env chimeric virus technology for evaluating human immunodeficiency virus susceptibility to entry inhibitors

We describe the development of chimeric virus technology (CVT) for human immunodeficiency virus (HIV) type 1 (HIV-1) env genes gp120, gp41, and gp160 for evaluation of the susceptibilities of HIV to entry inhibitors. This env CVT allows the recombination of env sequences derived from different strains into a proviral wild-type HIV-1 clone (clone NL4.3) from which the corresponding env gene has been deleted. An HIV-1 strain (strain NL4.3) resistant to the fusion inhibitor T20 (strain NL4.3/T20) was selected in vitro in the presence of T20. AMD3100-resistant strain NL3.4 (strain NL4.3/AMD3100) was previously selected by De Vreese et al. (K. De Vreese et al., J. Virol. 70:689-696, 1996). NL4.3/AMD3100 contains several mutations in its gp120 gene (De Vreese et al., J. Virol. 70:689-696, 1996), whereas NL4.3/T20 has mutations in both gp120 and gp41. Phenotypic analysis revealed that NL4.3/AMD3100 lost its susceptibility to dextran sulfate, AMD3100, AMD2763, T134, and T140 but not its susceptibility to T20, whereas NL4.3/T20 lost its susceptibility only to the inhibitory effect of T20. The recombination of gp120 of NL4.3/AMD3100 and gp41 of NL4.3/T20 or recombination of the gp160 genes of both strains into a wild-type background reproduced the phenotypic (cross-)resistance profiles of the corresponding strains selected in vitro. These data imply that mutations in gp120 alone are sufficient to reproduce the resistance profile of NL4.3/AMD3100. The same can be said for gp41 in relation to NL4.3/T20. In conclusion, we demonstrate the use of env CVT as a research tool in the delineation of the region important for the phenotypic (cross-)resistance of HIV strains to entry inhibitors. In addition, we obtained a proof of principle that env CVT can become a helpful diagnostic tool in assessments of the phenotypic resistance of clinical HIV isolates to HIV entry inhibitors.     

The CXCR4 antagonist AMD3100 efficiently inhibits cell-surface-expressed human immunodeficiency virus type 1 envelope-induced apoptosis

Infection by human immunodeficiency virus type 1 (HIV-1) has been associated with increased cell death by apoptosis in infected and uninfected cells. The envelope glycoprotein complex ([gp120/gp41](n)) of X4 HIV-1 isolates is involved in both infected and uninfected cell death via its interaction with cellular receptors CD4 and CXCR4. We studied the effect of the blockade of CXCR4 receptors by the agonist stromal derived factor (SDF-1alpha) and the antagonist bicyclam AMD3100 on apoptotic cell death of CD4(+) cells in different models of HIV infection. In HIV-infected CEM or SUP-T1 cultures, AMD3100 showed antiapoptotic activity even when added 24 h after infection. In contrast, other antiviral agents, such as zidovudine, failed to block apoptosis under these conditions. The antiapoptotic activity of AMD3100 was also studied in coculture of peripheral blood mononuclear cells or CD4(+) cell lines with chronically infected H9/IIIB cells. AMD3100 was found to inhibit both syncytium formation and apoptosis induction with 50% inhibitory concentrations ranging from 0.009 to 0.24 microg/ml, depending on the cell type. When compared to SDF-1alpha, AMD3100 showed higher inhibitory potency in all cell lines tested. Our data indicate that the bicyclam AMD3100 not only inhibits HIV replication but also efficiently blocks cell-surface-expressed HIV-1 envelope-induced apoptosis in uninfected cells.     

Reduced fitness of HIV-1 resistant to CXCR4 antagonists

HIV-1 strains with a syncytium-inducing phenotype that use CXCR4 (X4 strains) have been associated with faster disease progression and AIDS. Antiviral agents designed to block CXCR4 may prevent the emergence of X4 HIV strains but resistant strains that maintain the X4 phenotype can be raised by sequential passage in cell cultures. We have demonstrated that a laboratory adapted strain (NL4-3) and a cloned clinical isolate (CI-1) of HIV-1 cultured in the presence of the CXCR4 antagonist, AMD3100, became resistant to the compound without a change in co-receptor use. These strains became resistant through divergence with respect to the wild-type virus. Conversely, a clinical isolate made resistant to AMD3100 switched co-receptor use from X4 to R5 through a change in diversity from the original virus population. When dual infection competition/heteroduplex tracking assays were performed, all AMD3100-resistant strains, regardless of co-receptor use showed a significantly diminished fitness compared with the wild-type virus. Single virus infections, at a similar multiplicity of infection, also indicated that the wild-type strains possess better replicative ability than their corresponding resistant strains. Thus, viral resistance development to a CXCR4 antagonist such as AMD3100 is associated with reduced viral fitness.     

