Screening Of HIV-1 Entry Inhibitors And The Mechanism Study Of T-20

BACKGROUNDAcquired Immune Deficiency Syndrome (AIDS) was caused by human immunodeficiency virus (HIV-1). According to the UNAIDS/WHO report in 2009, an estimated 33.4 million people were living with HIV infection. It is also estimated that 2.7 million people became newly infected with HIV, and 2.0 million people died of AIDS-related illnesses in 2007. Nowadays, there are about 0.7 million HIV-1 infected people in China. The HIV-1 infection becomes a growing popular trend in many of provinces in our country, such as Yun-Nan, He-Nan, Xin-Jiang and Guang-Dong. From the statistical data in World Health Organization, the ratio of HIV-1 infection people to total people in our country is very low, but the real number of HIV-1 infected people is the second in Asia and the fourteenth in the world. This phenomenon became a serious social question in our life style.It is well known that developing vaccine is the best HIV-1 prevention strategy in all methods. But there is no effective HIV-1 vaccine in clinical treatment. Therefore, it is an important means to develop effective and safe drugs in treating HIV/AIDS. To this day, there are 28 kinds of anti-HIV drugs approved by FDA. These drugs were divided into three classes, such as reverse transcriptase inhibitors, protease inhibitors and HIV-1 entry inhibitors. But reverse transcriptase inhibitors and protease inhibitors could develop the anti-HIV-1 drug resistance mutations easily and showed strong adverse effect to human bodies. For these reasons more and more people could not tolerate these two group drugs. However, HIV-1 entry inhibitors, T-20 and Maraviroc, showed their anti-HIV activities to resistant-virus caused by reverse transcriptase inhibitors and protease inhibitors. It is a hot field to develop new anti-HIV drugs in these days.High-throughput drug screening is a hot strategy in current new-drug finding field. Since 1990s, Dr. Jiang found the first HIV-1 entry inhibitor which was derived from HIV-1 gp41 CHR. Our laboratory established a series of methods to screening HIV-1 entry inhibitors and found many active compounds targeting to gp41 or gp120.HIV-1 can cause serious infection, therefore, the researches on real virus could be only limited in P3 laboratory. So it is of great significance to identify the screening methods applying for common biological laboratory.The molecular weight of Fuzeon (also known as T-20 or enfuvirtide) is more 4000 dalton. It is easy degradated by endogenous protease. It can not be taken orally and can only be injected. It was too expensive for HIV-1 patients. So there is a hot research to find new small molecular drugs.In our previous studies, we found that even though it overlaps C34 sequence with 24 amino acids, T-20 has a different mechanism of action from C34. The interaction of T-20 with viral NHR region alone may not prevent the formation of the fusion active gp41 core. T-20-mediated anti-HIV activity can be significantly abrogated by peptides derived from the membrane-spanning domain in gp41 and coreceptor binding site in gp120. But the key sequence of interaction between T-20 and gp120 CXCR4 binding site is not known. Also it is not known whether T-20 can interact with the gp120-CD4 coreceptor binding site. Further elucidation of the mechanism of action of T-20 will provide new target(s) for development of novel HIV entry inhibitors. According to our laboratory research experience in HIV entry inhibitors, I focused on three projects in the present study. (1) Identify a method for for screening of HIV-1 fusion inhibitors and Tat-LTR interaction blockers by sequential application ofβ-galactosidase-and syncytium-based cell fusion assays. (2) Screening native source anti-HIV-1 compounds. (3) Identify the mechasim of T-20. For the first project, I intended to do following works:Using HL2/3 cells and Hela-CD4-LTR/β-gal cells as effector or target cells, respectively. Theβ-galactosidase staining kit was used to test the expression ofβ-galactosidase. At the same time, CHO/WT cells and MT-2 cells were selected as another pair of effector-target cells. Through the two systems, I can screen the HIV-1 entry inhibitor and Tat-LTR blocker rapidity. For the second project, I intended to do the following works:Screening the anti-HIV-1 activities and the mechanism of action of myriceric acid B and C isolated from Rhoiptelea chiliantha Diels et Hand.