Bromelain inhibits SARS‐CoV‐2 infection via targeting ACE‐2, TMPRSS2, and spike protein

Dear Editor, The new coronavirus, SARS-CoV-2, transmits rapidly from human-to-human resulting in the ongoing pandemic. SARS-CoV-2 infects angiotensin-converting enzyme 2 (ACE-2) expressing lung, heart, kidney, intestine, gall bladder, and testicular tissues of patients, leading to organ failure and sometimes death.1,2 Currently, COVID-19 patients are treated with different agents, including favilavir, remdesivir, chloroquine, hydroxychloroquine, lopinavir, darunavir, and tocilizumab.3,4 However, the safety and efficacy of those drugs against COVID-19 still need further confirmation by randomized clinical trials. Hence, there is an emergent need to repurpose the existing drugs or develop new virus-based and host-based antivirals against SARS-CoV-2. Bromelain is a cysteine protease isolated from pineapple stem and is used as a dietary supplement for treating patients with pain, inflammation,5 thrombosis,6 and cancer.7 Recently, studies have shown that SARS-CoV-2 homotrimeric viral spike protein (S1) binds to the Transmembrane Serine Protease 2 (TMPRSS2) primed host cell’s receptor ACE-2 for initial entry, followed by S2mediated membrane fusion.8 Of several normal and cancerous cells tested, VeroE6 and Calu-3 cells showed ACE-2 protein expression (Fig. 1A), as well as a basal level of TMPRSS2 protein (Fig. 1B). Since ACE-29 and TMPRSS2 (UniProtKB-O15393) contains cysteine residues with disulfide bonds to stabilize the protein structure, we investigated the effect of bromelain on ACE-2 and TMPRSS2 expression. Bromelain-induced a doseand time-dependent reduction of ACE-2 and TMPRSS2 expression in VeroE6 cells (Fig. 1C and D) but do not alter ACE-2 expression in Calu-3 cells (Fig. 1E). However, bromelain reduces the expression of TMPRSS2 in Calu-3 (Fig. 1E) and ACE-2 negative normal bronchial epithelial (BEAS-2B) and lung adenocarcinoma (A549) cells (Fig. 1F and G). Cysteine protease inhibitor (E-64) treatment further confirmed that bromelain’s cysteine protease

