PVP-coated silver nanoparticles block the transmission of cell-free and cell-associated HIV-1 in human cervical culture

Background  

Previous in vitro studies have demonstrated that polyvinylpyrrolidone coated silver nanoparticles (PVP-coated AgNPs) have antiviral activity against HIV-1 at non-cytotoxic concentrations. These particles also demonstrate broad spectrum virucidal activity by preventing the interaction of HIV-1 gp120 and cellular CD4, thereby inhibiting fusion or entry of the virus into the host cell. In this study, we evaluated the antiviral activity of PVP-coated AgNPs as a potential topical vaginal microbicide to prevent transmission of HIV-1 infection using human cervical culture, an in vitro model that simulates in vivo conditions.     

Anti-HIV-1 activity of resveratrol derivatives and synergistic inhibition of HIV-1 by the combination of resveratrol and decitabine

Ribonucleotide reductase inhibitors enhance the anti-HIV-1 activities of a variety of nucleoside analogs, including those that act as chain terminators and those that increase the HIV-1 mutation rate. However the use of these ribonucleotide reductase inhibitors is limited by their associated toxicities. The hydroxylated phytostilbene resveratrol has activity in a host of systems including inhibition of ribonucleotide reductase and has minimal toxicity. Here we synthesized derivatives of resveratrol and examined them for anti-HIV-1 activity and their ability to enhance the antiviral activity of decitabine, a nucleoside analog that decreases viral replication by increasing the HIV-1 mutation rate. The data demonstrates that six of the derivatives have anti-HIV-1 activity greater than resveratrol. However, only resveratrol acted in synergy with decitabine to inhibit HIV-1 infectivity. These results reveal novel resveratrol derivatives with anti-HIV-1 activity that may have mechanisms of action that differ from the drugs currently used to treat HIV-1.     

Targeting Spare CC Chemokine Receptor 5 (CCR5) as a Principle to Inhibit HIV-1 Entry

CCR5 binds the chemokines CCL3, CCL4, and CCL5 and is the major coreceptor for HIV-1 entry into target cells. Chemokines are supposed to form a natural barrier against human immunodeficiency virus, type 1 (HIV-1) infection. However, we showed that their antiviral activity is limited by CCR5 adopting low-chemokine affinity conformations at the cell surface. Here, we investigated whether a pool of CCR5 that is not stabilized by chemokines could represent a target for inhibiting HIV infection. We exploited the characteristics of the chemokine analog PSC-RANTES (N-α-(n-nonanoyl)-des-Ser(1)-[l-thioprolyl(2), l-cyclohexylglycyl(3)]-RANTES(4-68)), which displays potent anti-HIV-1 activity. We show that native chemokines fail to prevent high-affinity binding of PSC-RANTES, analog-mediated calcium release (in desensitization assays), and analog-mediated CCR5 internalization. These results indicate that a pool of spare CCR5 may bind PSC-RANTES but not native chemokines. Improved recognition of CCR5 by PSC-RANTES may explain why the analog promotes higher amounts of β-arrestin 2·CCR5 complexes, thereby increasing CCR5 down-regulation and HIV-1 inhibition. Together, these results highlight that spare CCR5, which might permit HIV-1 to escape from chemokines, should be targeted for efficient viral blockade.     

Mechanism of Multivalent Nanoparticle Encounter with HIV-1 for Potency Enhancement of Peptide Triazole Virus Inactivation

Entry of HIV-1 into host cells remains a compelling yet elusive target for developing agents to prevent infection. A peptide triazole (PT) class of entry inhibitor has previously been shown to bind to HIV-1 gp120, suppress interactions of the Env protein at host cell receptor binding sites, inhibit cell infection, and cause envelope spike protein breakdown, including gp120 shedding and, for some variants, virus membrane lysis. We found that gold nanoparticle-conjugated forms of peptide triazoles (AuNP-PT) exhibit substantially more potent antiviral effects against HIV-1 than corresponding peptide triazoles alone. Here, we sought to reveal the mechanism of potency enhancement underlying nanoparticle conjugate function. We found that altering the physical properties of the nanoparticle conjugate, by increasing the AuNP diameter and/or the density of PT conjugated on the AuNP surface, enhanced potency of infection inhibition to impressive picomolar levels. Further, compared with unconjugated PT, AuNP-PT was less susceptible to reduction of antiviral potency when the density of PT-competent Env spikes on the virus was reduced by incorporating a peptide-resistant mutant gp120. We conclude that potency enhancement of virolytic activity and corresponding irreversible HIV-1 inactivation of PTs upon AuNP conjugation derives from multivalent contact between the nanoconjugates and metastable Env spikes on the HIV-1 virus. The findings reveal that multispike engagement can exploit the metastability built into virus the envelope to irreversibly inactivate HIV-1 and provide a conceptual platform to design nanoparticle-based antiviral agents for HIV-1 specifically and putatively for metastable enveloped viruses generally.     

