DAS181 significantly reduced viral load in participants infected with influenza, thus warranting future clinical development of this novel host-directed therapy. CLINICAL TRIALS.GOV IDENTIFIER: NCT01037205.
Increasing resistance to currently available influenza antivirals highlights the need to develop alternate approaches for the prevention and/or treatment of influenza. DAS181 (Fludase), a novel sialidase fusion protein that enzymatically removes sialic acids on respiratory epithelium, exhibits potent antiviral activity against influenza A and B viruses. Here, we use a mouse model to evaluate the efficacy of DAS181 treatment against a highly pathogenic avian influenza H5N1 virus. When used to treat mice daily beginning 1 day before infection with A/Vietnam/ 1203/2004(H5N1) virus, DAS181 treatment at 1 mg/kg/day protected 100% of mice from fatal disease, prevented viral dissemination to the brain, and effectively blocked infection in 70% of mice. DAS181 at 1 mg/kg/day was also effective therapeutically, conferring enhanced survival of H5N1 virus-challenged mice when treatment was begun 72 h after infection. This notable antiviral activity underscores the potential utility of DAS181 as a new class of drug that is effective against influenza viruses with pandemic potential.
Protease nexin 1 (PN1) is a serine protease inhibitor (SERPIN) that acts as a suicide substrate for thrombin (Th) and urokinase-type plasminogen activator (uPA). PN1 forms 1:1 stoichiometric complexes with these proteases, which are then rapidly bound, internalized, and degraded. The low density lipoprotein receptor-related protein (LRP) is the receptor responsible for the internalization of protease-PN1 complexes. However, we found that the LRP is not significantly involved in the initial cell surface binding of thrombin-PN1, leading us to investigate what cellular component was responsible for this initial interaction. Since Th-PN1 complexes retain a high-affinity for heparin after complex formation, unlike several of the other SERPINs, we tested the possibility that cell surface heparins were involved in initial complex binding. Soluble heparin was found to be a potent inhibitor of the binding of Th-PN1 to the cell surface and greatly facilitated the dissociation of Th-PN1 complexes pre-bound in the absence of soluble heparin. To ascertain the role of cell surface heparins, further studies were done using complexes of thrombin and PN1(K7E), a variant of PN1 in which the heparin binding site was rendered non-functional. When added at equal initial concentrations of complexes, Th-PN1(K7E) was catabolized 5-to 10-fold less efficiently than Th-PN1, a direct result of the greatly diminished initial binding of the Th-PN1(K7E) complexes. These data demonstrate the sizable contribution of cell surface heparins to Thrombin-PN1 complex binding and support a model in which these heparins act to concentrate the complexes at the cell surface facilitating their subsequent LRP-dependent endocytosis.
Protease nexin 1 (PN1)1 is a 43-kDa serine protease inhibitor that is a member of the SERPIN superfamily (1). It is an important physiological regulator of thrombin in tissues, and like its plasma counter part, antithrombin III, it is activated by heparin (1, 2). Like other members of the SERPIN family, PN1 is proteolytically attacked by its target protease but arrests proteolytic cleavage resulting in the formation of a 1:1 stoichiometric complex with that protease (1, 2). Only PN1 that is in complex with a protease is internalized and degraded by cells at a significant rate (3). This is presumably due to the formation or unmasking of a receptor binding site in PN1 when in complex with a protease, which is absent in the free SERPIN. One candidate site within the PN1 sequence for this function has recently been identified (4). Mechanistically, this ensures that SERPINs will remain extracellular until they have become part of an inhibitory complex. Once they have formed an inhibitory complex with a protease, the rapid removal of protease-SERPIN complexes is important for two reasons. First, since there is evidence to suggest that protease-SERPIN complexes may not be covalently linked (5), the uninternalized complexes represent a potential source of active protease formed when the complexes dissociate. Second, in the case of one S...
The plasminogen receptors responsible for enhancing cell surface-dependent plasminogen activation expose COOH-terminal lysines on the cell surface and are sensitive to proteolysis by carboxypeptidase B (CpB). We treated U937 cells with CpB, then subjected membrane fractions to two-dimensional gel electrophoresis followed by ligand blotting with 125 I-plasminogen. A 54-kDa protein lost the ability to bind 125 I-plasminogen after treatment of intact cells and was purified by twodimensional gel electrophoresis and then sequenced by mass spectrometry. Two separate amino acid sequences were obtained and were identical to sequences contained within human and rat TIP49a. The cDNA for the 54-kDa protein matched the human TIP49a sequence, and encoded a COOH-terminal lysine, consistent with susceptibility to CpB. Antibodies against rat TIP49a recognized the plasminogen-binding protein on two-dimensional Western blots of U937 cell membranes. Human 125 I-Glu-plasminogen bound specifically to TIP49a protein, and binding was inhibited by ⑀-aminocaproic acid. A single class of binding sites was detected, and a K d of 0.57 ؎ 0.14 M was determined. TIP49a enhanced plasminogen activation 8-fold compared with the BSA control, and this was equivalent to the enhancement mediated by plasmin-treated fibrinogen. These results suggest that TIP49a is a previously unrecognized plasminogen-binding protein on the U937 cell surface.
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