Alpha and gamma mangostins inhibit wild-type B SARS-CoV-2 more effectively than the SARS-CoV-2 variants and the major target is unlikely the 3C-like protease.

BACKGROUND

Anti-SARS-CoV-2 and immunomodulatory drugs are important for treating clinically severe patients with respiratory distress symptoms. Alpha- and gamma-mangostins (AM and GM) were previously reported as potential 3C-like protease (3CL) and Angiotensin-converting enzyme receptor 2 (ACE2)-binding inhibitors .

OBJECTIVE

We aimed to evaluate two active compounds, AM and GM, from for their antivirals against SARS-CoV-2 in live virus culture systems and their cytotoxicities using standard methods. Also, we aimed to prove whether 3CL and ACE2 neutralization were major targets and explored whether any additional targets existed.

METHODS

We tested the translation and replication efficiencies of SARS-CoV-2 in the presence of AM and GM. Initial and subgenomic translations were evaluated by immunofluorescence of SARS-CoV-2 3CL and N expressions at 16 h after infection. The viral genome was quantified and compared with the untreated group. We also evaluated the efficacies and cytotoxicities of AM and GM against four strains of SARS-CoV-2 (wild-type B, B.1.167.2, B.1.36.16, and B.1.1.529) in Vero E6 cells. The potential targets were evaluated using cell-based anti-attachment, time-of-drug addition, 3CL activities, and ACE2-binding using a surrogated viral neutralization test (sVNT). Moreover, additional targets were explored using combinatorial network-based interactions and Chemical Similarity Ensemble Approach (SEA).

RESULTS

AM and GM reduced SARS-CoV-2 3CL and N expressions, suggesting that initial and subgenomic translations were globally inhibited. AM and GM inhibited all strains of SARS-CoV-2 at EC of 0.70-3.05 μM, in which wild-type B was the most susceptible strain (EC 0.70-0.79 μM). AM was slightly more efficient in the variants (EC 0.88-2.41 μM), resulting in higher selectivity indices (SI 3.65-10.05), compared to the GM (EC 0.94-3.05 μM, SI 1.66-5.40). GM appeared to be more toxic than AM in both Vero E6 and Calu-3 cells. Cell-based anti-attachment and time-of-addition suggested that the potential molecular target could be at the post-infection. 3CL activity and ACE2 binding were interfered with in a dose-dependent manner but were insufficient to be a major target. Combinatorial network-based interaction and chemical similarity ensemble approach (SEA) suggested that fatty acid synthase (FASN), which was critical for SARS-CoV-2 replication, could be a target of AM and GM.

CONCLUSION

AM and GM inhibited SARS-CoV-2 with the highest potency at the wild-type B and the lowest at the B.1.1.529. Multiple targets were expected to integratively inhibit viral replication in cell-based system.