EVALUATION OF INHIBITORY ACTIVITY OF SARS-COV-2 ANTIVIRAL CANDIDATES USING DIMER-BASED SCREENING SYSTEM AND CHEMICAL CROSS-LINKING METHODS

• Main Author: Angelina Putri T., Audrey
• Format: Theses
• Language: Indonesia
• Subjects: Teknologi
• Online Access: https://digilib.itb.ac.id/gdl/view/84499
• Institution: Institut Teknologi Bandung

Previous research has demonstrated the capability of the Dimer-based Screening System (DBSS) as a high-throughput screening system targeting the SARS-CoV-2 proteins, specifically the C-terminal domain (CTD) of the nucleocapsid (N) protein. This screening relies on the compatibility of compound structure to the dimerization interface of the AraC-CTD N fusion protein. However, there is a possibility of false positives if the compound interacts with AraC. Therefore, another method is needed to confirm the interaction between the antiviral compound and the target protein, such as the chemical cross-linking method, which allows direct observation of the interaction between antiviral compounds and the target protein. This research aims to screen antiviral candidate compounds using in silico approach and the DBSS method, while also confirm the obtained result using the chemical cross-linking method. The antiviral candidates used in this research are curcumin, porphyrin, and carbon-nanodots (curcumin-dots and porphyrin-dots). In silico screening of the compounds was conducted using molecular docking with AutoDock Vina, while in vitro testing was conducted using the previously developed DBSS system for SARS-CoV-2 N CTD. The best compound from the molecular docking and DBSS tests was further confirmed for their interaction and dimerization inhibition capability against SARS-CoV-2 N CTD using the cross-linking method, with optimization of BS3 crosslinker concentration and incubation duration. The recombinant protein was obtained from the expression plasmid pET23a(+)_CTD N (~3950 bp). Visualization of the cross-linking results were carried out using SDS-PAGE and westernblotting. The dimerization inhibition ability of the antiviral compound was confirmed by adding various concentrations of the compound to the cross-linking reaction. The in silico results showed the compound with highest percentage of interaction with the dimerization residues was curcumin-dots (28.57%), followed by porphyrin (14.28%) and curcumin (14.28%). These results were supported by DBSS, which showed the compound with highest dimerization inhibition ability (relative fluorescence value) was curcumin-dots (3417.2 RFU), followed by porphyrin-dots (2523.1 RFU), curcumin (2377.9 RFU), and porphyrin (2059.7 RFU). Based on these results, curcumin-dots was further validated with cross-linking. The cross-linking method successfully dimerize SARS-CoV-2 N CTD upon the addition of the BS3 crosslinker with the presence of dimer protein band (~29 kDa) on SDS-PAGE and western blotting. Confirmation of the antiviral compound’s ability showed that the addition of curcumin-dots (1000, 1500, and 2000 ?g/mL) to cross-linking reaction successfully reduced the presence of dimer protein (~29 kDa) compared to the control (0 ?g/mL). It can be concluded that the molecular docking and DBSS methods successfully demonstrated the dimerization inhibition ability of the CTD N of SARS-CoV-2 for all antiviral candidate compounds, and chemical cross-linking successfully confirmed the ability of curcumin-dots to interact with and inhibit the dimerization of the CTD N of SARS-CoV-2.