作用于HIV gp120的进入抑制剂研究进展

HIV-1病毒为包膜病毒,其感染靶细胞的第一步是由HIV包膜蛋白表面亚基gp120与靶细胞上的CD4分子和辅助受体（趋化因子受体CCR5或CXCR4等）结合,导致gp41的构型发生改变,启动病毒包膜与靶细胞膜的融合。与gp120相结合的一些抗体、蛋白、多糖、多肽和小分子化合物,都可能影响HIV-1病毒包膜和靶细胞膜融合的过程,从而起到抗HIV-1病毒的作用。该文对近年来以HIV gp120为靶点的HIV进入抑制剂的研究进展进行综述。     

A post-CD4-binding step involving interaction of the V3 region of viral gp120 with host cell surface glycosphingolipids is common to entry and infection by diverse HIV-1 strains

The V3-loop region in the envelope protein gp120 of HIV is critical for viral infection, but its interaction with the target cells is not clear. Using synthetic peptides, representing linear V3 sequences as reagents, we obtained evidence to show inhibition of infection by both T-cell- and macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1) (X4 and R5, respectively), without interfering with gp120-CD4 interaction, by the V3 peptides through binding to host cell membrane glycosphingolipids (GSL). Synthetic peptides mimicking the central 15-21 amino acid sequence of the V3-loop region in both X4 and R5 strains of HIV-1 competed with and blocked the entry of both types of HIV isolates. These HIV-inhibitory V3 peptides exhibited specific binding to target cells that was not competed by antibodies to either the primary receptor CD4 or the co-receptors CXCR-4 and CCR5. However, R15K, the V3 peptide from HIV-1 IIIB gp120 exhibited specific binding to three distinct cell surface GSL: GM3, Gb3, and GalCer. Further, R15K inhibited GSL binding of gp120 from both HIV-1 IIIB (X4, Gb3-binding strain) and HIV-1 89.6 (X4R5, GM3-binding strain). Together, these results suggest a critical V3-mediated post-CD4-binding event involving cell surface GSL binding represented by the HIV-inhibitory V3 peptides, that is common for the entry of diverse HIV-1 strains and may be targeted for the development of novel HIV therapeutics aimed at blocking viral entry.     

Potential drug targets on the HIV-1 envelope glycoproteins,gp120 and gp41

HIV-1 entry is an attractive target for anti-HIV-1 therapy. However, there are no entry inhibitors approved for the clinical treatment of HIV-1 infection. This is likely to be changed in the near future since promising HIV-1 entry inhibitors, such as T20 and some chemokine receptor antagonists, are in the pipeline to join the repertoire of anti-HIV-1 therapeutics. This review will focus on what might be potential targets on the key components of the viral entry machinery, gp120 and gp41. These two molecules are the viral proteins responsible for HIV-1 entry. Binding to CD4 induces a series of structural changes in gp120 and allows it to interact with chemokine receptors. The receptor binding eventually triggers conformational changes in gp41, which result in the formation of a fusion active molecule to attack the cell membrane. The structural and functional motifs that operate this delicate fusion machinery could become the Achilles' heel of the virus.     

HIV包膜蛋白的结构及其相应的病毒进入抑制剂

HIV-1病毒包膜蛋白gp120和gp41在病毒感染中起着重要的作用。在病毒进入细胞的过程中,gp120先和CD4分子结合,发生构象改变,进而导致gp41构象的变化,使病毒包膜和细胞膜融合而感染细胞。与gp120或者gp41相结合的多肽、大分子和小分子化合物,都可能影响HIV-1病毒包膜和靶细胞膜结合的过程,从而起到抗HIV-1病毒的作用。该文对gp120和gp41的结构及其相互作用,以及以HIV-1包膜糖蛋白为靶点的病毒进入抑制剂类抗艾滋病药物进行综述。     

Detection of functional galactosylceramide (GalCer) receptors on CD4-negative HIV-1 target cells

Summary HIV-1 infection in CD4-negative cells is now a well-established alternative route of infection for several cell types, including mucosal epithelial cells. Among different proposed mechanisms for HIV-1 entry into CD4-negative cells, the glycosphingolipid galactosylceramide (GalCer) has been characterized as a specific receptor for the surface envelope glycoprotein gp120 in cell lines of neural and epithelial origin. In this report we describe a protocol for identifying the GalCer receptor on the surface of CD4-negative cells. The development of this assay has been motivated by the unavailability of GalCer-specific antibodies, rendering the unambiguous immunological detection of this glycolipid on the cell surface impossible. For instance, we show that the commercially available anti-GalCer mAb (the R-mAb) can bind to the surface of both GalCer-positive and GalCer-negative human colon epithelial cell lines. This lack of specificity is due to a cross-reaction with several intestinal glycolipids that are not recognized by gp120. By contrast, gp120 only binds to the surface of GalCer-positive colon cells and the binding is specifically abolished by the R-mAb, as well as by compounds previously characterized as inhibitors of the GalCer-gp120 interaction. The specificity of gp120 binding was further demonstrated by incorporating sulfatide (the sulfated derivative of GalCer) in the membrane of GalCer-negative cells: in this case, gp120 binding was easily detectable by indirect immunofluorescence. Based on these data, we suggest the following protocol for assessing the expression of functional GalCer/sulfatide receptors on the surface of CD4-negative cells: (i) binding of gp120 revealed by indirect immunofluorescence; and (ii) inhibition of gp120 binding by the R-mAb. This would be preferable to using the R-mAb directly as a probe for GalCer/sulfatide expression.     

Small-molecule HIV-1 gp120 inhibitors to prevent HIV-1 entry: an emerging opportunity for drug development

The HIV-1 gp120 envelope protein is an essential component in the multi-tiered viral entry process. Despite the overall genetic heterogeneity of the gp120 glycoprotein, the conserved CD4 binding site provides an attractive antiviral target. Recently, increased efforts aimed at the development of inhibitors of gp120 have been reported. This review focuses primarily on small-molecule gp120 inhibitors and discusses key characteristics of compounds that appear to fall within this class. The preclinical profiles of compounds that prevent gp120 from assuming a conformation favorable for CD4 binding are described in this review. In addition, inhibitors possessing some common structural features, including at least one compound that exhibits sub-nanomolar potency in a cell fusion assay are discussed. A series of compounds that were designed to enhance immune responses to virus via alteration of the gp120 conformation after targeting the CD4 binding pocket are also described. The efficacy of gp120 inhibitors as a microbicide to prevent sexual HIV transmission in the rhesus macaque model is discussed. Results suggest that this class of compounds may have value if included in a microbicide cocktail with inhibitors of alternate mechanisms. Importantly, preliminary results from clinical studies of orally administered BMS-488043 demonstrate that antiviral efficacy can be achieved in humans with a CD4-attachment inhibitor that targets gp120.     

A novel class of HIV-1 inhibitors that targets the viral envelope and inhibits CD4 receptor binding

BMS-378806 is a prototype of a new class of small molecule HIV-1 inhibitors that blocks viral attachment to cells. This compound exhibits potent inhibitory activity against a panel of HIV-1 laboratory and clinical isolates (M- and T-tropic), selective for HIV-1 and inactive against HIV-2, SIV and a panel of other viruses. BMS-378806 exhibits no significant cytotoxicity and displays many attractive pharmacological properties such as low protein binding, minimal serum effect on anti-HIV-1 potency, good oral bioavailability in animal species and a clean safety profile in initial animal toxicology studies. The compound binds to gp120 and blocks the attachment of the HIV-1 envelope protein to cellular CD4 receptors via a specific and competitive mechanism. BMS-378806 binds directly to gp120 at an approximately 1:1 stoichiometry, with a binding affinity similar to that of soluble CD4. Further confirmation that this class of compounds 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 at or near the CD4 binding pocket of gp120. Recombinant HIV-1 carrying these two substitutions demonstrated significantly reduced susceptibility to inhibition. Using these HIV-1 gp120 resistant variants and gp120/CD4 contact site mutants, the potential BMS-378806 target site was localized to a specific region within the CD4 binding pocket of gp120. Together, the data show that BMS-378806 is the first of a new class of HIV inhibitors with the potential to become a valued addition to our current repertoire of antiretroviral drugs.     

Selection and characterization of viruses resistant to the dual acting pyrimidinedione entry and non-nucleoside reverse transcriptase inhibitor IQP-0410

The 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT)-like compounds with homocyclic moieties at the N-1 of the pyrimidinedione, including the highly potent lead compound IQP-0410, inhibit HIV-1 at sub-nanomolar concentrations primarily through a typical non-nucleoside mechanism involving allosteric inhibition at the hydrophobic binding pocket of the HIV-1 RT. Like all NNRTIs, the pyrimidinediones have no activity against HIV-2 RT. The pyrimidinediones, however, also possess a second mode of action involving inhibition of virus entry at nanomolar concentrations which extends their range of action to include HIV-2. Entry inhibition occurs through recognition of a complex conformational binding site formed upon interaction of the virus with target cells, but does not involve direct inhibition of gp120-CD4 binding. In order to further explore the means by which the pyrimidinediones act, resistant strains of HIV-1 and HIV-2 were selected in cell culture and molecularly and biologically characterized. With HIV-1, three phases of resistance selection occurred which involve an initial appearance of single amino changes in the NNRTI binding pocket, followed by changes in the envelope glycoproteins gp120 and gp41, and subsequent multiple additional changes in the RT, resulting in high level resistance to IQP-0410. With HIV-2, resistance to entry inhibition was achieved with no resistance-engendering mutations detected in the HIV-2 RT. Detailed molecular and biological characterization of IQP-0410-resistant viruses was performed to define the resistance-engendering mutations present in the RT and envelope and to quantify cross-resistance to other HIV inhibitors.     

Progress in targeting HIV-1 entry

Current HIV entry inhibitors target the binding of the viral envelope glycoprotein gp120 to cellular CD4 and co-receptors, or block a late stage of the fusogenic activation of adjacent gp41. New targets are suggested by the role of cell surface protein disulfide isomerase (PDI), which attaches to the primary receptor CD4 close to the gp120-binding site. This could enable PDI to reduce gp120 disulfide bonds, which triggers the major conformational changes in gp120 and gp41 required for virus entry. Inhibiting cell surface PDI prevents HIV-1 entry. The new potential targets outlined are PDI activity as well as the sites of PDI-CD4 and PDI-gp120 interaction.     

Structure-based design, synthesis, and characterization of dual hotspot small-molecule HIV-1 entry inhibitors

Cellular infection by HIV-1 is initiated with a binding event between the viral envelope glycoprotein gp120 and the cellular receptor protein CD4. The CD4-gp120 interface is dominated by two hotspots: a hydrophobic gp120 cavity capped by Phe43(CD4) and an electrostatic interaction between residues Arg59(CD4) and Asp368(gp120). The CD4 mimetic small-molecule NBD-556 (1) binds within the gp120 cavity; however, 1 and related congeners demonstrate limited viral neutralization breadth. Herein, we report the design, synthesis, characterization, and X-ray structures of gp120 in complex with small molecules that simultaneously engage both binding hotspots. The compounds specifically inhibit viral infection of 42 tier 2 clades B and C viruses and are shown to be antagonists of entry into CD4-negative cells. Dual hotspot design thus provides both a means to enhance neutralization potency of HIV-1 entry inhibitors and a novel structural paradigm for inhibiting the CD4-gp120 protein-protein interaction.     

A simple screening system for anti-HIV drugs: syncytium formation assay using T-cell line tropic and macrophage tropic HIV env expressing cell lines--establishment and validation.

The first step in cellular entry of HIV involves binding of the viral envelope glycoprotein complex (gp120/gp41) to specific receptor molecules on the target cells. The cell-cell fusion (syncytium formation) between env expressing cells and CD4+ cells mimics the viral infection of the host cells. To search for anti-HIV substances preventing this process, we constructed the recombinant cell lines, HeLa/CD4/Lac-Z and HeLa/T-env/Tat for T-cell tropic (HIV-1(NL4-3)) system, and HOS/CD4/CCR5/Lac-Z and HeLa/M-env/Tat for macrophage tropic (HIV-1(SF162)) system. When each pair of cells were co-incubated for 20 hours, the multinuclear giant cells (syncytia) were formed and beta-galactosidase was expressed. These systems are less biohazardous because no infectious virus particles are used. Their validity in screening for anti-HIV substances which inhibit syncytium formation was confirmed using various known HIV entry inhibitors.     

