Oxindole-curcumin hybrid compound enhances the transcription of γ-glutamylcysteine ligase

Abstract

Glutathione (GSH), which is particularly important for antioxidant defenses, is synthesized in two sequential enzymatic reactions catalyzed by γ-glutamylcysteine ligase (GCL) and GSH synthase. GCL comprises catalytic (GCLC) and regulatory subunits and catalyzes the rate-limiting step in de novo GSH synthesis. Accumulating evidence suggests that substances that stimulate GSH synthesis are therapeutic modalities for neurodegenerative disorders and schizophrenia, in which a deficit in brain GSH content has been observed. In the present study, we attempted to develop small organic compounds that increase GCLC transcription. Using HT22 cells stably expressing a luciferase reporter that contains rat GCLC promoter region (-1764 to +2), we assessed the effects of the novel neuroprotective compound oxindole and related compounds on GCLC promoter activity. Among approximately 220 synthesized compounds, five compounds increased GCLC promoter activity by >200% at a concentration of 50 μM, and 16 compounds increased promoter activity by approximately 150%. The most effective compound oxindole-curcumin hybrid GIF-2165X-G1 increased GCLC mRNA levels in HT22 mouse hippocampal cells, PC12 rat pheochromocytoma cells, and C6 rat glioma cells. Although GIF-2165X-G1 potently induced antioxidant response element (ARE)-driven transcription, the compound increased GCLC transcriptional activity through Sp1 pathway in a Keap1-Nrf2-ARE-independent manner. These results suggest that GIF-2165X-G1 itself and further modification of the compound are useful interventions for promoting neuronal survival by augmenting resistance to oxidative stress.

Keywords: Glutathione; HT22 cells; Oxindole; Oxytosis/ferroptosis; γ-Glutamylcysteine ligase.