In vitro study of BromAc on SARS-CoV-2 spike and envelope proteins shows synergy and disintegration at modest concentrations

Background and objectivesSARS-CoV-2 infection is the cause of a worldwide pandemic, currently with limited therapeutic options. Whilst vaccines are at the forefront of the therapeutic initiative, drug repurposing remains a promising approach for SARS-CoV-2 treatment. BromAc (Bromelain & Acetylcysteine) has synergistic action against glycoproteins by the synchronous breakage of glycosidic linkages and disulfide bonds. The spike protein of SARS-CoV-2, formed of glycoprotein and disulfide bridges for stabilization, represents an attractive target as it is essential for binding to the ACE2 receptor in host cells present in nasal mucosa. We sought to determine the effect of BromAc on the Spike and Envelope proteins and its potential to reduce infectivity in host cells. DesignRecombinant Spike and Envelope proteins were treated by single agent and combination BromAc at 50 and 100 {micro}g/20mg/mL and analyzed by electrophoresis. Ultraviolet analysis of disulfide bond reduction was performed for both Spike and Envelope proteins after treatment with Acetylcysteine. In vitro whole virus culture inactivation of pre-treated wild type and an S1/S2 Spike mutant SARS-CoV-2 with BromAc from 25 to 250 {micro}g/20mg/mL was measured by cytopathic effect, cell lysis assay, and replication capacity by RT-PCR. ResultsRecombinant Spike and Envelope SARS-CoV-2 proteins were fragmented by BromAc at both 50 and 100 {micro}g/20mg/mL whilst single agents had minimal effect. Spike and Envelope protein disulfide bonds were reduced by Acetylcysteine. In vitro whole virus culture of both wild type and Spike mutant SARS-CoV-2 demonstrated a concentration-dependent inactivation from BromAc treatment but not from single agents. ConclusionBromAc disintegrates SARS-CoV-2 Spike and Envelope proteins. In vitro tests on whole virus support this finding with inactivation of its replication capacity most strongly at 100 and 250 {micro}g/20mg/mL BromAc, even in Spike mutant virus. Clinical testing through nasal administration in patients with early SARS-CoV-2 infection is imminent. Author SummaryThere is currently no suitable therapeutic treatment for early SARS-CoV-2 aimed to prevent disease progression. BromAc is under clinical development by the authors for mucinous cancers due to its ability to alter complex glycoproteins structure. The potential of BromAc on SARS-CoV-2 Spike and Envelope glycoproteins stabilized by disulfide bonds was examined and found to disintegrate recombinant Spike and Envelope proteins whilst reducing disulfide stabilizer bridges. BromAc also showed an inhibitory effect on wild-type and Spike mutant SARS-CoV-2 by inactivation of its replication capacity in vitro. Hence, BromAc may be an effective therapeutic agent for early SARS-CoV-2 infection, despite mutations, and even have potential as a prophylactic in people at high risk of infection.