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.

A Tale of Two Glutaminases: Homologous Enzymes with Distinct Roles in Tumorigenesis

“…For most of the 80 years since glutaminase was discovered, the only chemical inhibitor of note was DON (6-diazo-5-oxo-l -norleucine), which is a glutamine mimetic and irreversibly binds to the catalytic serine of both glutaminase isozymes ( Figure 6A) [122,123]. While other small molecules were utilized to inhibit GLS (particularly membrane-impermeable molecules for determining subcellular localization), DON was the most extensively used inhibitor until the last decade [39].…”

Preventing Allograft Rejection by Targeting Immune Metabolism

“…6-Diazo-5-oxo-L-norleucine (DON) is a glutamine analog that inhibits multiple targets in the glutamine metabolic pathway, including glutaminase and glutamine transporters (Thomas et al, 2014b). To explore the role of glutaminase activity in T cell activation, naïve CD4 + T cells were stimulated with anti-CD3/CD28 in medium with or without metabolic inhibitors for 24 hrs.…”

“…Glutaminase activity measurement was adapted from previously described protocols (Thomas et al, 2014a). Briefly, T cells were lysed in ice-cold potassium phosphate buffer (45 mM, pH 8.2) containing protease inhibitors (Roche, Complete Protease Inhibitor Cocktail, 1 tablet in 50 ml) and incubated with 3 H-glutamine (0.03 μM, 0.91 μCi) for 90 min at room temperature.…”

Neuroglial Transmission

“…An exception to the anti-inflammatory role of neurotransmitters is glutamate, which when acting on ionotropic AMPA receptors on microglia can trigger activation (377) and release of the pro-inflammatory substance tumor necrosis factor (TNF)-␣ (287). Faced with proinflammatory stimuli, such as TNF-␣, microglia can release glutamate (403), and thus probably enter a vicious circle with increased generation of glutamate and TNF-␣. However, microglia seems to have developed also a negative-feedback loop, because when glutamate acts on group II and III metabotropic glutamate receptors it attenuates its own release from microglia (259).…”