Discovery of small-molecule human immunodeficiency virus type 1 entry inhibitors that target the gp120-binding domain of CD4

The interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and the CD4 receptor is highly specific and involves relatively small contact surfaces on both proteins according to crystal structure analysis. This molecularly conserved interaction presents an excellent opportunity for antiviral targeting. Here we report a group of pentavalent antimony-containing small molecule compounds, NSC 13778 (molecular weight, 319) and its analogs, which exert a potent anti-HIV activity. These compounds block the entry of X4-, R5-, and X4/R5-tropic HIV-1 strains into CD4(+) cells but show little or no activity in CD4-negative cells or against vesicular stomatitis virus-G pseudotyped virions. The compounds compete with gp120 for binding to CD4: either immobilized on a solid phase (soluble CD4) or on the T-cell surface (native CD4 receptor) as determined by a competitive gp120 capture enzyme-linked immunosorbent assay or flow cytometry. NSC 13778 binds to an N-terminal two-domain CD4 protein, D1/D2 CD4, immobilized on a surface plasmon resonance sensor chip, and dose dependently reduces the emission intensity of intrinsic tryptophan fluorescence of D1/D2 CD4, which contains two of the three tryptophan residues in the gp120-binding domain. Furthermore, T cells incubated with the compounds alone show decreased reactivity to anti-CD4 monoclonal antibodies known to recognize the gp120-binding site. In contrast to gp120-binders that inhibit gp120-CD4 interaction by binding to gp120, these compounds appear to disrupt gp120-CD4 contact by targeting the specific gp120-binding domain of CD4. NSC 13778 may represent a prototype of a new class of HIV-1 entry inhibitors that can break into the gp120-CD4 interface and mask the gp120-binding site on the CD4 molecules, effectively repelling incoming virions.     

Contribution of C/EBP proteins to Epstein-Barr virus lytic gene expression and replication in epithelial cells

The contribution of C/EBP proteins to Epstein-Barr virus (EBV) lytic gene expression and replication in epithelial cells was examined. Nasopharyngeal carcinoma cell lines constitutively expressed C/EBPbeta and had limited C/EBPalpha expression, while the AGS gastric cancer cell line expressed significant levels of both C/EBPalpha and C/EBPbeta. Induction of the lytic cycle in EBV-positive AGS/BX1 cells with phorbol ester and sodium butyrate treatment led to a transient stimulation of C/EBPbeta expression and a prolonged increase in C/EBPalpha expression. In AGS/BX1 cells, endogenous C/EBPalpha and C/EBPbeta proteins were detected associated with the ZTA and oriLyt promoters but not the RTA promoter. Electrophoretic mobility shift assays confirmed binding of C/EBP proteins to multiple sites in the ZTA and oriLyt promoters. The response of these promoters in reporter assays to transfected C/EBPalpha and C/EBPbeta proteins was consistent with the promoter binding assays and emphasized the relative importance of C/EBPs for activation of the ZTA promoter. Mutation of the oriLyt promoter proximal C/EBP site had little effect on ZTA activation of the promoter in a reporter assay. However, this mutation impaired oriLyt DNA replication, suggesting a separate replication-specific contribution for C/EBP proteins. Finally, the overall importance of C/EBP proteins for lytic gene expression was demonstrated using CHOP10 to antagonize C/EBP DNA binding activity. Introduction of CHOP10 significantly impaired induction of the ZTA, RTA, and BMRF1 proteins in chemically treated AGS/BX1 cells. Thus, C/EBPbeta and C/EBPalpha expression are associated with lytic induction in AGS cells, and expression of C/EBP proteins in epithelial cells may contribute to the tendency of these cells to exhibit constitutive low-level ZTA promoter activity.     

Attractive interhelical electrostatic interactions in the proline- and acidic-rich region (PAR) leucine zipper subfamily preclude heterodimerization with other basic leucine zipper subfamilies

Basic region-leucine zipper (B-ZIP) proteins homo- or heterodimerize to bind sequence-specific double-stranded DNA. We present circular dichroism (CD) thermal denaturation data on vitellogenin promoter-binding protein (VBP), a member of the PAR subfamily of B-ZIP proteins that also includes thyroid embryonic factor, hepatocyte leukemia factor, and albumin site D-binding protein. VBP does not heterodimerize with B-ZIP domains from C/EBP alpha, JUND, or FOS. We describe a dominant negative protein, A-VBP, that contains the VBP leucine zipper and an acidic amphipathic protein sequence that replaces the basic region critical for DNA binding. The acidic extension forms a coiled coil structure with the VBP basic region in the VBP.A-VBP heterodimer. This new alpha-helical structure extends the leucine zipper N-terminally, stabilizing the complex by 2.0 kcal/mol. A-VBP abolishes DNA binding of VBP in an equimolar competition assay, but does not affect DNA binding even at 100-fold excess of CREB, C/EBP alpha, or FOS/JUND. Likewise, proteins containing the acidic extension appended to seven other leucine zippers do not inhibit VBP DNA binding. We show that conserved g <--> e' or i, i' +5 salt bridges are sufficient to confer specificity to VBP by mutating the C/EBPalpha leucine zipper to contain the g <--> e' salt bridges that characterize VBP. A-VBP heterodimerizes with this mutant C/EBP, preventing it from binding to DNA. These conserved g <--> e' electrostatic interactions define the specificity of the PAR subfamily of B-ZIP proteins and preclude interaction with other B-ZIP subfamilies.     

Binding of ethidium to the nucleosome core particle. 2. Internal and external binding modes

We have previously reported that the binding of ethidium bromide to the nucleosome core particle results in a stepwise dissociation of the structure which involves the initial release of one copy each of H2A and H2B (McMurray & van Holde, 1986). In this report, we have examined the absorbance and fluorescence properties of intercalated and outside bound forms of ethidium bromide. From these properties, we have measured the extent of external, electrostatic binding of the dye versus internal, intercalation binding to the core particle, free from contribution by linker DNA. We have established that dissociation is induced by the intercalation mode of binding to DNA within the core particle DNA, and not by binding to the histones or by nonintercalative binding to DNA. The covalent binding of [3H]-8-azidoethidium to the core particle clearly shows that less than 1.0 adduct is formed per histone octamer over a wide range of input ratios. Simultaneously, analyses of steady-state fluorescence enhancement and fluorescence lifetime data from bound ethidium complexes demonstrate extensive intercalation binding. Combined analyses from steady-state fluorescence intensity with equilibrium dialysis or fluorescence lifetime data revealed that dissociation began when approximately 14 ethidium molecules are bound by intercalation to each core particle and less than 1.0 nonintercalated ion pair was formed per core particle.     

Fluorescence anisotropy decay of ethidium bound to nucleosome core particles. 2. The torsional motion of the DNA is highly constrained and sensitive to pH

The effects of pH on the torsional flexibility of DNA bound to nucleosome core particles were investigated by using time-resolved fluorescence anisotropy decays of intercalated ethidium. The decays were collected by using time-resolved single-photon counting and were fit to a model developed by J. M. Schurr [(1984) Chem. Phys. 84, 71-96] with a nonlinear least-squares-fitting algorithm developed for this purpose. As the torsional flexibility of DNA is affected by the presence of an intercalating dye, the decays were studied at different ethidium bromide to core particle binding ratios. Because we see large increases in DNA flexibility and in the rotational diffusion coefficient at binding ratios of 0.6 ethidium/core particle and above, we conclude that, under these conditions, the DNA begins to detach from the protein. At lower binding ratios, we observe only small changes in the anisotropy decay. The torsional parameters obtained are a function of N, the number of base pairs of DNA between points of attachment to the histone core. Only if N is greater than 30 base pairs is the torsional rigidity of DNA on a nucleosome core particle higher than that for DNA free in solution. Also, for reasonable values of N (less than 30), the friction felt by the DNA on a core particle is much higher than that felt by free DNA. This indicates that the region of the DNA to which the ethidium binds is highly constrained in its motions. pH changes nearly neutrality at moderate ionic strengths (100 mM) have a substantial effect on the fluorescence anisotropy decays, particularly at early times. These analyses indicated that the observed change on increasing pH can be attributed either to a loosening of the contacts between the DNA and the histone core (increasing N) or to a substantial relaxing of the torsional rigidity of the DNA.     

The use of fluorescence anisotropy decay of poly d(A-T) ethidium bromide complex to estimate the unwinding angle of the double helix.

We measured the fluorescence decay under polarized light, of ethidium bromide bound to the poly d(A-T) isolated from Cancer Pagurus. The decay of the whole fluorescence is a single exponential function revealing a good homogeneity of the binding sites. The anisotropy decay due to energy transfers between the ethidium bromide molecules bound to a same poly d(A-T) molecule has been analysed, with a Monte Carlo calculation. We found the dye unwinds the poly d(A-T) duplex by an angle of 17 degrees plus or minus 2 degrees. This result is in agreement with the value previously found in the case of calf thymus DNA-ethidium bromide complex, although the base compositions of the two nucleic acids are different.     

Circular dichroism studies of ethidium bromide binding to the isolated nucleolus.