Anti-HIV agents targeting the interaction of gp120 with the cellular CD4 receptor

Perhaps one of the most effective approaches to prevent and inhibit viral infections is to block host cell receptors that are used by viruses to gain cell entry. Major advances have been made over the past decade in the understanding of the molecular mechanism of HIV entry into target cells. A crucial step in this entry process is the interaction of the external HIV envelope glycoprotein, gp120, with the cellular CD4 receptor molecule. This binding step represents a potential target for new antiviral agents, and current efforts to develop safe and effective HIV entry inhibitors are focused on natural ligands and/or monoclonal antibodies that interfere with gp120/CD4 interaction. Also, small synthetic compounds obtained either by high-throughput screening of large compound libraries or by structure-guided rational design have recently entered the antiretroviral arena. In this review, the anti-HIV activity of novel entry inhibitors targeting gp120/CD4 interaction is outlined, and special attention is given to the cyclotriazadisulfonamide compounds, which are the most specific CD4-targeted antiviral drugs described so far.     

Anti-HIV agents targeting the interaction of gp120 with the cellular CD4 receptor

Perhaps one of the most effective approaches to prevent and inhibit viral infections is to block host cell receptors that are used by viruses to gain cell entry. Major advances have been made over the past decade in the understanding of the molecular mechanism of HIV entry into target cells. A crucial step in this entry process is the interaction of the external HIV envelope glycoprotein, gp120, with the cellular CD4 receptor molecule. This binding step represents a potential target for new antiviral agents, and current efforts to develop safe and effective HIV entry inhibitors are focused on natural ligands and/or monoclonal antibodies that interfere with gp120/CD4 interaction. Also, small synthetic compounds obtained either by high-throughput screening of large compound libraries or by structure-guided rational design have recently entered the antiretroviral arena. In this review, the anti-HIV activity of novel entry inhibitors targeting gp120/CD4 interaction is outlined, and special attention is given to the cyclotriazadisulfonamide compounds, which are the most specific CD4-targeted antiviral drugs described so far.     

Small-molecule HIV-1 entry inhibitors targeting gp120 and gp41: a patent review (2010-2015)

Introduction: It is essential to discover and develop small-molecule HIV-1 entry inhibitors with suitable pharmaceutical properties.

Areas covered: We review the development of small-molecule HIV-1 entry inhibitors as evidenced in patents, patent applications, and related research articles published between 2010 and 2015.

Expert opinion: HIV-1 Env gp120 and gp41 are important targets for development of HIV-1 entry inhibitors. The Phe43 pocket in gp120 and the highly conserved hydrophobic pocket on gp41 NHR-trimer are important targets for identification of HIV-1 attachment and fusion inhibitors, respectively. Compounds that bind to Phe43 pocket can block viral gp120 binding to CD4 on T cells, thus inhibiting HIV-1 attachment. However, most compounds targeting Phe43 pocket identified so far are HIV-1 entry agonists with the ability to enhance infectivity of HIV-1 in CD4-negative cells. Therefore, it is essential to identify HIV-1 entry antagonist-based HIV-1 attachment/entry inhibitors. Compounds binding to the gp41 hydrophobic pocket may inhibit CHR binding to the gp41 NHR trimer, thus blocking six-helix bundle formation and gp41-mediated virus-cell fusion. However, most lead compounds targeting this pocket have low potency, possibly because the pocket is too big or too deep. Therefore, it is necessary to identify other pockets in gp41 for developing HIV-1 fusion/entry inhibitors.     

