APLIKASI DIMER-BASED SCREENING SYSTEM (DBSS) DALAM MENYELEKSI KANDIDAT OBAT ANTI-HIV

• Main Author: Tsurayya, Nur
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/43314
• Institution: Institut Teknologi Bandung

In 2017, there were 631,635 patients with HIV in Indonesia. However, the high price of anti-HIV drugs (antiretroviral therapy, ART) on the market today, causes only 14.4% of the total HIV sufferers in Indonesia to receive treatment. On the other hand, HIV resistant strains to some ART have been found and the prevalence of this resistance tends to continue to increase. Therefore, the attempts to find new and effective anti-HIV drugs is an important thing to do. This study aims to select potential compounds for anti-HIV drugs using the DBSS (Dimer-based Screening System) method that has been developed and optimized in previous studies. This selection method uses fusion of DNA-binding domain AraC protein and HIV-1 protease as a regulator and Green Fluorescent Protein (GFP) as the reporting gene. The application of DBSS consists of 3 stages: confirmation of the DBSS plasmid (DBDAraC-Protease HIV-1 pRSET) through amplification by PCR and sequencing, confirmation of protein expression using the SDS-PAGE method, and testing with DBSS. The test results are then further analyzed by molecular docking and analysis of protein-ligand chemical interactions. Testing using the DBSS method was carried out on nine test compounds. In this test, Escherichia coli strain BL21 (DE3) + pRSET DBDAraC-Protease HIV-1 treated using organic solvent DMSO (Dimethyl Sulfoxide) acts as a baseline. BL21 (DE3) + DBDAraC pRSET, which is a recombinant protein that still has a regulatory protein domain but does not have a dimerized domain, used as positive control. As a comparison, darunavir, an active compound comercially used in the treatment of HIV, is used. Compound testing was carried out at concentrations of 2, 4, 6, 8, 10 ppm. PCR analysis showed the presence of ~ 1057 bp DNA bands which the fusion-protein coding genes. SDS PAGE analysis showed a ~ 24.2 kDa band which was thought to be a fusion-protein monomer. From DBSS testing of 9 compounds, there were 8 compounds that showed an increase in fluorescence value relative to negative controls. Three of the compounds named CAAM, CAMA, and CMMM showed fluorescence which was always higher than negative controls at every concentration (2, 4, 6, 8, 10 ppm). Meanwhile, 5 other compounds named CAMM, CMAA, CMMA, CMAM, and CMMA only showed positive results at certain concentrations. Overall, the highest increase in fluorescence was obtained from the treatment using CAMA compounds with a concentration of 8 ppm which gave a fluorescence value of 2.5 times compared to baseline. Based on the results of this study, it can be concluded that there are several compounds that potential to become anti-HIV drugs. However, further research is still needed to determine the effective concentration of each of these compounds.