losartan-potassium 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

Transcriptional activation of erythropoietin, glycolytic enzymes, and vascular endothelial growth factor occurs during hypoxia or in response to cobalt chloride (CoCl2) in Hep3B cells. However, neither the mechanism of cellular O2 sensing nor that of cobalt is fully understood. We tested whether mitochondria act as O2 sensors during hypoxia and whether hypoxia and cobalt activate transcription by increasing generation of reactive oxygen species (ROS). Results show (i) wild-type Hep3B cells increase ROS generation during hypoxia (1. 5% O2) or CoCl2 incubation, (ii) Hep3B cells depleted of mitochondrial DNA (rho0 cells) fail to respire, fail to activate mRNA for erythropoietin, glycolytic enzymes, or vascular endothelial growth factor during hypoxia, and fail to increase ROS generation during hypoxia; (iii) rho0 cells increase ROS generation in response to CoCl2 and retain the ability to induce expression of these genes; and (iv) the antioxidants pyrrolidine dithiocarbamate and ebselen abolish transcriptional activation of these genes during hypoxia or CoCl2 in wild-type cells, and abolish the response to CoCl2 in rho degrees cells. Thus, hypoxia activates transcription via a mitochondria-dependent signaling process involving increased ROS, whereas CoCl2 activates transcription by stimulating ROS generation via a mitochondria-independent mechanism.

Topics: Adenosine Triphosphate; Antioxidants; Azoles; Cell Hypoxia; Cell Line; Cobalt; DNA-Binding Proteins; DNA, Mitochondrial; Endothelial Growth Factors; Erythropoietin; Glycolysis; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Isoindoles; Lymphokines; Mitochondria, Liver; Nuclear Proteins; Organoselenium Compounds; Pyrrolidines; Reactive Oxygen Species; RNA, Messenger; Thiocarbamates; Transcription Factors; Transcriptional Activation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors