Study On The Mechanism And Function Of Anti - HIV Effect Of Marine Natural Products And The Study Of The Effect Of Ribonuclease H Activity On Anti - HIV Drugs In Vitro

Drug resistance is an obstacle for anti-retroviral therapy. Resistant viruses appear and accumulate after a certain time of therapy. Therefore, there is still a strong demand for new drugs development to the current resistant viruses. Reverse transcriptase plays a key role in HIV reverse transcription process, which is the most important target for anti-HIV therapy. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) play their roles by binding to the hydrophobic pocket close to the catalytic sites which leads to the active conformation change and the DNA polymerase activation. Marine sourced bioactive substances are an important part of natural source drugs. Abundant marine species have a number of various metabolic pathways which offer tons of structural unique compounds. Seeking from marine sourced compounds will provide us a clue of novel structural NNRTIs which may benefit to resistant viruses carrying patients.In this study over200marine sourced compounds were screened by VSVG/HIV recombinant viral replication system. We found an active compound GQQ-25, which could inhibit HIV-1replication with IC50of8.8μmol·L-1. Through adding the active compound at different time points after virus infection to detect failure time of compound (time of addition, TOA), the results showed GQQ-25act on the HIV reverse transcription step. By using ELISA and fluorescence method to detect the compound’s effect on RNA dependent DNA polymerase (RDDP) and RNase H activity of reverse transcriptase. The results showed that GQQ-25could inhibit RDDP activity with IC50of50μmol·L-1. Further study exhibited that GQQ-25could block replication of NNRTI resistent strains (HIV-1RT-K103N, HIV-1RT-L1001,K103N, HIV-1RT-K103N,V108I, HIV-1RT-K103N,G190A, and HIV-1RT-K103N,P225H) with IC50values of7.0,23.8,13.3,14.2,6.2,8.4μM, which was as good as HIV-1RT-WT (8.8μM). Molecular docking (SYBYL software) data showed that GQQ-25bind to RT hydrophobic pocket. It interacted directly with Tyr181and Tyr188by H-n force, but not with the Lys103. This could explain why GQQ-25are effective to HIV carrying RTK103N mutation, but not RTy181C or RTY188L.HIV-RT is a multi-functional enzyme. It includes an RNA-dependent DNA polymerase and a DNA-dependent DNA polymerase, which work together to perform transcription. In addition to DNA polymerase activity, it also has a RNase H activity to hydrolyze RNA. The completion of reverse transcription need the coordination of the two enzymatic activities. The FDA approved nucleoside and non-nucleoside reverse transcriptase drugs are indispensable in drug combination of anti-retroviral therapy. They block HIV-1replication by inhibiting the activity of polymerase. This study used HIV RNase H active sites inhibitor (2-hydroxyisoquinoline-1,3-dione, HQD) and RNase H activator (oligodeoxynucleotide, ODN) combined with FDA approved NRTIs and NNRTIs to test their interaction(synergism, addition or antagonism) on anti-HIV activity.