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. 2010 Mar;85(3):551-5.
doi: 10.1016/j.antiviral.2009.12.001. Epub 2009 Dec 6.

TACE antagonists blocking ACE2 shedding caused by the spike protein of SARS-CoV are candidate antiviral compounds

Shiori Haga  1 , Noriyo Nagata, Tadashi Okamura, Norio Yamamoto, Tetsutaro Sata, Naoki Yamamoto, Takehiko Sasazuki, Yukihito Ishizaka
Affiliations

TACE antagonists blocking ACE2 shedding caused by the spike protein of SARS-CoV are candidate antiviral compounds

Shiori Haga et al. Antiviral Res. 2010 Mar.

Abstract

Because outbreaks of severe acute respiratory syndrome coronavirus (SARS-CoV) might reemerge, identifying antiviral compounds is of key importance. Previously, we showed that the cellular factor TNF-alpha converting enzyme (TACE), activated by the spike protein of SARS-CoV (SARS-S protein), was positively involved in viral entry, implying that TACE is a possible target for developing antiviral compounds. To demonstrate this possibility, we here tested the effects of TACE inhibitors on viral entry. In vitro and in vivo data revealed that the TACE inhibitor TAPI-2 attenuated entry of both pseudotyped virus expressing the SARS-S protein in a lentiviral vector backbone and infectious SARS-CoV. TAPI-2 blocked both the SARS-S protein-induced shedding of angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV, and TNF-alpha production in lung tissues. Since the downregulation of ACE2 by SARS-S protein was proposed as an etiological event in the severe clinical manifestations, our data suggest that TACE antagonists block SARS-CoV infection and also attenuate its severe clinical outcome.