Mutations in both env and gag genes are required for HIV-1 resistance to the polysulfonic dendrimer SPL2923, as corroborated by chimeric virus technology

A drug-resistant NL4.3/SPL2923 strain has previously been generated by in vitro selection of HIV-1(NL4.3) in the presence of the polysulfonic dendrimer SPL2923 and mutations were reported in its gp120 gene (Witvrouw et al., 2000). Here, we further analysed the (cross) resistance profile of NL4.3/SPL2923. NL4.3/SPL2923 was found to contain additional mutations in gp41 and showed reduced susceptibility to SPL2923, dextran sulfate (DS) and enfuvirtide. To delineate to what extent the mutations in each env gene were accountable for the phenotypic (cross) resistance of NL4.3/SPL2923, the gp120-, gp41- and gp160-sequences derived from this strain were placed into a wild-type background using env chimeric virus technology (CVT). The cross resistance of NL4.3/SPL2923 towards DS was fully reproduced following gp160-recombination, while it was only partially reproduced following gp120- or gp41-recombination. The mutations in gp41 of NL4.3/SPL2923 were sufficient to reproduce the cross resistance to enfuvirtide. Unexpectedly, the reduced sensitivity towards SPL2923 was not fully reproduced after gp160-recombination. The search for mutations in NL4.3/SPL2923 in viral genes other than env revealed several mutations in the gene encoding the HIV p17 matrix protein (MA) and one mutation in the gene encoding the p24 capsid protein (CA). In order to analyse the impact of the gag mutations alone and in combination with the mutations in env on the phenotypic resistance towards SPL2923, we developed a novel p17- and p17/gp160-CVT. Phenotypic analysis of the NL4.3/SPL2923 p17- and p17/gp160-recombined strains indicated that the mutations in both env and gag have to be present to fully reproduce the resistance of NL4.3/SPL2923 towards SPL2923.     

Identification and characterization of UK-201844, a novel inhibitor that interferes with human immunodeficiency virus type 1 gp160 processing

More than 10(6) compounds were evaluated in a human immunodeficiency virus type 1 (HIV-1) high-throughput antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (UK-201844). UK-201844 exhibited antiviral activity against HIV-1 NL4-3 in MT-2 and PM1 cells, with 50% effective concentrations of 1.3 and 2.7 microM, respectively, but did not exhibit measurable antiviral activity against the closely related HIV-1 IIIB laboratory strain. UK-201844 specifically inhibited the production of infectious virions packaged with an HIV-1 envelope (Env), but not HIV virions packaged with a heterologous Env (i.e., the vesicular stomatitis virus glycoprotein), suggesting that the compound targets HIV-1 Env late in infection. Subsequent antiviral assays using HIV-1 NL4-3/IIIB chimeric viruses showed that HIV-1 Env sequences were critical determinants of UK-201844 susceptibility. Consistent with this, in vitro resistant-virus studies revealed that amino acid substitutions in HIV-1 Env are sufficient to confer resistance to UK-201844. Western analysis of HIV Env proteins expressed in transfected cells or in isolated virions showed that UK-201844 inhibited HIV-1 gp160 processing, resulting in the production of virions with nonfunctional Env glycoproteins. Our results demonstrate that UK-201844 represents the prototype for a unique HIV-1 inhibitor class that directly or indirectly interferes with HIV-1 gp160 processing.     

2-Aminothiazolones as Anti-HIV Agents That Act as gp120-CD4 Inhibitors

We report here the synthesis of 2-aminothiazolones along with their biological properties as novel anti-HIV agents. Such compounds have proven to act through the inhibition of the gp120-CD4 protein-protein interaction that occurs at the very early stage of the HIV-1 entry process. No cytotoxicity was found for these compounds, and broad antiviral activities against laboratory strains and pseudotyped viruses were documented. Docking simulations have also been applied to predict the mechanism, at the molecular level, by which the inhibitors were able to interact within the Phe43 cavity of HIV-1 gp120. Furthermore, a preliminary absorption, distribution, metabolism, and excretion (ADME) evaluation was performed. Overall, this study led the basis for the development of more potent HIV entry inhibitors.     

Novel agents for the treatment of HIV-2 infection

Many of the antiretrovirals used against HIV-1 are either ineffective or less effective in HIV-2 infection. There is in vitro evidence of the potency of maraviroc and several investigational agents against HIV-2. We conclude that, whilst specific boosted protease inhibitors combined with nucleoside analogues should still be considered the mainstays of HIV-2 treatment, maraviroc, T-1249, TAK-779 and AMD3100, as well as raltegravir, could contribute to regimens for treatment-experienced individuals. Factors bearing on the use and timing of these alternative agents are discussed.     

Anti-human immunodeficiency virus effects of dextran sulfate are strain dependent and synergistic or antagonistic when dextran sulfate is given in combination with dideoxynucleosides.

