The Haart Pharmacodynamics Study With Dolutegravir As The "Anchor" In Vitro And Natural Products’ Anti-HIV-1 Mechanism And Characteristics Study

Highly Active Antiretroviral Therapy (HAART) is the standard-of-care therapy of AIDS. It involves at least three drugs usage combination. Most antiretroviral therapy regiments list two nucleoside reverse transcriptase inhibitors (NRTIs) as the backbone with either a non-nucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor or an integrase inhibitor (INI) as the "anchor". Dolutegravir (DTG), an integrase inhibitor, was approved by FDA in 2013. Compared to the other INIs (Raltegravir and Elvitegravir), it has a favorable pharmacokinetic profile and a higher genetic barrier to viral resistance. This study was based on the first-line NRTIs recommenated in China and aimed to compare the pharmacodynamics of INIs (DTG or Raltegravir) with NNRTIs (Efavirenz or Nevirapine) as the "anchor". HIV-1RT-D67N,K70R,T215F and HIV-1IN-G140S,Q148H were resistant to NRTIs and INIs, respectively. We establised these two mutant HIV-1 replication models by using pseudotyped technique, i.e., a recombinant virus as env-deficient HIV-1 core packed with vesicular stomatitis virus glycoprotein replication model. The results showed that an INI as the "anchor" gave a stronger synergism against wild-type HIV-1 replication comparing to an NNRTI. Between the two INIs, DTG exhibited an equivalent CI to that of Raltegravir on wild type HIV-1 replication; but a greater synergy than Raltegravir against INIs-resistant HIV-1 replication. This result may provide experimental datas for clinical antiviral therapy in our country.Although HAART has dramatically increased the life expectancy of the HIV-infected individual, the healing efficacy is still impaired by poor drug tolerability and the resistant virus appearance after a period which leading to treatment failure. Therefore, novel anti-HIV drugs develepment is still greatly demanded. In this research, Salvia miltiorrhiza sourced compounds (NPB524, NPB527) and Tripterygium wilfordii Hook F sourced compounds (KET30, KET30C, KET31A and KET31C) were found as HIV-1 replication inhibitors by using VSVG/HIV-1 recombinant system. The results showed that NPB524 and NPB527 inhibited HIV-1 replication with IC50 at 1.6 nmol·L-1 and 0.38 μmol·L-1; KET30, KET30C, KET31A and KET31C exhibited anti-HIV activity with IC50 at 5.27 μmol·L-1,0.027 μmol·L-1,6.04 μmol·L-1 and 0.09 μmol·L-1, respectively.The study also researched the anti-HIV-1 mechanisms of NPB524 and NPB527. Firstly, we used Time-of-addition assay to identify the targets of compounds. However, comparing with FT50 of known drugs (AZT, EFV and RAL), both of these two compounds didplayed a longer failure time. This indicated that NPB524 and NPB527 might have a complex mechanism. They possibly act on a host factor. VSVG/HTV-1 recombinant system can only test for HIV-1 replication inhibitors including reverse transcriptase inhibitors, integrase inhibitors and transcription and translation inhibitors. We further tested the effect of the two compounds on reverse transcriptase activity, i.e., DNA polymerase activity and RNase H activity. The results showed that both NPB524 and NPB527 blocked HIV-1 reverse transcription by inhibiting its reverse transcriptase RDDP activity (IC50:70.6 μmol·L-1,63.5 μmol·L-1) and RNase H activity (IC50:49 μmol·L-1,26.2 μmol·L-1). The IC50 of NPB524 and NPB527 for enzyme modle were more than 69 times than that for cell modle. It indicated that reverse transcriptase was not the major target for NPB524 and NPB527. And we need further study to identify the major target for NPB524 and NPB527.In addition, the thesis researched the anti-HIV-1 functional characteristics by evaluating the effect of compounds on NNRTIs-resistant strains (HIV-1RT-k103N and HIV-1RT-Y181C). The results showed NPB524 and NPB527 exhibited equivalent inhibitory activity against HTV-1RT-k103N and HIV-1RT-Y181C with that of wild-type HIV-1, but stronger than that of NVP.