The replication of HIV-1 in infected patients can be reduced considerably by treatment with combinations of drugs with multiple viral drugs. But none of currently 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. For many decades, the discovery of drugs has depended on screening. Traditional screening was labor intensive and expensive. More and more high-throughput methods were developed and enabled to screening large pool of compounds. In this research, we expressed and purified HIV-1 protease. A cell-free screening method based on FRET was established. Another HIV-1 viral protein, NCp7, was also expressed and purified in this research. A method to test zinc ejection activity of compounds by TSQ zinc ion indicator was developed. Both methods are amenable to high-throughput screening. A large pool of samples, either natural products or synthesized compounds, were examined and some of them are found to inhibit protease or NCp7 in vitro. Structural modification to improve activity of an identified lead molecular is a cost effective way for drug discovery. A β-carboline compound, flazin isolated from Svillus granvlatvs has been shown weak anti-HIV-1 activity. Based on the structure of flazin, dehydroxymethylflazinamide (1-(2’-furyl)-β-carboline-3- formamide) and flazinamide [1-(5’-Hydromethyl-2’-furyl)-β-carboline-3- carboxamide] was synthesized and their anti-HIV activities were evaluated in the present study. The cytotoxicity of dehydroxymethyl- flazinamide was similar with that of flazin and cytotoxicity of flazinamide was about 4.1-fold lower. Dehydroxymethylflazinamide and flazinamide potently reduced syncytium formation induced by HIV-1IIIB with EC50 values of 0.31 μM and 0.38 μM, about 7-fold lower than that of flazin. Dehydroxymethylflazinamide and flazinamide also inhibited HIV-2ROD and HIV-2CBL-20 infection. Dehydroxymethylflazinamide and flazinamide reduced p24 antigen expression in HIV-1IIIB acute infected C8166 and in clinical isolated strain HIV-1KM018 infected PBMC. Neither suppressed HIV-1 replication in chronically infected H9 cells. Both of them blocked the fusion between normal cells and HIV-1 or HIV-2 chronically infected cells. They did not show virucide or attachment inhibitory activities. Dehydroxymethylflazinamide inhibited HIV-1 entry with EC50 value of 1.08 μM. Dehydroxymethylflazinamide and flazinamide weakly inhibited activities of recombinant HIV-1 reverse transcriptase at higher concentrations. Dehydroxymethylflazinamide reduced protease activity and flazinamide bound to intergrase in vitro. The structural modification of flazin markedly enhanced the antiviral activity. TI value of dehydroxymethyl- flazinamide increased from 12.1 to 88.19 and flazinamide to 312.2. In despite of structural similarity, they may possess distinct action mechanisms. Dehydroxymethylflazinamide might target entry step and flazinamide might interfere other steps in the early stage of HIV life cycle.
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