key: cord-0869799-dnj71g2g
authors: Wang, Runming; Chan, Jasper Fuk-Woo; Wang, Suyu; Li, Hongyan; Zhao, Jiajia; Ip, Tiffany Ka-Yan; Zuo, Zhong; Yuen, Kwok-Yung; Yuan, Shuofeng; Sun, Hongzhe
title: Orally administered bismuth drug together with N-acetyl cysteine as a broad-spectrum anti-coronavirus cocktail therapy
date: 2021-12-03
journal: Chemical science
DOI: 10.1039/d1sc04515f
sha: 27c553d44f404252c2875c6caa23dba1981b00d7
doc_id: 869799
cord_uid: dnj71g2g

The emergence of SARS-CoV-2 variants of concern compromises vaccine efficacy and emphasizes the need for further development of anti-SARS-CoV-2 therapeutics, in particular orally administered take-home therapies. Cocktail therapy has shown great promise in the treatment of viral infection. Herein, we reported the potent preclinical anti-SARS-CoV-2 efficacy of a cocktail therapy consisting of clinically used drugs, e.g. colloidal bismuth subcitrate (CBS) or bismuth subsalicylate (BSS), and N-acetyl-l-cysteine (NAC). Oral administration of the cocktail reduced viral loads in the lung and ameliorated virus-induced pneumonia in a hamster infection model. The mechanistic studies showed that NAC prevented the hydrolysis of bismuth drugs at gastric pH via the formation of the stable component [Bi(NAC)(3)], and optimized the pharmacokinetics profile of CBS in vivo. Combination of bismuth drugs with NAC suppressed the replication of a panel of medically important coronaviruses including Middle East respiratory syndrome-related coronavirus (MERS-CoV), Human coronavirus 229E (HCoV-229E) and SARS-CoV-2 Alpha variant (B.1.1.7) with broad-spectrum inhibitory activities towards key viral cysteine enzymes/proteases including papain-like protease (PL(pro)), main protease (M(pro)), helicase (Hel) and angiotensin-converting enzyme 2 (ACE2). Importantly, our study offered a potential at-home treatment for combating SARS-CoV-2 and future coronavirus infections.

For hamster experiments, 6-to-10-week-old male and female Syrian hamsters with body weight of 70-100 g, were obtained from the Chinese University of Hong Kong Laboratory Animal Service Centre through the HKU Centre for Comparative Medicine Research. The hamsters were kept in biosafety level 2 housing and given access to standard pellet feed and water ad libitum, as previously described 5, 6 . All experimental protocols were approved by the CULATR of the University of Hong Kong and were performed according to the standard operating procedures of the biosafety level 3 animal facilities (reference code: CULATR 5370- 20) .

intranasally inoculated with 10 5 p.f.u. of SARS-CoV-2 (SARS-CoV-2 HKU-001a) in 100 µL PBS under intraperitoneal ketamine (200 mg per kg body weight) and xylazine (10 mg per kg body weight) anaesthesia. From -2 day-post-infection (dpi) to 1 dpi, hamsters were orally administered once daily with water as vehicle, CBS (300 mg/kg) or BSS (300 mg/kg), NAC (370 mg/kg), CBS (300 mg/kg)+3NAC (370 mg/kg) or BSS (300 mg/kg)+3NAC (405 mg/kg), respectively, for four consecutive days. Animals were monitored twice daily for clinical signs of disease. Eight or four animals in each group were euthanized at 2 dpi. for virological and histolopathological analyses. Lung tissue samples were isolated. Viral yield in the tissue homogenates was detected by qRT-PCR methods. The cytokine and chemokine profiles of the hamster lungs were detected by the 2 −ΔΔCT method using probe-based one-step qRT-PCR (Qiagen). The tissue pathology of infected animals was examined by H&E and immunofluorescence staining in accordance with an established protocol 7 Immunofluorescence microscopy. Vero E6 cells were infected with SARS-CoV-2 (MOI=0.1) and exposed to the treatment of water as vehicle, CBS (1000 μM), NAC (1000 μM), and CBS (1000 μM)+3NAC (3000 μM), respectively, for 24 hours. Antigen expression in the infected cells was detected with an in-house rabbit antiserum against SARS-CoV-2-nucleocapsid protein (NP) of SARS-CoV-2. Cell nuclei were labelled with the 4,6-diamidino-2-phenylindole (DAPI) nucleic acid stain (Thermo Fisher Scientific). The Alexa Fluor secondary antibodies were obtained from Thermo Fisher Scientific. Mounting was performed with the Diamond Prolong AntiFade mountant from Thermo Fisher Scientific.