Dear Editor, The new coronavirus, SARS-CoV-2, transmits rapidly from human-to-human resulting in the ongoing pandemic. SARS-CoV-2 infects angiotensin-converting enzyme 2 (ACE-2) expressing lung, heart, kidney, intestine, gall bladder, and testicular tissues of patients, leading to organ failure and sometimes death. 1,2 Currently, COVID-19 patients are treated with different agents, including favilavir, remdesivir, chloroquine, hydroxychloroquine, lopinavir, darunavir, and tocilizumab. 3,4 However, the safety and efficacy of those drugs against COVID-19 still need further confirmation by randomized clinical trials. Hence, there is an emergent need to repurpose the existing drugs or develop new virus-based and host-based antivirals against SARS-CoV-2. Bromelain is a cysteine protease isolated from pineapple stem and is used as a dietary supplement for treating patients with pain, inflammation, 5 thrombosis, 6 and cancer. 7 Recently, studies have shown that SARS-CoV-2 homotrimeric viral spike protein (S1) binds to the Transmembrane Serine Protease 2 (TMPRSS2) primed host cell's receptor ACE-2 for initial entry, followed by S2mediated membrane fusion. 8 Of several normal and cancerous cells tested, VeroE6 and Calu-3 cells showed ACE-2 protein expression (Fig. 1A), as well as a basal level of TMPRSS2 protein (Fig. 1B). Since ACE-2 9 and TMPRSS2 (UniProtKB-O15393) contains cysteine residues with disulfide bonds to stabilize the protein structure, we investigated the effect of bromelain on ACE-2 and TMPRSS2 expression. Bromelain-induced a dose-and time-dependent reduction of ACE-2 and TMPRSS2 expression in VeroE6 cells ( Fig. 1C and D) but do not alter ACE-2 expression in Calu-3 cells (Fig. 1E). However, bromelain reduces the expression of TMPRSS2 in Calu-3 ( Fig. 1E) and ACE-2 negative normal bronchial epithelial (BEAS-2B) and lung adenocarcinoma (A549) cells ( Fig. 1F and G). Cysteine protease inhibitor (E-64) treatment further confirmed that bromelain's cysteine protease  (Fig. 1H). Surface plasmon resonance (SPR) analysis revealed that purified SARS-CoV-2 S-ectodomain binds with ACE-2 in a concentration-dependent manner and has a comparable binding affinity as control RBD (Fig. 1I). The calculated molecular weight of the purified S-ectodomain-GFP protein is ∼165 kDa; however, we observed a higher molecular weight of S-ectodomain (∼215 kDa), which may be due to heavy N-and O-linked glycosylation (Fig. 1I intent). A serological assay showed a significantly increased median fluorescent intensity (MFI) of purified S-ectodomain with COVID-19 positive patients' samples (Fig. 1J). These two results indicated that purified S-ectodomain is a properly folded and functionally active protein.
The S-ectodomain has 30 cysteine amino acids with 15 stabilizing disulfide bonds (UniProtKB: P0DTC2) ( Fig. 2A). The RBD domain alone has nine cysteine residues, eight of which form four disulfide linkages. Bromelain-induced a dose-and time-dependent cleavage of S-ectodomain in Tni insect cell supernatant ( Fig. 2B and C) and purified S-ectodomain (Fig. 2D). Heat inactivation and cysteine proteinase inhibitor (E-64) treatment inhibited bromelain mediated digestion of S-ectodomain ( Fig. 2E and F). Further, SARS-CoV-2 with bromelain treatment showed the loss of Spike protein on the viral surface ( Fig. 2G). Our docking studies between homotrimeric (RBD containing chain A) S protein and stem bromelain revealed that bromelain cleaves the S-protein equally likely at the 131-166 (50.4%) and 617-649 (49.6%) disulfide bonds (Fig. 2H). Though the catalytic site is not directly engaged, protein-protein docking places the enzyme in close proximity to these bonds. These results demonstrate that bromelain's cysteine protease activity is responsible for the cleavage of host cells' ACE-2 and SARS-CoV-2 S-protein.
Since bromelain digested ACE-2 and S-ectodomain, we investigated the effect of bromelain on the interactions  of S-ectodomain and SARS-CoV-2 with VeroE6 cells. Bromelain significantly reduced the binding of S-protein to VeroE6 cells ( Fig. 3A and B) and was further confirmed by cysteine protease inhibitor (E-64) treatment (Fig. 3C). Interestingly, bromelain pre-treatment significantly decreased SARS-CoV-2 viral binding in VeroE6 cells (P = .0021) (Fig. 3D). Most importantly, VeroE6 cells or SARS-CoV-2 or both with bromelain reduces the viral infection ( Fig. 3E and F). Additionally, we found significantly reduced SARS-CoV-2 viral RNA copies in bromelain-treated VeroE6 (P = .0010) and Calu-3 (P = .0099) cells ( Fig. 3G and H, respectively). Collectively, these results suggest that bromelain could inhibit SARS-CoV-2 binding and infection in VeroE6 and Calu-3 cells. Studies have demonstrated that SARS-CoV-2 S-protein has high homology among other coronaviruses (76% identity with SARS-CoV) with conserved cysteine amino acids (UniProtKB: P59594). This indicates that bromelain may be used as a broad antiviral agent against SARS-CoV-2 and other related family members.
In conclusion, the currently used drugs against SARS-CoV-2 have potential side effects. Vaccine development is a significant risk factor of multiorgan failure and death in COVID-19 patients. 10 Since bromelain inhibits SARS-CoV-2 infection, and its profound fibrinolytic activity 6 suggests that bromelain or bromelain-rich pineapple could be used as an antiviral against SARS-CoV-2 and future outbreaks of other coronaviruses.

C O N F L I C T O F I N T E R E S T
Satish Sagar and Prakash Radhakrishnan have ownership interest (including patents) in a pending patent.

E T H I C S A P P R O VA L A N D C O N S E N T T O PA R T I C I PAT E
The institutional review board (IRB) of the University of Nebraska Medical Center approved clinical samples used in this study.