Discovery of a Novel γ‐Glutamylcyclotransferase Inhibitor Plumbagin by Luminescence‐Based High‐Throughput Screening

γ-Glutamylcyclotransferase (GGCT) is a highly expressed protein in various cancers, and its knockdown and inhibitors suppress the growth of cancer cells in vitro and in vivo. Although GGCT is a promising target for anti-cancer strategies, no drugs have progressed to clinical availability yet. We established a novel target-based and cell-based screening method for GGCT inhibitors, enabling the efficient discovery of compounds that inhibit intracellular GGCT activity. Using the chemiluminogenic probe LISA-103 in response to GGCT enzymatic activity, high-throughput screening for GGCT inhibitors was performed in lung cancer SW1573 cells. The hit compound plumbagin was identified as a novel GGCT inhibitor that suppressed its activity intracellularly. Plumbagin directly bound to a GGCT-recombinant protein and inhibited its activity. Moreover, plumbagin up-regulated p21, a cyclin-dependent kinase inhibitor, and activated the tumor suppressor RB protein, inhibiting cell growth and cell cycle progression in breast MCF7 and colon HCT116 cancer cell lines. These phenomena induced by plumbagin mimicked the effect of GGCT depletion. The treatment with plumbagin suppressed tumor growth of HCT116 xenograft mice. These results suggest the potential of plumbagin in cancer therapy as a GGCT inhibitor.