HIV-1 PROTEASE DIMERIZATION INHIBITOR COMPOUND SCREENING SYSTEM DEVELOPMENT AND EXAMINATION

• Main Author: Dewa Agung Panji Dwipayana, I
• Format: Final Project
• Language: Indonesia
• Subjects: Ilmu hayati ; Biologi
• Online Access: https://digilib.itb.ac.id/gdl/view/27911
• Institution: Institut Teknologi Bandung

<p align="justify">In 2016, Indonesia had ± 630 thousand people with HIV (0.4%) and the number continues to grow. Unfortunately this problem is not balanced with the anti-HIV drugs (antiretroviral therapy / ART) penetration which only reaches 14% of the total patients. Expensive treatment cost also makes it difficult to control the HIV epidemics in Indonesia so that the search for new medicines is an important priority. Drugs from natural compounds can be a good alternative because Indonesia has so many natural compounds some of which may be a cheaper and more effective medicine. In the previous study, a dimer based screening system (DBSS) was developed to select new anti-HIV drug candidates. This system uses the fusion of AraC protein and HIV protease as a regulator and Green Fluorescent Protein (GFP) as the reporting gene. To be able to develop the system further, this study aims to optimize DBSS so it can be used and to test the ability of Darunavir and JH3 to inhibit the formation of HIV-1 protease dimers. The DBSS plasmid confirmation was done using PCR method and DNA sequencing. To observe how protein expression occurs in the transformant culture from the beginning of incubation until 18 hours of age, a growth curve is made and crude protein sample is taken from the culture once every three hours until it reached 18 hours of age. The growth curve was made by measuring the absorbance of the culture at OD600, then the crude protein samples were analyzed using the SDS-PAGE method. The inhibitory compound ability test were tested using Darunavir and JH3 with pDBD transformant was used as a positive control and pHxB transformant was used as a negative control. Both are grown without any compound addition. Darunavir test were performed at 5, 10, 15, 20, 25, 35, 45, 55, 85 and 100 ppm while JH3 test was carried out at 1, 5 and 10 ppm. PCR results showed the presence of ~ 1075 bp DNA band which based on its DNA sequencing results, was the result of amplification of the fusion protein coding gene. The results of SDS PAGE analysis on crude protein samples showed the presence of ~ 24.2 kDa band which is thought to be the fusion protein monomer. The growth curve and protein expression profiles showed that the 15-hour-old culture is thought to be the optimum age to be used in the system. Darunavir testing in the DBSS system showed a fluctuating flouresence increase compared to the negative control. Meanwhile, testing of JH3 compounds showed that it was also able to increase the flouresence value fluctuatively although not as high as the Darunavir-given culture. These results indicate that the DBSS system can already be used to test the ability of anti-HIV compounds and JH3 compound showed the ability to inhibit the HIV-1 protease dimer formation.<p align="justify">