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PLoS Pathog
2023 Aug 01;198:e1011328. doi: 10.1371/journal.ppat.1011328.
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Post-infection treatment with the E protein inhibitor BIT225 reduces disease severity and increases survival of K18-hACE2 transgenic mice infected with a lethal dose of SARS-CoV-2.
Ewart G
,
Bobardt M
,
Bentzen BH
,
Yan Y
,
Thomson A
,
Klumpp K
,
Becker S
,
Rosenkilde MM
,
Miller M
,
Gallay P
.
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The Coronavirus envelope (E) protein is a small structural protein with ion channel activity that plays an important role in virus assembly, budding, immunopathogenesis and disease severity. The viroporin E is also located in Golgi and ER membranes of infected cells and is associated with inflammasome activation and immune dysregulation. Here we evaluated in vitro antiviral activity, mechanism of action and in vivo efficacy of BIT225 for the treatment of SARS-CoV-2 infection. BIT225 showed broad-spectrum direct-acting antiviral activity against SARS-CoV-2 in Calu3 and Vero cells with similar potency across 6 different virus strains. BIT225 inhibited ion channel activity of E protein but did not inhibit endogenous currents or calcium-induced ion channel activity of TMEM16A in Xenopus oocytes. BIT225 administered by oral gavage for 12 days starting 12 hours before infection completely prevented body weight loss and mortality in SARS-CoV-2 infected K18 mice (100% survival, n = 12), while all vehicle-dosed animals reached a mortality endpoint by Day 9 across two studies (n = 12). When treatment started at 24 hours after infection, body weight loss, and mortality were also prevented (100% survival, n = 5), while 4 of 5 mice maintained and increased body weight and survived when treatment started 48 hours after infection. Treatment efficacy was dependent on BIT225 dose and was associated with significant reductions in lung viral load (3.5 log10), virus titer (4000 pfu/ml) and lung and serum cytokine levels. These results validate viroporin E as a viable antiviral target and support the clinical study of BIT225 for treatment and prophylaxis of SARS-CoV-2 infection.
Fig 1. Effect Of BIT225 On E protein channels In Xenopus oocytes.
A. Resting membrane potential in oocytes injected with E protein encoding cRNA versus non-injected oocytes. B. Current-voltage relationships in injected and control oocytes and the effects of BIT225 (10 μM) application.
Fig 2. Efficacy and dose-dependence of seven-day treatment of SARS-CoV-2 infected K18-hACE2 mice with BIT225.
BIT225 dosing started 12 hours before infection. (A) Dosing Scheme. (B) Body weight changes in individual animals (dashed lines) and mean body-weight changes (solid lines) as percent of baseline pre-infection weight and 95% confidence interval error bars (n = 5 per group). Vehicle control (black circles); BIT225 (100 mg/kg–red triangles); BIT225 (300 mg/kg–blue squares). (C) Day 7 analysis of lung viral load by qRT-PCR. (D) Day 7 analysis of lung virus titre by plaque assay. (E) Day 7 analysis of cytokines or chemokines in the lung. Symbols represent data for individual mice: Vehicle control (black circles); BIT225 (100 mg/kg–red triangles); BIT225 (300 mg/kg–blue squares). Horizontal lines and “+” indicate the group median and mean, respectively. Welch’s T-tests were used to compare the group means and P-values are indicated as: ns—P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig 3. Twelve-day treatment of SARS-CoV-2 infected K18-hACE2 mice with BIT225.
BIT225 dosing started 12 hours before infection. (A) Dosing Scheme. (B) Kaplan-Meier plot: vehicle control (black line, n = 7, 0% survival); BIT225 12-day treatment group (blue line, n = 7, 100% survival). (C) Body weight changes in individual animals. Inset: Satellite groups dosed for 5 days for viral load and inflammation analyses. (D) Day 5 and Day 12 analysis of lung viral load by qRT-PCR. (E) Day 5 and Day 12 analysis of lung virus titre by plaque assay. Vehicle control (black circles) and 5-day BIT225 (red triangles) treatment data are from the satellite group. 12-day data (blue squares) are from the surviving animals in the 12-day treatment group. (F) Day 5 and Day 12 analysis of cytokines or chemokines in the lung. Symbols represent data for individual mice. Horizontal lines and “+” indicate the group median and mean, respectively. Welch’s T-tests were used to compare the group means and P-values are indicated as: ns—P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.
