title: Small molecule glutaminase inhibitors block glutamate release from stimulated microglia.
abstract: Glutaminase plays a critical role in the generation of glutamate, a key excitatory neurotransmitter in the CNS. Excess glutamate release from activated macrophages and microglia correlates with upregulated glutaminase suggesting a pathogenic role for glutaminase. Both glutaminase siRNA and small molecule inhibitors have been shown to decrease excess glutamate and provide neuroprotection in multiple models of disease, including HIV-associated dementia (HAD), multiple sclerosis and ischemia. Consequently, inhibition of glutaminase could be of interest for treatment of these diseases. Bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and 6-diazo-5-oxo-l-norleucine (DON), two most commonly used glutaminase inhibitors, are either poorly soluble or non-specific. Recently, several new BPTES analogs with improved physicochemical properties were reported. To evaluate these new inhibitors, we established a cell-based microglial activation assay measuring glutamate release. Microglia-mediated glutamate levels were significantly augmented by tumor necrosis factor (TNF)-α, phorbol 12-myristate 13-acetate (PMA) and Toll-like receptor (TLR) ligands coincident with increased glutaminase activity. While several potent glutaminase inhibitors abrogated the increase in glutamate, a structurally related analog devoid of glutaminase activity was unable to block the increase. In the absence of glutamine, glutamate levels were significantly attenuated. These data suggest that the in vitro microglia assay may be a useful tool in developing glutaminase inhibitors of therapeutic interest.
authors.affiliation: Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States.
authors.forename: Ajit G
authors.initials: AG
authors.lastname: Thomas
authors.affiliation: Kennedy Krieger Institute, Johns Hopkins Medical Institutions, Baltimore, MD 21205, United States.
authors.forename: Cliona M
authors.initials: CM
authors.lastname: O'Driscoll
authors.affiliation: Kennedy Krieger Institute, Johns Hopkins Medical Institutions, Baltimore, MD 21205, United States.
authors.forename: Joseph
authors.initials: J
authors.lastname: Bressler
authors.affiliation: Kennedy Krieger Institute, Johns Hopkins Medical Institutions, Baltimore, MD 21205, United States; Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, United States.
authors.forename: Walter
authors.initials: W
authors.lastname: Kaufmann
authors.affiliation: Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States.
authors.forename: Camilo J
authors.initials: CJ
authors.lastname: Rojas
authors.affiliation: Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States; Department of Neurology and Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States. Electronic address: bslusher@jhmi.edu.
authors.forename: Barbara S
authors.initials: BS
authors.lastname: Slusher
mesh_terms: D015526:AIDS Dementia Complex / Q000201:enzymology; D000818:Animals; D001681:Biological Assay; D002545:Brain Ischemia / Q000201:enzymology; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical; D018698:Glutamic Acid / Q000378:metabolism*; D005972:Glutaminase / Q000037:antagonists & inhibitors*; D051379:Mice; D017628:Microglia / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D009103:Multiple Sclerosis / Q000201:enzymology; D018696:Neuroprotective Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D054852:Small Molecule Libraries / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D051193:Toll-Like Receptors / Q000819:agonists; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D018696:Neuroprotective Agents; D054852:Small Molecule Libraries; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D018698:Glutamic Acid; C016658:phorbolol myristate acetate; D005972:Glutaminase; D013755:Tetradecanoylphorbol Acetate
keywords: 6-diazo-5-oxo-l-norleucine; BPTES; CNS; CpG oligodeoxynucleotide; DON; GC; GLS; Glutamate; Glutaminase; Glutamine; HAD; HIV; HIV-associated dementia; HIV-associated dementia (HAD); HRP; Inflammation; LPS; Microglia; NF-κB; PMA; Pam3SK4; TLR; TNF-α; Toll-like receptor; bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethyl sulfide; central nervous system; glutaminase; horse radish peroxidase; human immunodeficiency virus; lipopolysaccharide; nuclear factor-κB; phorbol 12-myristate 13-acetate; poly I:C; polyinosinic–polycytidylic; tripalmitoyl-S-glyceryl-cysteine; tumor necrosis factor-α