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Figures

Fig. 1
Fig. 1
ACE2 shedding is inhibited by TACE inhibitors. (A) TACE inhibitors blocked SARS-S protein-induced processing of ACE2. Vero E6 cells were treated with two TACE inhibitors, TAPI-0 (100 nM, BIOMOL International, Plymouth Meeting, PA) or TAPI-2 (200 nM, BIOMOL International) for 1 h, and 100 μg/ml SARS-S protein (Haga et al., 2008) was added for 3 and 6 h. The proteins present in the culture supernatants were recovered by coprecipitation with ovalbumin (OVA) in trichloroacetic acid, and subjected to Western blot analysis, as previously described (Haga et al., 2008). Lane 1, control without the addition of SARS-S protein; lanes 2 and 3, saline with SARS-S protein; lanes 4 and 5, TAPI-0 with SARS-S protein; lanes 6 and 7, TAPI-2 with SARS-S protein. The culture supernatants were collected after 3 h (lanes 2, 4 and 6) or 6 h (lanes 3, 5 and 7). (B and C) TAPI-0 inhibits the release of ACE2 activity into the culture supernatants. The ACE2 activity was measured in the supernatants of Vero E6 cells cultured with or without 100 nM TAPI-0. The horizontal axis shows the incubation time of a fluorescence substrate of ACE2 (Mca-Y-V-A-D-A-P-K-Dnp-OH, amino acid depicted by single letters; R&D Systems, Minneapolis, MN) (B). The relative rate of inhibition calculated based on the data shown in (B) at 90 min (C). (D and E) TAPI-2 inhibits the release of ACE2 activity into the culture supernatants. The experiment was carried out by the same procedures as described in (B) and (C). TAPI-2 was used at a concentration of 200 nM.
Fig. 2
Fig. 2
TACE inhibitors block the SARS-S protein-induced TACE activity. HuH-7 cells were treated with TAPI-2 (200 nM) for 1 h and then SARS-S protein (100 μg/ml) was added. After 3 or 6 h, cellular extracts were prepared and incubated with a fluorescence substrate of TACE (Mca-P-L-A-Q-A-V-Dpa-R-S-S-S-R-NH2, amino acid depicted by single letters; BIOMOL International, Plymouth Meeting, PA). The TACE activity was measured every 15 min after adding the substrate. (A) Time course of the TACE activity. The fluorescent intensity of cleaved substrate was monitored in extracts of cells treated with or without TAPI-2. After 45 min, a difference was detected in the TACE activity of the control sample and the sample treated with TAPI-2. (B) TAPI-2 blocked the TACE activity. TACE activity was detected 6 h after adding SARS-S protein, and this was blocked completely by pretreatment with TAPI-2 (p < 0.01).
Fig. 3
Fig. 3
Effects of TACE inhibitors on viral entry in vitro. (A and B) TACE inhibitors suppressed SARS-S protein-dependent viral entry. Plasmid DNA encoding ACE2 was introduced into HEK293T cells, and then the cells were infected with the virus. The cells were treated with TAPI-0 (A) or TAPI-2 (B) for 1 h and then infected with the SARS-S pseudotyped lentivirus (Invitrogen, Carlsbad, CA) for 4 h. The intracellular p24 was measured using a Retro-Tek HIV-1 p24 ELISA kit (ZeptoMetrix, Buffalo, NY) according to the manufacturer's instructions. (C) TAPI-2 blocked the entry of infectious SARS-CoV. The viral infection efficiency was measured using real-time RT-PCR of viral mRNA, as described (Haga et al., 2008). Test samples were harvested and analyzed 4 h after infection with SARS-CoV. The data were normalized using 18S ribosomal RNA. In this experiment, TAPI-2 was used because TAPI-2 is water soluble, whereas TAPI-0 is water insoluble. TAPI-2 did not require control samples treated with of dimethylsulfoxide (DMSO), a solvent of TAPI-0.
Fig. 4
Fig. 4
TACE inhibitors attenuated SARS-S protein-dependent viral entry in vivo. (A) Inhibitory effects of TAPI-0 and TAPI-2 on viral entry. Left panel: male C57BL/6j mice (5–7-week old, A, left panel) or female Balb/c mice (28–32-week old) were inoculated intratracheally with 25 μl of a solution of the SARS-S pseudotyped lentivirus (4 μg/ml p24). To test the effects of TAPI-0, 25 μl of 0.3 μM of the compound dissolved in 0.0075% of DMSO or the same amount of diluted DMSO was administered intratracheally 2 h before the viral challenge (n = 3 per group, respectively). As additional control, the effects on the viral entry of VSV-G pseudotyped lentivirus were also examined. After 4 h, lung tissues were first refluxed with 50 ml of phosphate-based buffer, 50 ml of phosphate-based buffer containing 500 μg/ml collagenase, and 50 ml of phosphate-buffered saline containing 50 μg/ml trypsin, and then extracted. Extracted p24 was measured using a p24 ELISA kit. Right panel: For testing the TAPI-2, 25 μl of 2 μM of the compound in water or the same volume of distilled water was administered by the same method (right panel, n = 6 per group). We measured intracellular p24 by the similar procedures. (B) Effect of TAPI-2 on the production of TNF-α. After viral infection with TAPI-2 or water as control, lung tissues, prepared without the refluxing using collagenase and trypsin, were subjected to the analysis of ELISA of TNF-α (n = 4 per group) (C) Effect of TAPI-2 on the viral infection of the infectious SARS-CoV. Left panel: Protocol for SARS-CoV inoculation and TAPI-2 treatment. In this experiment, we used TAPI-2 because it is water soluble. Although TAPI-0 seemed to be more effective than TAPI-2, TAPI-2 was better for in vivo experiments, because it was not necessary to include additional control groups treated with DMSO, a solvent of TAPI-0. Seven-week-old female Balb/c mice were pretreated with 20 μl of 2 μM of TAPI-2 or saline via intranasal injection under anesthesia. Soon afterward, these mice were infected with 20 μl of 106.0 TCID50 mouse-passaged Frankfurt isolate of SARS-CoV (Nagata et al., 2008) via intranasal inoculation. Three hours and 1 day after virus inoculation, animals were treated with 20 μl of TAPI-2 or saline via intranasal injection under anesthesia. Right panel: Virus titers in the lung lavage fluids 3 days after inoculation with saline or TAPI-2 (n = 4 per group). Balb/c mice on the virus titers in the lung lavage fluids after inoculation. The detection limit was 101.5 CCID50/ml of fluid.
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