The genes GLB1 and GALC encode GLB1 isoform 1 and galactocerebrosidase, respectively, which exhibit β-galactosidase activity in human lysosomes. GLB1 isoform 1 has been reported to play roles in rare lysosomal storage diseases. Further, its β-galactosidase activity is the most widely used biomarker of senescent and aging cells; hence, it is called senescence-associated β-galactosidase. Galactocerebrosidase plays roles in Krabbe disease. We previously reported a novel β-galactosidase activity in the Golgi apparatus of human cells; however, the protein responsible for this activity could not be identified. Inhibitor-derived chemical probes can serve as powerful tools to identify the responsible protein. In this study, we first constructed a cell-based high-throughput screening (HTS) system for Golgi β-galactosidase inhibitors, and then screened inhibitors from two compound libraries using the HTS system, in vitro assay, and cytotoxicity assay. An isoflavone derivative was identified among the final Golgi β-galactosidase inhibitor compound hits. Molecular docking simulations were performed to redesign the isoflavone derivative into a more potent inhibitor, and six designed derivatives were then synthesized. One of the derivatives, ARM07, exhibited potent inhibitory activity against β-galactosidase, with an IC50 value of 14.8 µM and competitive inhibition with Ki value of 13.3 µM. Furthermore, the in vitro and cellular inhibitory activities of ARM07 exceeded those of deoxygalactonojirimycin. ARM07 may contribute to the development of affinity-based chemical probes to identify the protein responsible for the newly discovered Golgi β-galactosidase activity. The therapeutic relevance of ARM07 against lysosomal storage diseases and its effect on senescent cells should be evaluated further.
Keywords: inhibitor screening; isoflavone; lysosomal storage disease; senescence-associated β-galactosidase; β-galactosidase.