Leupeptin hemisulfate is a protease inhibitor with cell membrane-permeable, reversible, competitive, and oral activities. Leupeptin hemisulfate inhibits the activity of Cathepsin B, Cathepsin H, and Cathepsin L, and blocks fusion of amphipathic lysosomes. Leupeptin hemisulfate also has anti-inflammatory activity.

METHODS: SARS-CoV-2 infected Vero cells were treated with Leupeptin hemisulfate (0.06-200 µM) for 72 h, and the viral RNA level was detected by RT-PCR.
RESULTS: Leupeptin hemisulfate inhibited SARS-CoV-2 RNA levels in Vero cells with an EC50 value of 42.34 µM. [1]
METHODS: SARS-CoV-2 pseudovirus-infected Huh7 cells were treated with Leupeptin hemisulfate (0.1-100 µM) for 24 h. Pseudovirus infection was detected by luciferase activity assay.
RESULTS: Leupeptin hemisulfate inhibited pseudovirus infection in Vero cells with an EC50 value of 39.29 µM. [2]

METHODS: To determine macrophage autophagic flux, Leupeptin hemisulfate (9-40 mg/kg in 0.5 mL PBS) was administered to C57BL/6NCrl mice by single intraperitoneal injection.
RESULTS: Leupeptin hemisulfate treatment resulted in the highest accumulation of LC3b in the liver and the lowest in the spleen. LC3a, ATG8/LC3b homologs, and the LC3b-interacting protein p62 were degraded with kinetics similar to those of LC3b. [3]
METHODS: To examine whether cold activates autophagic flux, Leupeptin hemisulfate (40 mg/kg) was administered intraperitoneally to C57B6 mice in a single injection, and then the mice were cold-exposed at 4 ℃ for 1 h. The mice were then exposed to cold for 1 h.
RESULTS: LC3-II flux in brown fat was increased >2-fold in cold-exposed mice. [4]

Determination of BET Protein Binding Affinities to I-BET726: For determination of binding affinities to BET protein bromodomains, I-BET726 is titrated against truncates containing both BD1 and BD2 of BRD2 (10 nM), BRD3 (10 nM), and BRD4 (10 nM) in 50 mM HEPES pH7.5, 150 mM NaCl, 5% Glycerol, 1 mM DTT and 1 mM CHAPS in the presence of an Alexa 647 derivative (50 nM) of fluorescent ligand. After equilibrating for 1 h, the bromodomain protein: ligand interaction is detected using Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) following the addition of 1.5 nM europium chelate labeled anti-6His antibody. Plates are read using an Envision Plate reader (λEX = 337 nm, λEM = 615 nm, λEM = 665 nm; dual dichroic = 400 nm & 630 nm). These data are fitted to a four parameter IC50 model using Graphit data analysis software.