Interaction of arsenite with a zinc finger CCHC peptide: evidence for formation of an As-Zn-peptide mixed complex

The interaction of arsenite with a Cys₃His (CCHC) zinc finger model (34-51) HIV-1 nucleocapsid protein p7 (NCp7) peptide in the absence and presence of Zn^II was studied using fluorescence spectroscopy, CD (circular dichroism) and ESI-MS (Electrospray Ionization Mass Spectrometry). We found that arsenic forms different complexes with the free peptide and the zinc finger peptide. In the former case the peptide conformation differed greatly from that of the zinc finger, whereas in the second case a mixed As-Zn-peptide complex was formed with partial preservation of zinc finger conformation. An apparent stability constant was estimated for the mixed As-Zn-peptide complex (K = 2083 M^− 1 and 442 M^− 1 at 25 °C and pHs 6 and 7, respectively). Our study also shows that the interaction of arsenic with the CCHC motif is facilitated by glutathione (GSH), through formation of a GS-As-peptide conjugate.     

Zinc finger proteins as templates for metal ion exchange: Substitution effects on the C-finger of HIV nucleocapsid NCp7 using M(chelate) species (M = Pt, Pd, Au)

The interactions of monofunctional [MCl(chelate)] compounds (M = Pt(II), Pd(II) or Au(III) and chelate = diethylenetriamine, dien or 2,2′,2″-terpyridine, terpy) with the C-terminal finger of the HIV nucleocapsid NCp7 zinc finger (ZF) were studied by mass spectrometry and circular dichroism spectroscopy. In the case of [M(dien)] species, Pt(II) and Pd(II) behaved in a similar fashion with evidence of adducts caused by displacement of Pt-Cl or Pd-Cl by zinc-bound thiolate. Labilization, presumably under the influence of the strong trans influence of thiolate, resulted in loss of ligand (dien) as well as zinc ejection and formation of species with only Pd(II) or Pt(II) bound to the finger. For both Au(III) compounds the reactions were very fast and only “gold fingers” with no ancillary ligands were observed. For all terpyridine compounds ligand scrambling and metal exchange occurred with formation of [Zn(terpy)]²⁺. The results conform well to those proposed from the study of model Zn compounds such as N,N′-bis(2-mercapto-ethyl)-1,4-diazacycloheptanezinc(II), [Zn(bme-dach)]₂. The possible structures of the adducts formed are discussed and, for Pt(II) and Pd(II), the evidence for possible expansion of the zinc coordination sphere from four- to five-coordinate is discussed. This observation reinforces the possibility of change in geometry for zinc in biology, even in common “structural” sites in metalloenzymes. The results further show that the extent and rate of zinc displacement by inorganic compounds can be modulated by the nature (metal, ligands) of the reacting compound.     

Novel epidithiodiketopiperazines as anti-viral zinc ejectors of the Feline Immunodeficiency Virus (FIV) nucleocapsid protein as a model for HIV infection

Focused libraries of multi-substituted epidithiodiketopiperazines (ETP) were prepared and evaluated for efficacy of inhibiting the nucleocapsid protein function of the Feline Immunodeficiency Virus (FIV) as a model for HIV. This activity was compared and contrasted to observed toxicity utilising an in-vitro cell culture approach. This resulted in the identification of several promising lead compounds with nanomolar potency in cells with low toxicity and a favorable therapeutic index.     

HIV chemokine receptor inhibitors as novel anti-HIV drugs

The chemokine receptors CXCR4 and CCR5 are the main coreceptors used by the T-cell-tropic (CXCR4-using, X4) and macrophage-tropic (CCR5-using, R5) HIV-1 strains, respectively, for entering their CD4⁺ target cells. In this review, we focus on the function of these chemokine receptors in HIV infection and their role as novel targets for viral inhibition. Besides some modified chemokines with antiviral activity, several low-molecular weight CCR5 and CXCR4 antagonistic compounds have been described with potent antiviral activity. The best CXCR4 antagonists described are the bicyclam derivatives, which consistently block X4 but also R5/X4 viral replication in PBMCs. We believe that chemokine receptor antagonists will become important new antiviral drugs to combat AIDS. Both CXCR4 and CCR5 chemokine receptor inhibitors will be needed in combination and even in combinations of antiviral drugs that also target other aspects of the HIV replication cycle to obtain optimum antiviral therapeutic effects.     

Evaluation of the antiviral efficacy of bis[1,2]dithiolo[1,4]thiazines and bis[1,2]dithiolopyrrole derivatives against the nucelocapsid protein of the Feline Immunodeficiency Virus (FIV) as a model for HIV infection

A diverse library of bis[1,2]dithiolo[1,4]thiazines and bis[1,2]dithiolopyrrole derivatives were prepared for evaluation of activity against the nucleocapsid protein of the Feline Immunodeficiency Virus (FIV) as a model for HIV, using an in vitro cell culture approach, yielding nanomolar active compounds with low toxicity.     

Evaluation of multibranched peptides as inhibitors of HIV infection

Summary Synthetic polymeric constructions (SPCs) containing the consensus sequence of the HIV-1 surface envelope gycoprotein gp120 V3 loop (GPGRAF) block the fusion between HIV-1- and HIV-2-infected cells and CD4⁺-uninfected lymphocytes. By testing the activity of a series of SPC analogs in a cell-to-cell fusion assay, we found that the most active construction is an eight-branched SPC with the hexapeptide motif GPGRAF. This compound is also able to inhibit the infection of human lymphocytes and macrophages by unrelated isolates of HIV-1 and HIV-2. This antiviral activity is specific, since no toxicity was observed at the concentrations that inhibit HIV replication and syncytia formation. These data suggest that V3 SPCs may represent a new class of therapeutic anti-HIV agents, able to neutralize a wide range of viral isolates in infected individuals.     

HIV coreceptors: role of structure, posttranslational modifications, and internalization in viral-cell fusion and as targets for entry inhibitors

The human immunodeficiency virus (HIV) envelope glycoprotein forms trimers on the virion surface, with each monomer consisting of two subunits, gp120 and gp41. The gp120 envelope component binds to CD4 on target cells and undergoes conformational changes that allow gp120 to interact with certain G-protein-coupled receptors (GPCRs) on the same target membranes. The GPCRs that function as HIV coreceptors were found to be chemokine receptors. The primary coreceptors are CCR5 and CXCR4, but several other chemokine receptors were identified as “minor coreceptors”, indicating their ability support entry of some HIV strains in tissue cultures. Formation of the tri-molecular complexes stabilizes virus binding and triggers a series of conformational changes in gp41 that facilitate membrane fusion and viral cell entry. Concerted efforts are underway to decipher the specific interactions between gp120/CD4, gp120/coreceptors, and their contributions to the subsequent membrane fusion process. It is hoped that some of the transient conformational intermediates in gp120 and gp41 would serve as targets for entry inhibitors. In addition, the CD4 and coreceptors are primary targets for several classes of inhibitors currently under testing. Our review summarizes the current knowledge on the interactions of HIV gp120 with its receptor and coreceptors, and the important properties of the chemokine receptors and their regulation in primary target cells. We also summarize the classes of coreceptor inhibitors under development.     

Synthesis and comparison of substituted 1,2,3-dithiazole and 1,2,3-thiaselenazole as inhibitors of the feline immunodeficiency virus (FIV) nucleocapsid protein as a model for HIV infection

We report the first biological evaluation the 1,2,3-thiaselenazole class of compound and utilising a concise synthetic approach of sulfur extrusion, selenium insertion of the 1,2,3-dithiazoles. We created a small diverse library of compounds to contrast the two ring systems. This approach has highlighted new structure activity relationship insights and lead to the development of sub-micro molar anti-viral compounds with reduced toxicity. The 1,2,3-thiaselenazole represents a new class of potential compounds for the treatment of FIV and HIV.     

New 5'-phosphonates, modified through the nucleoside sugar residue, as inhibitors of HIV replication]

A number of nucleoside 5'-phosphonates and nucleoside 5'-methylphosphonates were synthesised, to study their ability to inhibit reproduction of HIV-1. Three compounds, 5'-hydrogen phosphonates of 3'-azido-2',3'-dideoxythymidine (AZT-HP, IVc), 3'-fluoro-2',3'-dideoxythymidine (FLT-HP, IVa) and 2',3'-dideoxyadenosine (ddA-HP, I), exhibit potent anti-HIV-1 activity with selectivity indices similar to or better of those of their parent nucleosides.     

A platform for designing HIV integrase inhibitors. Part 2: a two-metal binding model as a potential mechanism of HIV integrase inhibitors

We propose a two-metal binding model as a potential mechanism of chelating inhibitors against HIV integrase (HIV IN) represented by 2-hydroxy-3-heteroaryl acrylic acids (HHAAs). Potential inhibitors would bind to two metal ions in the active site of HIV IN to prevent human DNA from undergoing the integration reaction. Correlation of the results of metal (Mg²⁺ and Mn²⁺) titration studies with HIV IN inhibition for a series of active and inactive compounds provides support for the model. Results suggest Mg²⁺ is an essential cofactor for chelating inhibitors.     

Minimum structural requirements for inhibitors of the zinc finger protein TRAF6

Zinc fingers have rarely been regarded as drug targets. On the contrary, the zinc-binding site of enzymes has often been considered a target of inhibitors. We previously developed a dithiol compound called SN-1 that binds to the zinc finger protein tumor necrosis factor receptor-associated factor 6 (TRAF6) and suppresses downstream nuclear factor-κB (NF-κB) signaling. To determine the minimal structure requirements of TRAF6 inhibitors based on SN-1, NF-κB inhibitory activity and cytotoxicity of its derivatives including new compounds were examined. SN-2, an oxidative type of prodrug of SN-1 with 2-nitrophenylthio groups via disulfide, has the minimum structure for an inhibitor of TRAF6, as seen with cellular experiments. The importance of two side chains with a thiol group was shown with molecular modelling. This study may lead to development of selective TRAF6 inhibitors in the near future.     

Modulation of apoptosis by HIV protease inhibitors

Advances in treatment have transformed the Human Immunodeficiency Virus (HIV) infection from a progressive and ultimately fatal disease to one that can be managed effectively by chronic suppressive antiretroviral therapy. The drugs now used to treat HIV infection not only inhibit viral replication but also have effects on cellular metabolism and homeostasis. Of particular interest to cellular immunologists, members of the HIV Protease Inhibitor (PI) class of antiretroviral agents possess intrinsic immunomodulatory and antiapoptotic properties. This review focuses on the development and use of PI together with their impact on HIV disease, immunity, and apoptosis.     

6-Benzylamino 4-oxo-1,4-dihydro-1,8-naphthyridines and 4-oxo-1,4-dihydroquinolines as HIV integrase inhibitors

SAR studies on the quinolone carboxylic acid class of HIV-1 integrase inhibitors focused on improving the metabolic stability and led to the discovery of 27 and 38.     

Gene expression,X-inactivation,and methylation during spermatogenesis: The case of Zfa,Zfx, and Zfy in mice

While it has become clear that X-inactivation in the female soma is complete in mouse (in contrast to being “patchy” in man), the degree of X-inactivation in the testes has not been ascertained. We have compared autosomal and X-linked zinc finger homolog expression and X-linked and Y-linked zinc finger homolog methylation in an attempt to elucidate this question. Using RTPCR, we have extended earlier studies of Zfx and Zfa expression in developing testes and find that Zfa expression starts at the time of X-inactivation while Zfx expression is continuous. Cell separation studies did not preclude continued expression of Zfx in adult germ cells. The methylation status of four CCGG residues in the Zfx promoter was studied using PCR bridging this region before and after DNA digestion with the isoschizomers Msp I and Hpa II, the latter being methylation sensitive. Hpa II resistant Zfx promoter DNA was found in all female tissues, but not in male tissues, including the testes. Previous studies have shown that Zfy is expressed at meiosis (like Zfa and unlike Zfx). Despite its expression, the Zfy gene is adjacent to, or contains, highly methylated CCGG sites since hybridization after Msp I digestion detected multiple small fragments that were not released after DNA digestion with Hpa II. Thus, Zfx is not methylated in sperm, while Zfy is, in contrast to their apparent patterns of expression. © 1993 Wiley-Liss, Inc.     

Identification by high throughput screening of small compounds inhibiting the nucleic acid destabilization activity of the HIV-1 nucleocapsid protein

Due to its highly conserved zinc fingers and its nucleic acid chaperone properties which are critical for HIV-1 replication, the nucleocapsid protein (NC) constitutes a major target in AIDS therapy. Different families of molecules targeting NC zinc fingers and/or inhibiting the binding of NC with its target nucleic acids have been developed. However, their limited specificity and their cellular toxicity prompted us to develop a screening assay to target molecules able to inhibit NC chaperone properties, and more specifically the initial NC-promoted destabilization of the nucleic acid secondary structure. Since this destabilization is critically dependent on the properly folded fingers, the developed assay is thought to be highly specific. The assay was based on the use of cTAR DNA, a stem–loop sequence complementary to the transactivation response element, doubly labelled at its 5′ and 3′ ends by a rhodamine 6G fluorophore and a fluorescence quencher, respectively. Addition of NC(12-55), a peptide corresponding to the zinc finger domain of NC, to this doubly-labelled cTAR, led to a partial melting of the cTAR stem, which increases the distance between the two labels and thus, restores the rhodamine 6G fluorescence. Thus, positive hits were detected through the decrease of rhodamine 6G fluorescence. An “in-house” chemical library of 4800 molecules was screened and five compounds with IC₅₀ values in the micromolar range have been selected. The hits were shown by mass spectrometry and fluorescence anisotropy titration to prevent binding of NC(12-55) to cTAR through direct interaction with the NC folded fingers, but without promoting zinc ejection. These non-zinc ejecting NC binders are a new series of anti-NC molecules that could be used to rationally design molecules with potential anti-viral activities.     

