The Combination of Bromelain and Acetylcysteine (BromAc) Synergistically Inactivates SARS-CoV-2

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection is the cause of a worldwide pandemic, currently with limited therapeutic options. The spike glycoprotein and envelope protein of SARS-CoV-2, containing disulfide bridges for stabilization, represent an attractive target as they are essential for binding to the ACE2 receptor in host cells present in the nasal mucosa. Bromelain and Acetylcysteine (BromAc) has synergistic action against glycoproteins by breakage of glycosidic linkages and disulfide bonds. We sought to determine the effect of BromAc on the spike and envelope proteins and its potential to reduce infectivity in host cells. Recombinant spike and envelope SARS-CoV-2 proteins were disrupted by BromAc. Spike and envelope protein disulfide bonds were reduced by Acetylcysteine. In in vitro whole virus culture of both wild-type and spike mutants, SARS-CoV-2 demonstrated a concentration-dependent inactivation from BromAc treatment but not from single agents. Clinical testing through nasal administration in patients with early SARS-CoV-2 infection is imminent.

Keywords: Acetylcysteine; BromAc; Bromelain; SARS-CoV-2; drug repurposing.

Figure 1

(A) Bromelain and Acetylcysteine present a synergistic effect on severe acute respiratory syndrome coronavirus (SARS-CoV-2) spike and envelope protein destabilization. SDS-PAGE of the recombinant SARS-CoV-2 spike protein S1 + S2 subunits (150 kDa) and envelope protein (25 kDa). Proteins were treated with 20 mg/mL Acetylcysteine alone, 100 and 50 µg/mL Bromelain alone, and a combination of 100 and 50 µg/20 mg/mL BromAc. (B) Disulfide reduction of recombinant SARS-CoV-2 spike protein by Acetylcysteine. The differential assay between Acetylcysteine (Ac) and Dithiothreitol (DTT) for the reduction of disulfide bonds found on the spike protein indicates that Acetylcysteine reduces 42% of the disulfide bonds before the addition of DTT. The remaining bonds are reduced by DTT to produce the chromogen detected at 310 nm. (C) Disulfide reduction of recombinant SARS-CoV-2 envelope protein by Acetylcysteine. The differential assay between Acetylcysteine (Ac) and Dithiothreitol (DTT) for the reduction of disulfide bonds found on the envelope protein indicates that Acetylcysteine reduces 40% of the bonds before the addition of DTT.

Figure 2

Cell lysis assays demonstrated in vitro inactivation potential of Acetylcysteine and Bromelain combined (BromAc) against SARS-CoV-2. Cell viability was measured by cell staining with Neutral Red, where optical density (OD) is directly proportional to viable cells. Low OD would signify important cell lysis due to virus replication. The wild-type (WT) SARS-CoV-2 strain at 5.5 and 4.5 log₁₀TCID₅₀/mL titers (A and B, respectively) showed no inhibition of cytopathic effect (CPE) for single agent treatment, compared to the mock treatment virus control condition. BromAc combinations were able to inhibit CPE, compared to the mock infection cell controls. Treatment of a SARS-CoV-2 spike protein variant (∆S) with a mutation at the S1/S2 junction at 5.5 and 4.5 log₁₀TCID₅₀/mL titers (C and D, respectively) showed similar results. Bars represent the average of each quadruplicate per condition, illustrated by white circles. Ordinary one-way ANOVA was performed, using the mock treatment virus control as the control condition (**** p < 0.0001, *** p < 0.0005, ** p < 0.003, and * p < 0.05).

Figure 3

Threshold matrix of log₁₀ reduction values (LRV) of in vitro virus replication 96 h after BromAc treatment on WT and ∆S SARS-CoV-2 strains at 5.5 and 4.5 log₁₀TCID₅₀/mL titers. LRV were calculated with the following formula: LRV = (RT-PCR Ct of treatment—RT-PCR Ct virus control)/3.3; as 1 log10 ≈ 3.3 Ct. The color gradient matrix displays the number of quadruplicates per condition yielding an LRV > 4, corresponding to a robust inactivation according to the WHO. WT = wild-type; ∆S = S1/S2 spike mutant.

Figure 4

SARS-CoV-2 replication capacity of WT and ∆S SARS-CoV-2 measured by Real-Time Cell Analysis. Data points correspond to area under the curve analysis of normalized cell index (electronic impedance of RTCA established at time of inoculation) at 12-h intervals. Cell viability was then determined by normalizing against the corresponding cell control. WT = wild-type; ∆S = S1/S2 spike mutant.