Antiviral activity of Rwandan medicinal plants against human immunodeficiency virus type-1 (HIV-1)

Selected plants used in Rwandan traditional medicine for the treatment of infections and/or rheumatoid diseases were investigated for antiviral activity in vitro against human immunodeficiency virus type-1 (HIV-1). Of the 38 tested 80% ethanolic extracts, belonging to plants of 21 different families only the extracts from the leaves of Aspilia pluriseta (Asteraceae) and Rumex bequaertii (Polygonaceae) had interesting selectivity indices (SI = ratio of the 50% cytotoxic concentration to the 50% effective antiviral concentration) higher than 1. Further fractionation of the initially antivirally inactive ethanolic extract of Tithonia diversifolia, however, led to an aqueous fraction with a high anti-HIV-1 activity (SI > 461), indicating that the cytotoxicity of some plant components may mask the antiviral properties of the active plant substances in total plant extracts.     

Simian immunodeficiency virus engrafted with human immunodeficiency virus type 1 (HIV-1)-specific epitopes: replication, neutralization, and survey of HIV-1-positive plasma

To date, only a small number of anti-human immunodeficiency virus type 1 (HIV-1) monoclonal antibodies (MAbs) with relatively broad neutralizing activity have been isolated from infected individuals. Adequate techniques for defining how frequently antibodies of these specificities arise in HIV-infected people have been lacking, although it is generally assumed that such antibodies are rare. In order to create an epitope-specific neutralization assay, we introduced well-characterized HIV-1 epitopes into the heterologous context of simian immunodeficiency virus (SIV). Specifically, epitope recognition sequences for the 2F5, 4E10, and 447-52D anti-HIV-1 neutralizing monoclonal antibodies were introduced into the corresponding regions of SIVmac239 by site-directed mutagenesis. Variants with 2F5 or 4E10 recognition sequences in gp41 retained replication competence and were used for neutralization assays. The parental SIVmac239 and the neutralization-sensitive SIVmac316 were not neutralized by the 2F5 and 4E10 MAbs, nor were they neutralized significantly by any of the 96 HIV-1-positive human plasma samples that were tested. The SIV239-2F5 and SIV239-4E10 variants were specifically neutralized by the 2F5 and 4E10 MAbs, respectively, at concentrations within the range of what has been reported previously for HIV-1 primary isolates (J. M. Binley et al., J. Virol. 78:13232-13252, 2004). The SIV239-2F5 and SIV239-4E10 epitope-engrafted variants were used as biological screens for the presence of neutralizing activity of these specificities. None of the 92 HIV-1-positive human plasma samples that were tested exhibited significant neutralization of SIV239-2F5. One plasma sample exhibited >90% neutralization of SIV239-4E10, but this activity was not competed by a 4E10 target peptide and was not present in concentrated immunoglobulin G (IgG) or IgA fractions. We thus confirm by direct analysis that neutralizing activities of the 2F5 and 4E10 specificities are either rare among HIV-1-positive individuals or, if present, represent only a very small fraction of the total neutralizing activity in any given plasma sample. We further conclude that the structures of gp41 from SIVmac239 and HIV-1 are sufficiently similar such that epitopes engrafted into SIVmac239 can be readily recognized by the cognate anti-HIV-1 monoclonal antibodies.     

Structural requirements for and consequences of an antiviral porphyrin binding to the V3 loop of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp120

Several porphyrin derivatives were reported to have anti-HIV-1 activity. Among them, meso-teta(4-carboxyphenyl)porphine (MYCPP) and other carboxyphenyl derivatives were the most potent inhibitors (EC₅₀ < 0.7 μM). MTCPP bound to the HIV-1 enveloope glycoprotein gp120 and to full-length V3 loop peptides corresponding to several HIV-1 isolates but not to other peptides from gp120+gp41. However, it remained possible that MTCPP bound to HIV-1 envelop glycoprotein gp120 and to full-length V3 loop peptides corresponding to several HIV-1 isolates but not to other peptides from gp120+gp41. However, it remained possible that MTCPP bound to regions on gp120 which cannot be mimicked by peptides. Further characterization of the binding domain for MTCPP is important for understanding the antiviral activity of porphyrins and for the design of anit-HIV-1 drugs interfering with functions of the virus envelope. Results presented here show that: (i) deletion of the V3 loop from the gp120 sequence resulted in drastically diminished MTCPP binding, suggesting that the V3 loop is the dominant if not the only target site on gp120; (ii) this site was only partially mimicked by full-length V3 loop peptides; (iii) MTCPP binding to the gp120 V3 loop elicited allosteric effects resulting in decreased accessibility of the CD4 receptor binding site; (iv) the binding site for MTCPP lies within the central portion of the V3 loop (KSIHIGPGRAFY for the HIV-1 subtype B consensus sequence) and does not involve directly the GPG apex of the loop. These results may help in designing antiviral compounds with improved activity.     

