Human chromosome 7 ORF 24 (C7orf24) was discovered as a tumor-related protein, and later identified as a γ-glutamyl cyclotransferase (GGCT) involved in the glutathione homeostasis cycle.¹ C7orf24 was found to be over-expressed in a range of cancers and silencing of the gene by small interfering RNA (siRNA) showed an antiproliferative effect on cancer cell lines.

GGCT converts γ-glutamyl amino acids into pyroglutamate (pyroGlu) and the corresponding amino acids. This singular substrate preference of the enzyme hampered its chemical probe development, and no fluorogenic ligand had been reported. Here we report the first fluorogenic dipeptide ligand “LISA-4”, which should contribute toward further understanding of GGCT.

LISA-4 [γ-Glu-Ser(CO-MU)] contains the Ser derivative with a carbonate bond-linked 4-methylumbelliferone (MU). The Ser derivative possesses an α-carboxyl group necessary for primary enzyme recognition, and it is designed to liberate the inherent fluorophore “MU” via an intramolecular O-to-N acyl migration-type reaction upon disengagement of the α-amino groups by GGCT (i.e. the enzyme-triggered chemical reaction).

In the molecule of LISA-4, the inherent fluorescence of MU was completely masked by its O-acylation. When LISA-4 was cleaved into pyroGlu and the Ser derivative by GGCT, however, the successive O-to-N acyl migration-type reaction released the intact MU from the Ser derivative. This intramolecular reaction was quick and clean. As a result, the enzyme reaction could be readily monitored with a fluorescence meter. The use of LISA-4 should accelerate future GGCT inhibitor screening and we hope that it can become a new tool in the cancer-related researches.²