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^{pro}) and Angiotensin-converting enzyme receptor 2 (ACE2)-binding inhibitors in silico . Objective We aimed to evaluate two active compounds, AM and GM, from Garcinia mangostana 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^{pro} 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^{pro} 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, in vitro 3CL^{pro} 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^{pro} and N expressions, suggesting that initial and subgenomic translations were globally inhibited. AM and GM inhibited all strains of SARS-CoV-2 at EC 50} of 0.70–3.05 μM, in which wild-type B was the most susceptible strain (EC 50} 0.70–0.79 μM). AM was slightly more efficient in the variants (EC 50} 0.88–2.41 μM), resulting in higher selectivity indices (SI 3.65–10.05), compared to the GM (EC 50} 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^{pro} 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.
【저자키워드】 Target identification, 3C-like protease, COVID-19 drug, antiviral drug discovery, Alpha mangostin, Gamma mangostin, SARS-CoV-2 drugs,