocitrate Dehydrogenase; D005972:Glutaminase; C011145:zaprinast
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