Abstract
BackgroundGlioblastoma (GBM) is a universally lethal tumor with frequently overexpressed or mutated epidermal growth factor receptor (EGFR). NADPH quinone oxidoreductase 1 (NQO1) and glutathione-S-transferase Pi 1 (GSTP1) are commonly upregulated in GBM. NQO1 and GSTP1 decrease the formation of reactive oxygen species (ROS), which mediates the oxidative stress and promotes GBM cell proliferation.MethodsHigh-throughput screen was used for agents selectively active against GBM cells with EGFRvIII mutations. Co-crystal structures were revealed molecular details of target recognition. Pharmacological and gene knockdown/overexpression approaches were used to investigate the oxidative stress in vitro and in vivo.ResultsWe identified a small molecular inhibitor, “MNPC,” that binds to both NQO1 and GSTP1 with high affinity and selectivity. MNPC inhibits NQO1 and GSTP1 enzymes and induces apoptosis in GBM, specifically inhibiting the growth of cell lines and primary GBM bearing the EGFRvIII mutation. Co-crystal structures between MNPC and NQO1, and molecular docking of MNPC with GSTP1 reveal that it binds the active sites and acts as a potent dual inhibitor. Inactivation of both NQO1 and GSTP1 with siRNA or MNPC results in imbalanced redox homeostasis, leading to apoptosis and mitigated cancer proliferation in vitro and in vivo.ConclusionsThus, MNPC, a dual inhibitor for both NQO1 and GSTP1, provides a novel lead compound for treating GBM via the exploitation of specific vulnerabilities created by mutant EGFR.
Highlights
Glioblastoma (GBM) is one of the most aggressive and malignant human brain tumors with a mean survival rate of 12 months [1] despite the standard therapeutic regiment of maximal surgical resection, radiation, andLei et al J Hematol Oncol (2020) 13:141 clinical trials have not proven the efficacy of this strategy
High‐throughput screening for small molecules that selectively inhibit proliferation of U87MG/EGFRvIII cells versus U87MG cells To search for the pharmacological agents for potential GBM therapeutics, we employed an ultra-high-throughput screen in a 1536-well plate format and testing funnels to screen and identify the small molecules, which differentially inhibited the growth of U87MG/EGFRvIII cells against U87MG (PTEN mutant) parental cells
In our study, we show that 5-Methyl-N-(5-nitro-thiazol-2-yl)-3-phenylisoxazole -4-carboxamide (MNPC) simultaneously inhibits both NADPH quinone oxidoreductase 1 (NQO1) and glutathione-S-transferase Pi 1 (GSTP1) enzyme activities via binding to both enzymes’ active sites (Fig. 2 and Additional file 1: Supplementary Fig. 2)
Summary
Glioblastoma (GBM) is one of the most aggressive and malignant human brain tumors with a mean survival rate of 12 months [1] despite the standard therapeutic regiment of maximal surgical resection, radiation, andLei et al J Hematol Oncol (2020) 13:141 clinical trials have not proven the efficacy of this strategy. One strategy for patient-specific therapeutics is to take advantage of the downstream consequences of the activation of oncogenic mutations. Mounting evidence suggests that EGFRvIII activation correlates to the level of cellular oxidative stress [5, 6]. Subsequent studies revealed that EGFRvIII overexpression in glioblastoma cells caused increased levels of reactive oxygen species (ROS), DNA strand break accumulation and genome instability [8]. ROS are derived from enzymatic reactions involving NADPH-dependent oxidases NAD(P)H: quinone oxidoreductase 1 (NQO1), which is a cytosolic reductase, and it plays important roles in the cellular response to numerous stresses and is upregulated in many human cancers compared to adjacent normal tissues [12]. NQO1 and GSTP1 decrease the formation of reactive oxygen species (ROS), which mediates the oxidative stress and promotes GBM cell proliferation
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