Leupeptin

Abstract: The Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host proteases for virus entry into lung cells. In the current study, Vero cells constitutively expressing type II transmembrane serine protease (Vero-TMPRSS2 cells) showed larger syncytia at 18 h after infection with MERS-CoV than after infection with other coronaviruses. Furthermore, the susceptibility of Vero-TMPRSS2 cells to MERS-CoV was 100-fold higher than that of non-TMPRSS2-expressing parental Vero cells. The serine protease inhibitor camostat, which inhibits TMPRSS2 activity, completely blocked syncytium formation but only partially blocked virus entry into Vero-TMPRSS2 cells. Importantly, the coronavirus is thought to enter cells via two distinct pathways, one mediated by TMPRSS2 at the cell surface and the other mediated by cathepsin L in the endosome. Simultaneous treatment with inhibitors of cathepsin L and TMPRSS2 completely blocked virus entry into Vero-TMPRSS2 cells, indicating that MERS-CoV employs both the cell surface and the endosomal pathway to infect Vero-TMPRSS2 cells. In contrast, a single camostat treatment suppressed MERS-CoV entry into human bronchial submucosal gland-derived Calu-3 cells by 10-fold and virus growth by 270-fold, although treatment with both camostat and (23,25)-trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester, a cathepsin inhibitor, or treatment with leupeptin, an inhibitor of cysteine, serine, and threonine peptidases, was no more efficacious than treatment with camostat alone. Further, these inhibitors were not efficacious against MERS-CoV infection of MRC-5 and WI-38 cells, which were derived from lung, but these characters differed from those of mature pneumocytes. These results suggest that a single treatment with camostat is sufficient to block MERS-CoV entry into a well-differentiated lung-derived cell line.

Abstract: 'Wash-out' and inactivation of the Ca current were examined in dialysed, voltage-clamped neurones of Helix aspersa under conditions that isolate the Ca current virtually free of other currents. EGTA or other internal Ca2+ chelators were routinely omitted from the dialysate. The time-dependent loss, or wash-out, of Ca current was slowed by addition to the dialysing solution of agents, such as dibutyryl adenosine 3'-5'-cyclic monophosphate (dibutyryl cyclic AMP), Mg adenosine 5'-triphosphate (ATP) and the catalytic subunit of cyclic-AMP-dependent protein kinase, that promote protein phosphorylation and by EGTA. However, neither the phosphorylation-promoting agents nor internal EGTA prevented wash-out entirely, nor did they significantly restore previously 'washed-out' current. With phosphorylating agents in the dialysing solution, the irreversible development of wash-out was greatly reduced by introduction of leupeptin, an inhibitor of protease activity. Thus, the irreversible component of wash-out appears to result from a Ca-dependent proteolytic process. In the presence of leupeptin alone, Ca current amplitude continued to decline: however, the current could be largely or fully restored with addition of catalytic subunit, dibutyryl cyclic AMP, and Mg ATP to the dialysing solution. Thus, inhibition of proteolysis revealed a reversible component of wash-out that appears to result from dephosphorylation. During perfusion with leupeptin, Mg ATP, dibutyryl cyclic AMP and catalytic subunit the Ca current remained stable for up to several hours without addition of internal Ca2+ buffer. The rate of inactivation of the current that occurs during a depolarizing step showed only a very gradual decline during this time. Under these conditions, perfusion with calcineurin, a Ca-calmodulin-dependent phosphatase, caused a significant increase in the rate of Ca current inactivation. This inactivation was virtually eliminated by introduction of EGTA or by replacement of external Ca2+ with Ba2+, which is consistent with the ion dependency for calmodulin-dependent activation of calcineurin. When ATP in the dialysate was replaced with ATP-gamma-S (adenosine 5'-O-(thiotriphosphate], an analogue that donates a thiophosphate group resistant to hydrolysis, the rate of inactivation slowed. Since Ca-dependent inactivation during step depolarizations is enhanced by conditions that promote dephosphorylation, and Ca current wash-out is slowed by conditions that promote phosphorylation, inactivation and reversible wash-out appear to be related.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract: Tryptase, a protease unique to the mast cell secretory granule, is released in substantial quantities into the respiratory tract of patients with inflammatory disease of the airways. We have investigated the potential of tryptase to act as a mitogen for bronchial epithelial cells and to stimulate release of IL-8 and expression of ICAM-1. Tryptase was isolated from extracts of human lung tissue using ammonium sulphate precipitation, octyl agarose, and heparin agarose chromatography. Purified tryptase stimulated DNA synthesis in the human epithelial cell line H292, as measured by [3H] thymidine incorporation. Maximal growth was observed after 24 h using 25 mU/ml of tryptase (where 1 micron is defined as that which can hydrolyze 1 mumol of the peptide substrate N-alpha-benzoyl-DL-arginine p-nitroanilide hydrochloride per minute at 25 degrees C), a concentration that is likely to be achieved in vivo. Inhibitors of tryptase activity, including leupeptin and benzamidine hydrochloride, significantly decreased tryptase-induced stimulation of DNA synthesis, indicating the requirement for an active catalytic site. Tryptase stimulated a catalytic site-dependent release of IL-8 from epithelial cells after 24 h, and this was associated with up-regulation of ICAM-1 expression, as revealed by FACS analysis. Tryptase may play a critical role in epithelial repair and in the recruitment of granulocytes following mast cell activation.