-Mazz, caffeoyl glucose compounds from Balanophora japonica Makino and Betulinic acid-derivated compounds. For the third project, I intended to do the following works:Synthesizing the serial peptides which overlap gp120 sequence from residue 409 to residue 431. Finding the key motif which could eliminate the activity of T-20 caused by gp120 co-receptor binding site.OBJECTIVES:a) Viral envelope glycoprotein (Env)-mediated membrane fusion and Tat-LTR interaction are two critical steps of HIV-1 entry and replication. In this experiment we want to develop a novel strategy to screen anti-HIV-1 agents that block HIV-1 fusion and Tat-LTR interaction by sequential use ofβ-galactosidase(gal)-and syncytium-based cell fusion assays.b) Establish the HIV-1 Env pseudovirus cell model to screen small molecular compounds. Try to clarify the detail mechanism of small molecular compounds.c) To identify the detail anti-HIV-1 mechanism of action of T-20.METHODS:a) HL2/3 Cells and Hela-CD4-LTR/β-gal cells were act as effector or target cells, respectively. Theβ-gal expressing cells were revealed by using aβ-gal staining set.b) Establish the system of testing HIV-1 pseudovirus in cell level:The plasmids encoding envelope proteins of HIV-1 (pHXB2) and VSV (pVSV-G) were cotransfected 293T cells with pNL4-3.Luc.R-E-to produce HIV-1 Env pseudovirus and VSV-G pseudovirus, respectively, which were used for testing the antiviral activities of these compounds. U87.CD4.CXCR4 cells were act as target cells; ELIS A and molecular docking were used to study the mechanism of action of the active compounds, such as Myriceric acid B derivatived from Rhoiptelea chiliantha Diels et Hand.-Mazz, caffeoyl glucose compound derived from Balanophora japonica Makino and Betulinic acid-derivated compounds.c) Synthesize serial peptides which overlap gp120 sequence from residue 409 to residue 431. Try to find the key motif which could eliminate the activity of T-20 caused by gp120 co-receptor binding site and the sequence of T-20-gp120 interaction. Construct HIV-1 Env pseudovirus to test the activity of T-20.RESULTS:a) Coculture of HeLa-CD4-LTR-B-gal cells with HL2/3 cells resulting in expression ofβ-galactosidase. To develop a B-gal-based cell-cell fusion assay, we selected HeLa-CD4-LTR-β-gal cells as the target cells and HL2/3 cells as the effector cells. HL2/3 cells also express HIV-1 Env and Tat proteins. After fusion of HeLa-CD4-LTR-B-gal cells with HL2/3 cells, the LTR-linkedβ-gal gene could be activated by the Tat protein in HL2/3 cells, resulting in expression of B-galactosidase in the fused cells which become visible after B-gal staining. A number of syncytia were revealed in the coculture of CHO-WT and MT-2 cells, while no syncytium was seen when CHO-WT and MT-2 cells were cultured separately. All of the three HIV-1 fusion inhibitors, T-20, C34 and ADS-Jl could inhibited, in dose-dependent manner, both B-gal-based cell fusion between HeLa-CD4-LTR-B-gal cells and HL2/3 cells and syncytium-based cell fusion between CHO-WT and MT-2 cells. However, two inhibitors of Tat-LTR interaction, curcumin and chlorpromazine, could only effectively inhibit B-gal-based cell fusion between HeLa-CD4-LTR-B-gal cells. Their IC50 values were 31.7 and 54.9μM, respectively.b) Myriceric acid B could significantly inhibit the infection of HIV-1 Env pseudovirus with an IC50 of 8.3±0.2μg·ml-1. The carbonoxyl group at C-28 position and the hydroxyl group at the C-3 position of myriceric acid B are important for its anti-HIV-1 activity. Like other HIV-1 entry inhibitors targeting gp41 (e.g., ADS-J1 and NB-64), myriceric acid B could also block the gp41 six-helix bundle formation. Molecular docking analysis suggests that myriceric acid B may bind to the hydrophobic cavity of the gp41 N-trimeric coiled coil. TCGP and DCGGP could inhibit HIV-1 Env pseudovirus infection. The IC50 values of these two compounds were 5.5±0.2μg·ml-1 and 5.3±0.1μg·ml-1 respectively. These two compounds could also block the gp41 six-helix bundle formation. They might bind to the hydrophobic cavity of the gp41 N-trimeric coiled-coil. Betulinic acid-derivated compounds, LY7 and LY14, can inhibit the HIV env pseudovirus entry. The value of IC50 is 7.96±1.54μg·ml-1 and 5.67±0.40μg·ml-1, respectively.c) MN-6312, MN-6313, SW-1012 and SW-1013, which which derived from gp120, can block the activity of T-20. Biotin-6312 can not interact with gp41-derived peptides. MN-6312, MN-6313, SW-1010 and SW-1012, which derived from gp120, are able to increase the HIV-1 Env pseudovirus infectivity on target cells. But they can not enhance the infection abilities of VSV-G pseudovirus.CONCLUSIONS:a) The cells used in both cell-cell assays are non-infectious and can be used in a general biology laboratory. This approach has great potential for rapid identification of HIV-1 fusion inhibitors and/or Tat-LTR interaction blockers.b) Myriceric acid B, TCGP, DCGGP, Betulinic acid-derivated compounds, LY7 and LY14, showed good activities of anti-HIV pseudovirus. They are potent HIV-1 entry inhibitors and can serve as a lead compound for developing novel anti-HIV-1 drug.c) T-20 can not interact with gp120. The peptides derived from gp 120 are able to enhance HIV-1 Env pseudovirus infection to target cells.