A Novel Assay System for Anti-Human Immunodeficiency Virus Type 1 (HIV-1) Activity Using a Subclone of a Monocytic Cell Line,U937

A subclonal cl.1–14 cell was established from a monocytic cell line U937 by a limiting dilution method. The anti-HIV-1 activity of some antiviral compounds was evaluated in HIV-1-infected cl.1–14 cells. The results demonstrated that although AZT was a potent inhibitor of HIV-1 replication in cl.1–14 cells, its 50% effective concentration (EC₅₀) values was 80 times higher than that in HIV-1 infected MT-4 cells; the EC₅₀ of AZT was 0.16 μM and 0.002 μM in cl.1–14 and MT-4 cells, respectively. In contrast, the anti-HIV-1 activity of ddA, ddI and ddC in cl.1–14 cells was comparable to that in MT-4 cells. The antiviral activity of nevirapine, dextran sulfate, curdlan sulfate and T22 did not differ significantly between the cl. 1–14 and MT-4 cells. The antiviral activity of several compounds in the HIV-1-infected cl.1–14 cells was similar to that in the HIV-1jr -fl -infected human peripheral macrophages. Our results suggest that cl.1–14 cell cultures are very useful for estimating antiviral activity and more advantageous than the use of peripheral blood macrophages.     

Virtual screening based identification of novel small-molecule inhibitors targeted to the HIV-1 capsid

The hydrophobic cavity of the C-terminal domain (CTD) of HIV-1 capsid has been recently validated as potential target for antiviral drugs by peptide-based inhibitors; however, there is no report yet of any small molecule compounds that target this hydrophobic cavity. In order to fill this gap and discover new classes of ant-HIV-1 inhibitors, we undertook a docking-based virtual screening and subsequent analog search, and medicinal chemistry approaches to identify small molecule inhibitors against this target. This article reports for the first time, to the best of our knowledge, identification of diverse classes of inhibitors that efficiently inhibited the formation of mature-like viral particles verified under electron microscope (EM) and showed potential as anti-HIV-1 agents in a viral infectivity assay against a wide range of laboratory-adapted as well as primary isolates in MT-2 cells and PBMC. In addition, the virions produced after the HIV-1 infected cells were treated with two of the most active compounds showed drastically reduced infectivity confirming the potential of these compounds as anti-HIV-1 agents. We have derived a comprehensive SAR from the antiviral data. The SAR analyses will be useful in further optimizing the leads to potential anti-HIV-1 agents.     

Amine substituted N-(1H-benzimidazol-2ylmethyl)-5,6,7,8-tetrahydro-8-quinolinamines as CXCR4 antagonists with potent activity against HIV-1

Several novel amine substituted N-(1H-benzimidazol-2ylmethyl)-5,6,7,8-tetrahydro-8-quinolinamines were synthesized which had potent activity against HIV-1. The synthetic approaches adopted allowed for variation of the substitution pattern and resulting changes in antiviral activity are highlighted. This led to the identification of compounds with low and sub-nanomolar anti-HIV-1 activity.     

Polyvalent side chain peptide-synthetic polymer conjugates as HIV-1 entry inhibitors

This report describes the synthesis and properties of a series of polyvalent side chain peptide-synthetic polymer conjugates designed to block the CD4 binding site on gp120 and inhibit HIV-1 entry into a host cell. The peptide sequences in the conjugates are based on the CDR H3 region of the neutralizing anti-HIV-1 antibody IgG1 b12. Using a consecutive ester-amide/thiol-ene postpolymerization modification strategy, a library of polymer conjugates was prepared. Evaluation of the HIV-1 inhibitory properties revealed that midsized polymer conjugates displayed the highest antiviral activity, while shorter and longer conjugates proved to be less efficacious inhibitors. The lower molecular weight conjugates may not have sufficient length to span the distance between two neighboring gp120 containing spikes, while the higher molecular weight conjugates may be compromised due to a higher entropic penalty that would accompany their binding to the viral envelope. Although the IC(50) values for these polymer conjugates are higher than that of the parent IgG1 b12 antibody, the strategy presented here may represent an interesting antiviral approach due to the attractive properties of such polymer therapeutics (relatively inexpensive production and purification costs, high thermal and chemical stability in storage conditions, long half-life in biological tissues, low immunogenicity, and protection from proteolytic degradation).     