Peptide nucleic acids as epigenetic inhibitors of HIV-1

Summary The advent of highly active antiretroviral therapy (HAART) was once perceived to have transformed deadly HIV/AIDS into a treatable, chronic infectious disease. However, mounting evidence now suggests that the prevalence of multi-drug resistant HIV (MDR-HIV) infection is steadily rising among newly infected individuals in the HAART-experienced countries, raising a concern for a future outbreak of MDR-HIV/AIDS. Our global fight against AIDS must include sustained effort to search and discover a new therapeutic modality for HIV infection. Of plausible viral targets explored to date, HIV gene-targeting approach has not yet seen a considerable success in vivo. The pursuit of anti-HIV gene intervention should include the identification of critical gene targets as well as the optimization of biomolecules that can effectively interact with the intended targets. Using unmodified peptide nucleic acids (PNA) as a biomolecular tool, we discovered a potentially critical HIV gene segment within gag-pol encoding gene. Antisense PNA targeting this specific region effectively disrupted a translation of HIV gag-pol mRNA, abolishing the virion production from chronically HIV-infected cells. This exemplifies the possibility that epigenic HIV inhibitors may be developed in the coming years, if emerging novel technologies permit sufficient and stable in vivo delivery of PNA or other similarly effective biomolecules.     

Failure to detect active virus replication in mast cells at various tissue sites of HIV patients by immunohistochemistry

A recent report postulated that the mast cell population is a significant reservoir for persistent HIV infection. Our study attempted to validate this hypothesis by quantitatively comparing the distribution of mast cells and cells expressing the HIV protein p24 in HIV infected patients. Consecutive sections of paraffin-embedded human tissues from various tissue sites were subjected to immunohistochemistry with monoclonal antibodies to mast cell tryptase, viral protein p24, and other molecules. The sub-cellular distribution of these molecules was examined, to determine whether immunoreactivities to these molecules would be co-localized within the same cells. Our study revealed that, in two immediate adjacent sections immunostained for mast cell tryptase and p24, respectively, all or nearly all tryptase and p24 expressing cells were distributed at different areas. In the single section double immunostained for mast cell tryptase and p24, 5 (1.1%) of 460 large p24 expressing cell clusters encountered showed a single or few mast cells within or adjacent to p24 expressing cell clusters, but no distinct co-localization of these two proteins was observed. Similarly, no distinct co-localization was observed in any of over 500 isolated individual mast cells and p24 expressing cells. In contrast, macrophages were consistently intermixed with or adjacent to p24 expressing cells, and p24 immunostaining were seen in the cytoplasm of a subset of macrophages. These findings suggest that tissue mast cells do not show evidence for active virus replication by the techniques employed.     

Peptide nucleic acids as epigenetic inhibitors of HIV-1

Summary The advent of highly active antiretroviral therapy (HAART) was once perceived to have transformed deadly HIV/AIDS into a treatable, chronic infectious disease. However, mounting evidence now suggests that the prevalence of multi-drug resistant HIV (MDR-HIV) infection is steadily rising among newly infected individuals in the HAART-experienced countries, raising a concern for a future outbreak of MDR-HIV/AIDS. Our global fight against AIDS must include sustained effort to search and discover a new therapeutic modality for HIV infection. Of plausible viral targets explored to date, HIV gene-targeting approach has not yet seen a considerable success in vivo. The pursuit of anti-HIV gene intervention should include the identification of critical gene targets as well as the optimization of biomolecules that can effectively interact with the intended targets. Using unmodified peptide nucleic acids (PNA) as a biomolecular tool, we discovered a potentially critical HIV gene segment within gag-pol encoding gene. Antisense PNA targeting this specific region effectively disrupted a translation of HIV gag-pol mRNA, abolishing the virion production from chronically HIV-infected cells. This exemplifies the possibility that epigenic HIV inhibitors may be developed in the coming years, if emerging novel technologies permit sufficient and stable in vivo delivery of PNA or other similarly effective biomolecules.