Nephrotoxicity test. Groups of mice (n=4 per group) were orally administered with water as vehicle, CBS (500 mg/kg) and CBS (500 mg/kg)+3NAC (580 mg/kg) 4 consecutive days, respectively. Mice were sacrificed at 1, 7, 14, 28 day post last dosing and mice serum was collected for the blood urea nitrogen test (ThermoFisher, USA) and creatinine test (Cayman Chemical, MI, USA) according to the manufacturer's instruction. Serum isolated from untreated mice were used as control. Plaque reduction assay. Plaque reduction assay was performed to estimate the half maximal effective concentration (EC 50 ) as previously described with slight modifications 6, 11 . Briefly, VeroE6 cells were seeded at 4×10 5 cells/well in 12-well tissue culture plates on the day before the assay was performed. After 24 hour of incubation, 50 plaque-forming units (PFU) of SARS-CoV-2 were added to the cell monolayer with or without the addition of CBS, NAC or CBS+3NAC at varying concentrations. The plates were further incubated for 1 hour at 37°C in 5% CO 2 before removal of unbound viral particles by aspiration of the media and washing once with DMEM. Monolayers were then overlaid with media containing 1% low melting agarose (Cambrex Corporation, New Jersey, USA) in DMEM and appropriate concentrations of trichostatin A, inverted and incubated as above for another 72 hours. The wells were then fixed with 10% formaldehyde (BDH, Merck, Darmstadt, Germany) overnight. After removal of the agarose plugs, the monolayers were stained with 0.7% crystal violet (BDH, Merck) and the plaques were counted. The percentage of plaque inhibition relative to the control (i.e. without the addition of compound) wells was determined for each concentration of drug compound. EC 50 was calculated using Sigma plot (SPSS) in an Excel add-in ED50V10. The plaque reduction assay experiments were performed in triplicate and repeated twice for confirmation.

was used to monitor the levels of bismuth in all investigated subjects. A quadrupole-based inductively coupled plasma mass spectroscopy (ICP-MS) (Agilent 7700x, Agilent Technologies, CA), equipped with a glass concentric nebulizer was used in this study. The samples were diluted to an appropriate concentration, sprayed into aerosols using microconcentric nebulizer and introduced into the ICP directly for time-resolved ICP-MS measurements. Samples were further diluted when the measured signals exceeded the liner range of standard curve. Bismuth contents (Bi 209 ) in the investigated substance were calculated according to the standard curve in 1% nitric acid or respective blank control solution of organ and blood. Only one isotope was monitored in each measurement.

The main parameters were listed as follows: RF power (1300 kW); spray chamber (Scott spray chamber); nebulizer (MicroMist nebulizer); lens: (Ni); nebulizer gas flow (0.8 mL/min); In vitro Caco-2 permeability assay. The in vitro permeability of CBS in the absence or presence of NAC was evaluated by using the Caco-2 permeability assay according to a method as described previously 12 .

Briefly, Caco-2 cells with 80-90% confluence was sub-cultured by trypsinization with 0. phenol red ] and loaded in the donor (apical) chamber in a 1.5 mL aliquot, respectively, followed by adding 2.5 mL transport buffer in receiver (basolateral) chamber. Aliquots of 0.1 mL samples was withdrawn from the receiver chamber at different time intervals (10, 20, 30, 40 , 50, 60 min) and equal volume of blank transport buffer was supplemented in receiver chamber immediately. The assay was performed in triplicate. Samples collected from the transport study were diluted to appropriate concentrations with 1% HNO 3 followed by ICP-MS measurement of bismuth content transported from donor side to receiver side.

At the end of transport study, Caco-2 cells on the monolayer were also collected after washing with PBS for six times, and the numbers of cells were counted by hemocytometer under an optical microscope. The resulting cell pellets were acidified with 69% HNO 3 , and diluted appropriately for the measurement of bismuth accumulation in cells. The apparent permeability coefficients (P app , cm/s) of CBS from different treatment groups were calculated through the following equation 13 : For ACE2 activity inhibition, a modified assay was performed according to the manufactory's instruction (ab273373, Abcam) with a synthetic MCA based peptide substrate to release a free fluorophore. Recombinant ACE2 (0.1 uM) was incubated with CBS, NAC and CBS+3NAC at varying concentrations, respectively, for 30 min in ACE2 assay buffer at room temperature. The substrates (2 μM) was added to the protein solutions. After another 1hour incubation, fluorescence (λ ex = 320 nm, λ em = 420 nm) was measured to determine ACE2 activity. The relative ACE2 activity was the ratio between the activity of the samples in the presence of drugs and the activity of the control, and is therefore expressed as a percentage. The assay was performed in triplicate. Ellman's assay. Amount of free cysteine was assayed spectrophotometrically with DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)] according to a previously described method 21 shown as mean ± SD. No difference in statistical significance was found among groups using an unpaired two-tailed Student's t-test. 

A) UV-Vis spectral profile of NAC in the absence and presence of increasing amounts of

The stochiometry of Bi 3+ to NAC was determined to be 1:3.05 from the overall absorbance changes at 350 nm, indicative of the formation of