Envelope conformational changes induced by human immunodeficiency virus type 1 attachment inhibitors prevent CD4 binding and downstream entry events.

BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes.

A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and inhibits CD4 receptor binding.

BMS-378806 is a recently discovered small molecule HIV-1 inhibitor that blocks viral entrance to cells. The compound exhibits potent inhibitory activity against a panel of R5-(virus using the CCR5 coreceptor), X4-(virus using the CXCR4 coreceptor), and R5/X4 HIV-1 laboratory and clinical isolates of the B subtype (median EC50 of 0.04 microM) in culture assays. BMS-378806 is selective for HIV-1 and inactive against HIV-2, SIV and a panel of other viruses, and exhibits no significant cytotoxicity in the 14 cell types tested (concentration for 50% reduction of cell growth, >225 microM). Mechanism of action studies demonstrated that BMS-378806 binds to gp120 and inhibits the interactions of the HIV-1 envelope protein to cellular CD4 receptors. Further confirmation that BMS-378806 targets the envelope in infected cells was obtained through the isolation of resistant variants and the mapping of resistance substitutions to the HIV-1 envelope. In particular, two substitutions, M426L and M475I, are situated in the CD4 binding pocket of gp120. Recombinant HIV-1 carrying these two substitutions demonstrated significantly reduced susceptibility to compound inhibition. BMS-378806 displays many favorable pharmacological traits, such as low protein binding, minimal human serum effect on anti-HIV-1 potency, good oral bioavailability in animal species, and a clean safety profile in initial animal toxicology studies. Together, the data show that BMS-378806 is a representative of a new class of HIV inhibitors that has the potential to become a valued addition to our current armamentarium of antiretroviral drugs.

Overexpression of human genes in Drosophila melanogaster by using GAL4 UAS system.

Many human genes determined by genomic sequencing have only few information about their functions. To fill this knowledge gap, the powerful Drosophila genetics was set as a model to elucidate human gene functions effectively. By using germline transformation together with GAL4-UAS system, we studied the possibility of expressing and functionally characterization of human genes in Drosophila. Fifty-four transgenic fly lines corresponding to 10 human genes have been established. When expressed individually by crossing to an array of 6 different GAL4 driver lines, one of these genes, the translation elongation factor 1 alpha 1 (EF1 alpha-1), resulted in abnormal notum and rough eye phenotypes. This study implies the feasibility of systematically screening human gene functions by overexpression in Drosophila.

[Effect of tumor necrosis factor alpha and beta on ionizing radiation-induced apotosis].

Present paper described the effect of human tumor necrosis factor alpha (hTNFalpha) and beta (hTNFbeta) on apoptosis induced by ionizing radiation in human embryonic lung cell line 2BS diploid cells and human lung cancer cell line A549 and SPC cells. The results showed that both hTNFalpha and hTNFbeta significantly inhibited 7Gy gamma-ray-induced apoptosis in 2BS cells. In contrast, hTNFalpha or hTNFbeta can increase the sensitivity of tumor cell lines (A549 and SPC cells) to radiation. Therefore, the results suggested that TNFalpha and TNFbeta had potential as a therapeutic agent to protect normal cell from the radiation-induced apoptosis and to sensitize the tumour cells for the damage effect of ionizing radiation.

Aggregation of the Intracellular Domain of the Human Thrombopoietin Receptor c-MPL.

Thrombopoietin (TPO) is the major cytokine which is involved in platelet production and exerts its effects via the receptor c-MPL. The yeast two-hybrid system has been used to study the aggregation of the intracellular domain of c-MPL in TPO signal transduction. First, the cDNA fragment of MPLP intracellular domain was amplified and cloned by using RT-PCR method from the total RNA of HEL cells. Then the cDNA fragment was sequenced and subcloned into two-hybrid vectors pAS2 and pGAD424, respectively, and the recombinants are named as pASMM and pGADMM. Co-transformation of these plasmids into yeast activated his3 and lacZ reporter genes, demonstrating in vivo interaction of the MPLP receptor intracellular domain itself. The aggregation may be important in TPO signal transduction.

Cloning and Expression of a Fused Gene Encoding TNFRI Death Domain and Chloramphenicol Acetyltransferase.

By designing four primers for an overlapping PCR, we created a fusion gene Ddlcat encoding human TNF receptor I death domain and chloramphenicol acetyltransferase (CAT). By DNA sequencing, the whole sequence of the fusion gene is confirmed to be correct. Two hours after induction with IPTG, we could see a 39 kD extra protein band on SDS-PAGE pattern. We proved that this 39 kD protein band is DdLcat protein by Western blotting. Then we purified this protein to the purity of 95% through Q-Sepharose chromatography.

High Expression of a cDNA Coding for Human Lymphotoxin alpha Derivative in E. coli and Purification of Its Protein Product.

Using PCR, a cDNA coding for human lymphotoxin alpha derivative (hLTalphaDa) lacking 27 amino acids at N-terminal of natural hLT was constructed. The expression construct was expressed in E. coli BL21 (DE3). The product of expression was in the form of inclusion bodies, and accounted for 60%-80% of total bacterial proteins. The product protein was purified to over 95% by treatments of inclusion bodies. The specific activity of hLTalphaDa was above 2x10(8) IU(per mg protein). Its cytotoxic activity can be neutralized by monoclone antibody against hLT. The anti-tumor effects of hLTalphaDa were also tested in vitro and in vivo.