Studies Of A High-throughput Screening Method For Identification Of HIV Fusion Inhibitors Targeting Gp41 And The Drug Screening From Natural Origin

An increasing portion of patients with HIV infection and/or AIDS can not use currently FDA-approved anti-HIV drugs, including the reverse transcriptase and protease inhibitors, due to the adverse effects and the emergence of drug resistance. Thus, it is essential to develop new anti-HIV agents with a target different from the HIV reverse transcriptase and protease.HIV gp41, a transmemberane envelope glycoprotein subunit, is responsible for HIV entry into cells by mediating fusion between viral and cellular membranes. Therefore, gp41 plays a crucial role in the early steps of viral entry into target cells and may serve as an important target for the development of anti-HIV compound. The N- and C-terminal heptad repeat (designated NHR and CHR, respectively) regions of gp41 extracellular domain can form a six-helix bundle by interaction, which brings both the viral and target cell membranes into proximity for fusion. Any compound, if it can inhibit the interaction of NHR and CHR as well as disturb the formation of six-helix bundle core structure, may block viral entry into target cells and protect the HIV infection of immunocytes Objective:To modify and improve a screening assay so that it becomes more convenient, economic and adaptable in China for high-throughput screening of HIV fusion inhibitors targeting gp41. Using this high-throughput screening method to screen HIV fusion inhibitors from complex sample, such as microorganism cultured broth, herbs, snake venoms and other natural products. Methods and Results: 1. Studies on a high-throughput screening method for identification of HIV fusioninhibitors targeting gp41The original screening method reported by Jiang et al (J Virol Methods. 1999;80:85-96) was modified by: 1) using a conformation-specific monoclonal antibody to replace a polyclonal antibody for coating plates; 2) simplifying the procedures; 3) using parts of the reagents produced in China. The modified screening assay is simpler, more convenient, and more economic than the original assay, but its sensitivity is comparable to and specificity is a little better than the original method. The optimal concentration for NC-1 coating is 2 ng/ml, for N36 and C34 are equimolor concentration into 1 wnol/ml, and for rabbit anti-gp41 IgGis 5 ng/ml. A pH range of 6~9 has no influence to the screening method. Organic solvent also has no influence if it was diluted to 10% (vol/vol) by water or ethanol. So, the modified screening assay can be used in China for high-throughput screening of HIV fusion inhibitors from complex sample, such as microorganism cultured broth, herbs and other natural products. 2. Drug screening from natural originA large number of samplesfron different origins, including 985 actinomyces fermentation samples, 9 Chinese herbs and 6 snake venoms, were screened for HIV fusion inhibitor targeting gp41. Among them, 118 actinomyces fermentation products, 2 Chinese herbs and 5 snake venoms can inhibit the gp41 six-helix bundle formation above 80%. 1). HIV fusion inhibitor screening from actinomyces originAmong the 118 actinomyces fermentation samples, 83 samples could inhibit gp41 six-helix bundle formation effectively, but did not block the mAb NC-1 binding to the pre-formed six-helix bundles. Six samples could inhibit the cell fusion and HIV-1 induced cytopathic effect (CPE), and the sample of 25-04 was the strongest. The strain of 25-04 is streptomyces hygnoscopicus. We selected a culture medium formula from 7 formulas for deep fluid fermentation, which is glucose 2%, starch 1%, peptone 0.5%, corn steep liquor 1.5%, NaCI 0.5%, CaCO3 0.5%, foam inhibitor 0.035%, pH 7.2-7.4. We found that the nitrogen source easy to use, such as (NH4)2SO4, can inhibit the production of active components. The deep fluid fermentation of 25-04 showed that the active components are mostly in cultured broth, but not in mycelium. The MW of the active components is above 3000. It is not stable by heating above 60癈 and in pH range out of 5-9. Protease inhibitors can make the active components lose its...