ABSTRACT
The COVID-19 endemic remains a global concern. The search for effective antiviral candidates is still needed to reduce disease risk. However, the availability of high biosafety level laboratory facilities for drug screening is limited in number. To address this issue, a screening system that could be utilized at lower biosafety levels remains essential. This study aimed to develop a novel SARS-CoV-2 main protease (Mpro) dimer-based screening system (DBSS) utilizing synthetic biology in Escherichia coli BL21(DE3). We linked the SARS-CoV-2 Mpro with the DNA-binding domain of AraC regulatory protein, which regulates the reporter gene expression. Protein modeling and molecular docking showed that saquinavir could bind to AraC-Mpro both in its monomer and dimer forms. The constructed DBSS assay indicated the screening system could detect saquinavir inhibitory activity at a concentration range of 4-10 µg/mL compared to the untreated control (P ≤ 0.05). The Vero E6 cell assay validated the DBSS result that saquinavir at 4-10 µg/mL exhibited antiviral activity against SARS-CoV-2. Our DBSS could be used for preliminary screening of numerous drug candidates that possess a dimerization inhibitor activity of SARS-CoV-2 Mpro and also minimize the use of a high biosafety level laboratory.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Saquinavir/pharmacology , Molecular Docking Simulation , Dimerization , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Protease Inhibitors/pharmacology , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Synthetic Biology , Molecular Dynamics SimulationABSTRACT
BACKGROUND: Since effective antiviral drugs for COVID-19 are still limited in number, the exploration of compounds that have antiviral activity against SARS-CoV-2 is in high demand. Porphyrin is potentially developed as a COVID-19 antiviral drug. However, its low solubility in water restricts its clinical application. Reconstruction of porphyrin into carbon dots is expected to possess better solubility and bioavailability as well as lower biotoxicity. METHODS AND RESULTS: In this study, we investigated the antiviral activity of porphyrin and porphyrin-derived carbon dots against SARS-CoV-2. Through the in silico analysis and assessment using a novel drug screening platform, namely dimer-based screening system, we demonstrated the capability of the antivirus candidates in inhibiting the dimerization of the C-terminal domain of SARS-CoV-2 Nucleocapsid. It was shown that porphyrin-derived carbon dots possessed lower cytotoxicity on Vero E6 cells than porphyrin. Furthermore, we also assessed their antiviral activity on the SARS-CoV-2-infected Vero E6 cells. The transformation of porphyrin into carbon dots substantially augmented its performance in disrupting SARS-CoV-2 propagation in vitro. CONCLUSIONS: Therefore, this study comprehensively demonstrated the potential of porphyrin-derived carbon dots to be developed further as a promisingly safe and effective COVID-19 antiviral drug.
ABSTRACT
Even entering the third year of the COVID-19 pandemic, only a small number of COVID-19 antiviral drugs are approved. Curcumin has previously shown antiviral activity against SARS-CoV-2 nucleocapsid, but its poor bioavailability limits its clinical uses. Utilizing nanotechnology structures, curcumin-derived carbon-dots (cur-CDs) were synthesized to increase low bioavailability of curcumin. In-silico analyses were performed using molecular docking, inhibition of SARS-CoV-2 nucleocapsid C-terminal domain (N-CTD) and antiviral activity were assessed in dimer-based screening system (DBSS) and in vitro respectively. Curcumin bound with the N-CTD at ΔG = -7.6 kcal/mol, however modifications into cur-CDs significantly improved the binding affinity and %interaction. Cur-CDs also significantly increased protection against SARS-CoV-2 in both DBSS and in vitro at MOI = 0.1. This study demonstrated the effect of post-infection treatment of curcumin and novel curcumin-derived carbon-dots on SARS-CoV-2 N-CTD dimerization. Further investigation on pre-infection and in-vivo treatment of curcumin and cur-CDs are required for a comprehensive understanding on the carbon-dots enhanced antiviral activity of curcumin against SARS-CoV-2.
