curcumin and ebselen

Given their medical importance, proteases have been studied by diverse approaches and screened for small molecule protease inhibitors. Here, we present a multiplexed microsphere-based protease assay that uses high-throughput flow cytometry to screen for inhibitors of the light chain protease of botulinum neurotoxin type A (BoNTALC). Our assay uses a full-length substrate and several deletion mutants screened in parallel to identify small molecule inhibitors. The use of multiplex flow cytometry has the advantage of using full-length substrates, which contain already identified distal-binding elements for the BoNTALC, and could lead to a new class of BoNTALC inhibitors. In this study, we have screened 880 off patent drugs and bioavailable compounds to identify ebselen as an in vitro inhibitor of BoNTALC. This discovery demonstrates the validity of our microsphere-based approach and illustrates its potential for high-throughput screening for inhibitors of proteases in general.

Topics: Antigens, Bacterial; Azoles; Bacterial Toxins; Botulinum Toxins, Type A; Drug Evaluation, Preclinical; Flow Cytometry; Fluorescence Resonance Energy Transfer; High-Throughput Screening Assays; Isoindoles; Metalloproteases; Microspheres; Organoselenium Compounds; Protease Inhibitors

Effects of new complex curcuminoids (cassumunin A and cassumunin B) isolated from tropical ginger, Zingiber cassumunar, were examined in dissociated rat thymocytes suffering from oxidative stress induced by 3 mM hydrogen peroxide by using a flow cytometer and ethidium bromide. The effects were compared with those of curcumin, a natural antioxidant, whose chemical structure is included in those of cassumunins A and B. Pretreatment of rat thymocytes with the respective cassumunins at concentrations ranging from 100 nM to 3 microM dose-dependently prevented the hydrogen peroxide (H2O2)-induced decrease in cell viability. It had the same action, although less effective, against the treatment with cassumunin A or B (3 microM) immediately after or 60 min after start of the oxidative stress. Respective potencies of cassumunins A and B in protecting the cells suffering from H2O2-induced oxidative stress were greater than that of curcumin. It is suggested that cassumunins A and B may possess a potent protective action on living cells suffering from oxidative stress.

Topics: Animals; Antioxidants; Azoles; Cell Line; Cell Survival; Curcumin; Deferoxamine; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Ethidium; Flow Cytometry; Hydrogen Peroxide; Isoindoles; Organoselenium Compounds; Oxidative Stress; Rats; Rats, Wistar; Thymus Gland; Zingiberales