The effects of three molecular weight ranges of dextran sulfate on five different human immunodeficiency virus (HIV) isolates (from patients with acquired immunodeficiency syndrome), alone and in combination with dideoxynucleosides, were investigated in vitro. The higher the molecular weight range of dextran sulfate, the more potent the activity as assessed by a quantitative syncytium formation assay. Although all five HIV isolates had similar susceptibilities to the inhibitory effects of dideoxynucleosides, the two clinical isolates of HIV (HIV type 1 [HIV-1] TM and SP) exhibited a pattern of reduced susceptibility to dextran sulfate when compared with the two cloned isolates (HIV-1 WMF and HIV-2 ROD) and a prototype laboratory strain (HIV-1 IIIB). In combination with dideoxynucleosides, the high-molecular-weight range of dextran sulfate (500,000) resulted in an antagonistic response directed against the two clinical isolates of HIV (HIV-1 TM and SP) when the antiviral concentrations of dextran sulfate were in the ineffective range. Additive or synergistic effects were seen with the other three HIV isolates and all five HIV isolates when the low-molecular-weight range of dextran sulfate (8,000) was used. The results of these studies raise issues on the impact of drug-resistant strains on disease progression and the use of dextran sulfate in combination with nucleoside analogs for the clinical management of HIV disease.     

N-substituted pyrrole derivatives as novel human immunodeficiency virus type 1 entry inhibitors that interfere with the gp41 six-helix bundle formation and block virus fusion

A recently approved peptidic human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, T-20 (Fuzeon; Trimeris Inc.), has shown significant promise in clinical application for treating HIV-1-infected individuals who have failed to respond to the currently available antiretroviral drugs. However, T-20 must be injected twice daily and is too expensive. Therefore, it is essential to develop orally available small molecule HIV-1 fusion inhibitors. By screening a chemical library consisting of "drug-like" compounds, we identified two N-substituted pyrroles, designated NB-2 and NB-64, that inhibited HIV-1 replication at a low micromolar range. The absence of the COOH group in NB-2 and NB-64 resulted in a loss of anti-HIV-1 activity, suggesting that this acid group plays an important role in mediating the antiviral activity. NB-2 and NB-64 inhibited HIV-1 fusion and entry by interfering with the gp41 six-helix bundle formation and disrupting the alpha-helical conformation. They blocked a d-peptide binding to the hydrophobic pocket on surface of the gp41 internal trimeric coiled-coil domain. Computer-aided molecular docking analysis has shown that they fit inside the hydrophobic pocket and that their COOH group interacts with a positively charged residue (K574) around the pocket to form a salt bridge. These results suggest that NB-2 and NB-64 may bind to the gp41 hydrophobic pocket through hydrophobic and ionic interactions and block the formation of the fusion-active gp41 core, thereby inhibiting HIV-1-mediated membrane fusion and virus entry. Therefore, NB-2 and NB-64 can be used as lead compounds toward designing and developing more potent small molecule HIV-1 fusion inhibitors targeting gp41.     

Coreceptors of HIV infection and the development of HIV entry inhibitors: overview and targets]

In 1996 CXCR4 was identified as a coreceptor for HIV-1. This finding has lead to further identification of more than ten G-protein-coupled receptors (GPCRs) as coreceptors for HIV/SIV. Cell tropisms and coreceptor uses of HIV during the course of HIV infection are summarized. Promiscuous properties of correlations between chemokines and their chemokine receptor uses and also between variable amino acid sequences in the V3 region of HIV gp120 Env and HIV coreceptor uses are discussed. This promiscuous property of HIV-1 is claimed to be a possible cause of a difficulty in developing highly effective entry inhibitors and in addition to allow rapid appearance of immune escape HIV mutants. Representative agents that inhibit HIV entry with a special reference to inhibitors of coreceptor use and gp41 function are summarized. gp41 is discussed as a promising target for the development of effective entry inhibitors.     

Phosphorothioate oligonucleotides inhibit human immunodeficiency virus type 1 fusion by blocking gp41 core formation

Several studies have shown that phosphorothioate oligodeoxynucleotides (PS-ONs) have a sequence-independent antiviral activity against human immunodeficiency virus type 1 (HIV-1). It has also been suggested that PS-ONs inhibit HIV-1 by acting as attachment inhibitors that bind to the V3 loop of gp120 and prevent the gp120-CD4 interaction. Here we show that PS-ONs (and their fully 2'-O-methylated derivatives) are potent inhibitors of HIV-1-mediated membrane fusion and HIV-1 replication in a size-dependent, phosphorothioation-dependent manner. PS-ONs interact with a peptide derived from the N-terminal heptad repeat region of gp41, and the HIV-1 fusion-inhibitory activity of PS-ONs is closely correlated with their ability to block gp41 six-helix bundle formation, a critical step during the process of HIV-1 fusion with the target cell. These results suggest that the increased hydrophobicity of PS-ONs may contribute to their inhibitory activity against HIV-1 fusion and entry, because longer PS-ONs (>or=30 bases) which have a greater hydrophobicity are more potent in blocking the hydrophobic interactions involved in the gp41 six-helix bundle formation and inhibiting the HIV-1-mediated cell-cell fusion than shorter PS-ONs (<30 bases). This novel antiviral mechanism of action of long PS-ONs has implications for therapy against infection by HIV-1 and other enveloped viruses with type I fusion proteins.