Fig 4. Treatment of SARS-CoV-2 infected K18-hACE2 mice with BIT225 starting 24 h before (pre), or 24 h or 48 h after (post) infection.
(A) Dosing Scheme. (B) Kaplan-Meier plot: Vehicle control (black line, 0% survival), BIT225 treatment starting 12 h pre infection (blue line, PRE, 100% survival), starting 24 h post infection (purple line with “o” symbols, POST (+24 h), 100% survival) or starting 48 h post infection (dark red line with “+” symbols, POST (+48 h), 80% survival). (C) Body weight changes in individual animals. (D) Day 12 analysis of lung viral load by qRT-PCR. (E) Day 12 analysis of lung virus titre by plaque assay. (F) Day 12 analysis of cytokines or chemokines in the lung. Symbols represent data for individual mice. Horizontal lines and “+” indicate the group median and mean, respectively. Welch’s T-tests were used to compare the group means and P-values are indicated as: ns—P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.
S2 Fig.
Effect Of BIT225 on E protein ion channel in Xenopus oocytes.
Change in currents for n = 5 individual oocytes after application of BIT225 (10 μM) at -85 mV holding potential: Injected (left panel) and uninjected (right panel).
S3 Fig.
No effect of BIT225 on Ca2+-induced endogenous currents in Xenopus oocytes.
Empty oocytes were exposed to 5 mM Ca2+ solutions in the absence (P1) and presence (P2) of drug and the Ca2+-induced currents were recorded. (A) The effect of drug treatment on the ratio of P1 and P2 currents relative to untreated control. (B) Representative traces of Ca2+-induced currents in the absence (P1) and presence (P2) of the indicated compound.
S4 Fig.
No effect of BIT225 on TMEM16A ion channel in HEK293 cells.
The Xenopus Ca2+-induced ion channel TMEM16A was transiently expressed in HEK293 cells. Ion channel activity was measured by manual patch clamp in the absence and presence of inhibitors as indicated.
S5 Fig.
Resting membrane potential in X. laevis oocytes injected with 20 ng KChIP2.1, 50 nl water and un-injected. n = 10–11.
S7 Fig.
Dose-dependent viral load, infectious titre and cytokine responses in serum of mice treated with BIT225 for 7 days.
Serum samples were harvested at Day 7 and analysed for; (A) viral load by pRT-PCR assay (left panel) or infectious titre by plaque assay (right panel); and (B) concentration of the indicated cytokines or chemokine by sandwich ELISA assay, as described in Methods. Symbols represent data for individual mice: Vehicle control (black circles); BIT225 (100 mg/kg–red triangles); BIT225 (300 mg/kg–blue squares). Horizontal lines and “+” indicate the group median and mean, respectively. Welch’s T-tests were used to compare the group means and P-values are indicated as: ns—P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.
S8 Fig.
Cytokine levels in serum of mice treated with BIT225 for 5 or 12 days.
Serum samples were harvested at Day 5 or Day 12 and analysed for concentration of the indicated cytokines or chemokine by sandwich ELISA assay, as described in Methods. Vehicle control and 5-day BIT225 treatment samples were from the satellite group. 12-day data are from the animals that survived to the end of the study. Symbols represent data for individual mice: Vehicle control (black circles); BIT225 5-day group (red triangles); BIT225 12-day group (blue squares). Horizontal lines and “+” indicate the group median and mean, respectively. Welch’s T-tests were used to compare the group means and P-values are indicated as: ns -; P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.
S9 Fig.
Cytokine levels at Day 12 post infection in serum of mice treated with BIT225 starting 24 h before (pre), or 24 h or 48 h after (post) infection.
Serum samples were harvested from surviving animals at Day 12 post infection and analysed for concentration of the indicated cytokines or chemokine by sandwich ELISA assay, as described in Methods. No vehicle control animals survived. Symbols represent data for individual mice: Pre-treated group (blue line and squares); 24 h post treated group (purple line and open circles); 48 h post treated group (red line and “+” symbols). Horizontal lines indicate the group median. Welch’s T-tests were used to compare the group means and P-values are indicated as: ns—P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.
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