| 其他摘要 | The replication of HIV-1 in infected patients can be reduced considerably by treatment with combinations of drugs with multiple anti-viral drugs. But none of currently these available drug or combinations could eradicate HIV-1 from patients completely. The long-term clinical effectiveness of approved anti-HIV drugs has been hampered by the ascendance of drug-resistant mutants in response to antiretroviral therapies. The rates of success of HAART are predicated to decrease gradually with the increase in the emergence of drug resistant strains. Therefore, it is essential to develop drugs targeting alternative steps of the viral replication cycle. Chondroitin sulfate is a sulfated glycosaminoglycan (glycosaminoglycans, GAG), which exists in many animal tissues and is abundant in many marine organisms, a number of studies show that the compound can anti-clotting, anti-tumor, anti-virusa, including the inhibition of HIV. FuCS-1 was synthesized based on the structure of thenenatan, a compound purified from Thelenata ananas. The cytotoxicities of FuCS-1 on C8166、H9、Hela、Jurkat、K562、MT-4、PBMC and H9/HIV-1ⅢB were assessed by MTT and the results manifested that FuCS-1 don’t show cytotoxicity to cultured cells even 25mg/mL. Anti-HIV activities of FuCS-1 were evaluated in the present study. The activities of FuCS-1 against 3 groups of human immunodeficiency virus (HIV) stains were determined in vitro. These were laboratory-derived virus (HIV-1IIIB and HIV-1RF), drug-resistant virus NNRTIs-resisant strain (HIV-1A17), PIs-resisant strain (HIV-1RF/V82F/I84V), T-20 sensitive strain (pNL4-3gp41(36G)N42S) and T-20 resistant strains pNL4-3gp41(36G)V38E,N42S, pNL4-3gp41(36G)V38A,N42D, pNL4-3gp41(36G)V38A,N42T) , pNL4-3gp41(V38E/N42S) and low-passage clinical isolated virus (HIV-1KM018 and HIV-1TC-2). In p24 antigen inhibition test, the EC50s of FuCS-1 against HIV-1IIIB and HIV-1RF infection in C8166 cells were 0.73 and 1.14μg/mL, respectively, and with selective index of >32426.57and >21929.82. FuCS-1 inhibit HIV-1KM018 and HIV-1TC-2 infection in PBMC with EC50 23.75 and 31.86μg/mL , SI >1052.63 and 784.68 respectively; FuCS-1 can against many kinds of HIV drug-resistant virus including NNRTIs-resisant strain (HIV-1A17), PIs-resisant strain (HIV-1RF/V82F/I84V) with EC50s1.09, 0.95μg/ml, respectively, T-20 sensitive strain (pNL4-3gp41(36G)N42S) and T-20 resistant strains pNL4-3gp41(36G)V38E,N42S, pNL4-3gp41(36G)V38A,N42D, pNL4-3gp41(36G)V38A,N42T) with EC50s 0.79, 0.94, 0.76, 1.13μg/mL, respectively, while the control drug T-20 only can inhibt the sensitive strain replication with EC50 226.16μg/mL but can’t inhibt the resisant strains at the highest concentration 1000ng/mL. FuCS-1 also can against HIV-2 CBL-20 and HIV-2R0D with EC50s 71.76μg/mL and 97.63μg/mL, respectively. All of these results indicatied that FuCS-1 has good anti-HIV activities in vitro. It not only can against laboratory-derived virus, but also can inhibit drug-resistant virus, clinical isolated virus and HIV-2. So we examine FuCS-1’s the mechanism of anti-HIV. The results show can’t inhibit HIV-1 protentase but can inhibit activities of recombinant HIV-1 reverse transcriptase weakly. FuCS-1 effectively inhibited HIV-1 replication at the early stages, blocked the fusion between normal cells and HIV-1 chronically infected cells and inhibit HIV virus enter thethe target cells. Further studies indicatied that FuCS-1 can bind to the recombinant HIV-1 gp120 protein potently. The interaction between FuCS-1 and gp120 interfered gp120 functions，so it can effectively inhibit HIV enter the the target cells. Indole is a natural substance with a number of biologically activities. Indole compounds can anti-inflammatory, antitumor, antibacterial, antiviral, anti-HIV and so on. So one of important direction of development new active substances is modificating the indoles. Twelone N-arylsulfonyl-3-acetylindoles (4a-4u) anti-HIV-1 activites were evaluated in the present study. The verified that there are several compounds inhibited HIV-1IIIB induced cytopathic effects (syncytia assay) in C8166 cell. Especially N-p-ethyl-benzenesulfonyl-3-acetyl-6-methylindole had the highest anti-HIV-1 activity with EC50 values of syncytium formation inhibition is 0.38 μg/mL and SI values is >543.78. The N-p-ethyl-benzenesulfonyl-3-acetyl-6-methylindole with the best activity deserved to be further study. The potential cell toxicity of N-p-ethyl-benzenesulfonyl-3-acetyl-6-methylindole to human peripheral blood mononuclear cells (PBMCs) and other cell lines was evaluated. The results showed that it was not significantly cytotoxic to these cells. In the cellular level, by observing cytopathic effects assay and ELISA method for HIV p24 antigen assay, compound was tested a panel of different HIV-1 strains, including laboratory-derived viruses (HIV-1IIIB and HIV-1RF), clinical isolated viruses (HIV-1KM018, HIV-1TC-1, HIV-1TC-2 and HIV-1wan) and drug-resistant viruses (HIV-1A17, HIV-1 RF/V82F/I84Vand pNL4-3gp41(36G)V38E,N42S), the compound also showed good anti-HIV-1 effect on these different HIV-1 strains, which exhibited potent and broad-spectrum anti-HIV-1 activities, as well as on the HIV-2 CBL-20. The mechanism of action in inhibiting HIV-1 by N-p-ethyl-benzenesulfonyl-3-acetyl-6-methylindolewas tested through a time-of-addition experiment, chronically infected H9 and uninfected C8166 cell fusion inhibition assay. The compound can inhibit HIV in 0-12 hour after compound interfering but did not interfere with virus fusion to target cells. This compound exerted its active mechanism through blocking the production of virus from chronically infected cells containing integrated proviral DNA.These results suggest its key mechinsm of N-p-ethyl-benzenesulfonyl-3-acetyl-6-methylindole is inhibition HIV-1 replication at the early stages. Furthermore, Mechanistic studies revealed that it can inhibit HIV-1 reverse transcriptase and HIV protease, but N-p-ethyl-benzenesulfonyl-3-acetyl-6-methylindole can’t inhibit HIV-1 integrase nuclear translocation. The structure-activity relationship (SAR) of these 21 compounds is also surveyed. It was found that antivities of the compounds with the electron- drawinggroups substituted on indole ring are not better than the compounds with electron-withdrawing groups, such as N-benzenesulfonyl-3-acetyl-6-methyl indoles (e.g., 4h and 4r vs 4j, 4g and 4q vs 4k, 4i and 4p vs 4j). The SAR research will pave the way for the design of indole derivatives as anti-HIV-1 drugs in the future. |
修改评论