Circular dichroism in the 300-360 nm region and fluorescence induced by intercaltating binding of ethidum bromide to both DNA and RNA components were studied in isolated HeLa nucleoli. Both DNA and RNA compoents contribute to the induced dichroic elliticity. Digestion of nucleoli by RNase or DNase shows that most of the induced ellipticity comes from the DNA component. In nucleoli with an RNA/DNA = 0.8/1.0 the RNA component gives only 20% of the total ellipticity when measured at an ethidium bromide/DNA = 0.25. Spectro-fluorometric titration shows that ethidium bromide intercalates mostly into DNA in nucleoli. Both circular dichroism and fluorescence studies indicate that both DNA and RNA components in isolated nucleoli are less accessible to intercalating binding by ethidium bromide when compared to purified nucleolar DNA, DNA in chromatin or purified ribosomal RNA. Circular dichroic measurements of intercalating binding of ethidium bromide to to nucleoli may be used to study changes in nucleoli under different physiological or pathological conditions.     

HDAC1 promotes liver proliferation in young mice via interactions with C/EBPbeta

HDAC1 (histone deacetylase 1) regulates a number of biological processes in cells. Our previous studies revealed that HDAC1 inhibits proliferation of the livers in old mice. We have surprisingly observed that HDAC1 is also increased in young livers proliferating after partial hepatectomy (PH) and in human liver tumors. Increased levels of HDAC1 after PH lead to its interaction with a member of the C/EBP family, C/EBPbeta, which is also elevated after PH. At early time points after PH, the HDAC1-C/EBPbeta complex binds to the C/EBPalpha promoter and represses expression of C/EBPalpha. A detailed analysis of the role of HDAC1 and C/EBPbeta proteins in the regulation of C/EBPalpha promoter showed that, whereas C/EBPbeta alone activates the promoter, HDAC1 represses the promoter in a C/EBPbeta-dependent manner. The inhibition of HDAC1 in the livers of young mice inhibits liver proliferation after PH, which is associated with high levels of C/EBPalpha. Consistent with the positive role of HDAC1-C/EBPbeta complexes in liver proliferation, we have found that the CUGBP1-HDAC1-C/EBPbeta pathway is activated in human tumor liver samples, suggesting that HDAC1-C/EBPbeta complexes are involved in the development of liver tumors. The causal role of the CUGBP1-HDAC1 pathway in liver proliferation was examined in CUGBP1 transgenic mice, which display high levels of the CUGBP1-eIF2 complex. We have demonstrated that elevation of the HDAC1-C/EBPbeta complexes in CUGBP1 transgenic mice reduces expression of C/EBPalpha and increases the rate of liver proliferation. Thus, these studies have identified a new pathway that promotes liver proliferation in young mice and might contribute to the malignant transformations in the liver.     

TCDD suppresses insulin-responsive glucose transporter (GLUT-4) gene expression through C/EBP nuclear transcription factors in 3T3-L1 adipocytes

TCDD is known to reduce significantly the level of the functionally active form of glucose transporter type 4 (GLUT4) in vivo in adipose tissue and muscles. To study the mechanistic basis of this phenomenon, we conducted transient transfection and DNA deletion analysis in 3T3-L1 cells using chloramphenicol acetyltransferase (CAT) reporter plasmids containing the GLUT4 promoter joined to the bacterial CAT. It was found that in transfected control samples, CAT activity was significantly higher in cells transfected with p469CAT and p273CAT than those with p78CAT, indicating that the region between -78 and -273 contained elements that play major roles in transactivation of this gene. Treatment with TCDD decreased CAT activity with p469CAT and p273CAT, but not with p78CAT, indicating the same region to contain the element(s) affected by TCDD. A gel-shift (EMSA) analysis result indicated that TCDD shows the profound effect only on the nuclear proteins binding to the [(32)P]-labeled probe containing C/EBP response element equivalent of the -265 to -242 stretch of the GLUT4 promoter. The results of supershift analysis showed that TCDD caused a decrease in the tier of C/EBPalpha and an increase in that of C/EBPbeta among the proteins bound to this C/EBP response element. We studied the effect of TCDD in cells overexpressing either C/EBPalpha, C/EBPbeta, or C/EBPdelta through transient transfection of p273CAT or p469CAT. The results clearly showed that the effect of TCDD to suppress the CAT activity of p273 or p469 disappeared in those cells overexpressing C/EBPalpha or C/EBPbeta. These results implicate the C/EBP proteins to be the main mediator of suppressive action of TCDD on GLUT4 gene expression in 3T3-L1 cells.