Heparin and its derivatives bind to HIV-1 recombinant envelope glycoproteins, rather than to recombinant HIV-1 receptor, CD4

We have employed a direct radiolabel binding assay to investigate the interaction between3H-heparin and recombinant envelope glycoproteins, rgp120s, derived from several different isolates of HIV-1. Comparable dose-dependent binding is exhibited by rgp120s from isolates IIIB, GB8, MN and SF-2. Under identical experimental conditions the binding of3H- heparin to a recombinant soluble form of the cellular receptor for gp120, CD4, is negligible. The binding of3H-heparin to rgp120 is competed for by excess unlabeled heparin and certain other, but not all, glycosaminoglycan and chemically modified heparins. Of a range of such polysaccharides tested, ability to compete with3H-heparin for binding was strictly correlated with inhibition of HIV-1 replication in vitro. Those possessing potent anti-HIV-1 activity were effective competitors, whereas those having no or little anti-HIV-1 activity were poor competitors. Scatchard analysis indicates that the K d of the interaction between heparin and rgp120 is 10 nM. Binding studies conducted in increasing salt concentrations confirm that the interaction is ionic in nature. Synthetic 33-35 amino acid peptides based on the sequence of the V3 loop of gp120 also bind to heparin with high affinity. V3 loop peptides that are cyclized due to terminal cysteine residues show more selective binding than their uncyclized counterparts. Overall, these data demonstrate further that heparin exerts its anti-HIV-1 activity by binding to the envelope glycoprotein of HIV-1, rather than its cellular receptor, CD4. This study confirms that the V3 loop of gp120 is the site at which heparin exerts its anti- HIV-1 activity. Moreover, it reveals that high affinity binding to heparin is shared by all four rgp120s examined, despite amino acid substitutions within the V3 loop.      

Human neutrophil alpha-defensin 4 inhibits HIV-1 infection in vitro

Human neutrophil alpha-defensin 4 (HNP4) is more effective than HNP1-3 in protecting human peripheral blood mononuclear cells from infection by both X4 and R5 HIV-1 strains. HNP4 binds to both CD4 and gp120 approximately two orders of magnitude weaker than does HNP1, and is less effectively sequestered by glycosylated serum proteins than HNP1. These results suggest that the HIV-1 inhibition by HNP4 stems at least partially from a unique and lectin-independent property of HNP4 with CD4 and/or gp120. Our finding identifies an anti-HIV-1 property of HNP4 and may have implications in the development of new antiviral agents for AIDS therapy.     

193 Computer-aided design of novel HIV-1 entry inhibitors based on glycosphingolipids

Novel HIV-1 entry inhibitors targeting the envelope gp120 V3 loop were designed by computer modeling based on glycosphingolipid β-galactosylceramide (β-GalCer) forming on the surface of some susceptible host cells the primary receptor for HIV-1 alternative to CD4, which is used by the virus to enter macrophages and T-lymphocytes (e.g. Fantini et al., 1993). To achieve this goal, 3D structures of the twelve water-soluble analogs of β-GalCer containing different substitutes of its fatty acid residue were determined by quantum chemical calculations and evaluation of their potential anti-HIV-1 activity was carried out by molecular docking, molecular dynamics, and free energy simulations. Analysis of the structural complexes of these β-GalCer derivatives with the HIV-1 V3 loops from the five diverse viral strains makes it clear that, in all of the cases of interest, the third variable domain of gp120 forms two potential binding sites for glycolipids concerning the immunogenic tip and the base of V3. At the same time, nonconventional XH … π hydrogen bonds between XH sugar groups (X designates C or O) and overlapping π-orbitals of the conserved Phe-20, Tyr-21, and His-34 residues of the V3 loop were shown to play a key role in specific binding of the designed glycosphingolipids to the above conserved structural motifs of V3 that include residues critical for cell tropism (Andrianov et al., 2011; Andrianov, Kornoushenko, et al., 2012). These findings testifying to the ability of the simulated chemicals to specifically and effectively interact with the functionally important sites of V3 were confirmed by those on molecular dynamics and calculating the free energy of formation of the complexes for these β-GalCer analogs with the HIV-1 V3 loops from different viral modifications. Finally, the majority of the designed molecules were found to form much more stable complexes with V3, compared to that of the β-GalCer-based anti-HIV-1 agent developed previously (Andrianov, Anishchenko, et al., 2012) and used in the calculations as a control. In the light of the data obtained, these potential HIV-1 entry inhibitors present the promising basic compounds for the development of novel, potent, and broad antiviral drugs.