HIV entry inhibitors targeting gp41: from polypeptides to small-molecule compounds

HIV envelope glycoprotein transmembrane subunit gp41 plays a critical role in the fusion between viral and target cell membranes. Upon gp120 binding to CD4 and a coreceptor (CCR5 or CXCR4), gp41 changes its conformation by forming N-helix trimer between N-heptad repeats (NHRs) and then six-helix bundle between the N-trimer and the C-heptad repeats (CHRs). Peptides derived from the NHR and CHR of gp41 extracellular region have demonstrated potent inhibitory activity on the HIV mediated cell fusion. One of these peptides, T-20, became the first success of a new class of anti-HIV agents, named HIV entry inhibitors. However, a relatively long peptide such as T-20 suffers from several limitations including lack of oral bioavailability and high cost of production. Great efforts have been made to develop alternative peptides and proteins with improved anti-HIV-1 activity, increased bioavailability and reduced cost of production. The most promising approach is the development of small molecule HIV entry inhibitors targeting gp41. Any molecule that blocks the process of NHR homotrimerization and the six-helix bundle formation by targeting the gp41 NHR, NHR trimer and CHR may inhibit HIV-mediated membrane fusion. The progress in development of those anti-HIV agents targeting gp41, from polypeptides to small-molecule compounds, is reviewed.     

Targeting HIV-1 gp41-induced fusion and pathogenesis for anti-viral therapy

HIV gp41 is a metastable protein whose native conformation is maintained in the form of a heterodimer with gp120. The non-covalently associated gp41/gp120 complex forms a trimer on the virus surface. As gp120 engages with HIV's receptor, CD4, and coreceptor, CXCR4 or CCR5, gp41 undergoes several conformational changes resulting in fusion between the viral and cellular membranes. Several lipophilic and amphiphilic domains have been shown to be critical in that process. While the obvious function of gp41 in viral entry is well-established its role in cellular membrane fusion and the link with pathogenesis are only now beginning to appear. Recent targeting of gp41 via fusion inhibitors has revealed an important role of this protein not only in viral entry but also in bystander apoptosis and HIV pathogenesis. Studies by our group and others have shown that the phenomenon of gp41-mediated hemifusion initiates apoptosis in bystander cells and correlates with virus pathogenesis. More interestingly, recent clinical evidence suggests that gp41 mutants arising after Enfuvirtide therapy are associated with CD4 cell increase and immunological benefits. This has in turn been correlated to a decrease in bystander apoptosis in our in vitro as well as in vivo assays. Although a great deal of work has been done to unravel HIV-1 gp41-mediated fusion mechanisms, the factors that regulate gp41-mediated fusion versus hemifusion and the mechanism by which hemifusion initiates bystander apoptosis are not fully understood. Further insight into these issues will open new avenues for drug development making gp41 a critical anti-HIV target both for neutralization and virus attenuation.     

Viral entry as the primary target for the anti-HIV activity of chicoric acid and its tetra-acetyl esters

The antiviral activity of L-chicoric acid against HIV-1 has been attributed previously to the inhibition of HIV-1 integration. This conclusion was based on the inhibition of integrase activity in enzymatic assays and the isolation of a resistant HIV strain with a mutation (G140S) in the integrase gene. Here we show that the primary antiviral target of L-CA and its analogs in cell culture is viral entry. L- and D-chicoric acid (L-CA and D-CA) and their respective tetra-acetyl esters inhibit the replication of HIV-1 (III(B) and NL4.3) and HIV-2 (ROD) in MT-4 cells at a 50% effective concentration (EC(50)) ranging from 1.7 to 70.6 microM. In a time-of-addition experiment, L-CA, D-CA, L-CATA, and D-CATA were found to interfere with an early event in the viral replication cycle. Moreover, L-CA, D-CA, and their analogs did not inhibit the replication of virus strains that were resistant toward polyanionic and polycationic compounds at subtoxic concentrations. Furthermore, HIV-1 strains resistant to L-CA and D-CA were selected in the presence of L-CA and D-CA, respectively. Mutations were found in the V2, V3, and V4 loop region of the envelope glycoprotein gp120 of the L-CA and D-CA-resistant NL4.3 strains that were not present in the wild-type NL4.3 strain. Recombination of the gp120 gene of the L-CA and D-CA resistant strain in a NL4.3 wild-type molecular clone fully rescued the phenotypic resistance toward L-CA and D-CA. No significant mutations were detected in the integrase gene of the drug-resistant virus strains. Although inhibition of HIV integrase activity by L-CA and its derivatives was confirmed in an oligonucleotide-driven assay, integrase carrying the G140S mutation was inhibited to the same extent as the wild-type integrase.