Screening, Synthesis, And Evaluation Of Novel Isoflavone Derivatives As Inhibitors Of Human Golgi Beta-Galactosidase

The genes GLB1 and GALC encode GLB1 isoform 1 and galactocerebrosidase, respectively, which exhibit beta-galactosidase activity in human lysosomes. GLB1 isoform 1 has been reported to play roles in rare lysosomal storage diseases. Further, its beta-galactosidase activity is the most widely used biomarker of senescent and aging cells; hence, it is called senescence-associated beta-galactosidase. Galactocerebrosidase plays roles in Krabbe disease. We previously reported a novel beta-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 beta-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 beta-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 beta-galactosidase, with an IC50 value of 14.8 mu M and competitive inhibition with K-i value of 13.3 mu M. 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 beta-galactosidase activity. The therapeutic relevance of ARM07 against lysosomal storage diseases and its effect on senescent cells should be evaluated further.